miR-135a和miR-183对3T3-L1前脂肪细胞分化及脂肪形成的调控作用研究
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摘要
本论文旨在研究miRNA对脂肪细胞分化的影响,探讨miRNA调控脂肪细胞分化和脂肪形成的分子机制。我们以5月龄大白猪和梅山猪的背部脂肪组织为研究对象,分别构建背部脂肪组织的小RNA文库,应用高通量的Solexa测序技术和生物信息学分析软件鉴定大白猪和梅山猪背部脂肪组织的miRNAs,并进行差异表达和预测新的miRNAs等一系列分析。对这些差异表达的miRNAs进行了鉴定、靶基因预测和功能分析,从中筛选与脂肪细胞分化、脂肪形成和脂肪代谢潜在相关的miRNAs。预测分析结果发现miR-135a和miR-183可能通过调控经典的Wnt/β-catenin信号通路来影响脂肪的形成。据此,本研究通过合成寡核苷酸片段miRNA mimics和miRNA inhibitor分别模拟“功能获得”和“功能缺失”来研究miRNAs对3T3-L1脂肪细胞的甘油三酯含量和分化标记基因表达水平的影响,并通过双荧素酶报告基因检测、QPCR及Western blotting分析来证实预测的靶基因。最后,通过生物信息学软件对小鼠miR-183的侧翼序列进行预测、分离并克隆其核心启动子、ChiP-PCR试验证实GATA3是小鼠nir-183的转录因子,GATA3与mir-183核心启动子的结合可能负调控pri-miR-183的转录从而抑制成熟miR-183的表达。主要研究结果如下：
1.猪背部脂肪组织miRNAs的筛选和鉴定
Solexa高通量测序分析结果发现：①在大白猪和梅山猪的背部脂肪组织样品中分别获得21,911,830和22,384,940条小RNA原始序列,其中高质量序列各为19,510,268和19,952,323条,分别占小RNA序列的89.04%和89.13%。得到的纯净小RNA读数(clean reads)分别为19,271,999条(占高质量序列的98.78%)和19,613,350条(占高质量序列的98.30%)。②在两样品库中共鉴定出121个成熟miRNAs,包括72个已知的猪miRNAs和49个同源的猪中新的miRNA*.在9个差异表达显著的保守miRNAs, ssc-miR-135\ssc-miR-224、ssc-miR-146b和ssc-miR-215在大白猪中高表达,而ssc-miR-1a、ssc-miR-122、ssc-miR-133a、ssc-miR-183和ssc-miR-204则在梅山猪中高表达。③在两样品库中共发现了140个新的候选miRNAs(对应156个基因组位点),大白猪样品中87个,梅山猪样品中102个。其中miR-1343、miR-2320、miR-2326、miR-2411和miR-2483只与牛同源保守,其他都是猪特异的miRNAs。
2.猪背部脂肪组织niRNAs的表达分析和功能预测
对6个差异表达显著的保守miRNAs及4个新的候选miRNAs进行stem-loop QPCR试验验证。分析结果显示,除miR-la以外,其余miRNAs的表达趋势与Solexa测序结果一致。
多种靶基因软件预测结果显示,小鼠的miR-135a与APC基因的3'UTR具有两个潜在的结合位点；GSK3β也可能是miR-135a的靶标；LRP6是miR-183的潜在靶基因。KEGG软件分析结果表明APC、GSK3fl和LRP6是经典Wnt/β-catenin信号通路中的重要成员。据此推测,miR-135a和miR-183有可能分别通过靶向作用APC、GSK3β和LRP6参与经典的Wnt/β-catenin信号通路来调控脂肪的形成。
3.miR-135a对3T3-L1前脂肪细胞的分化及脂肪形成的影响
与未分化的3T3-L1前脂肪细胞相比,成脂诱导分化过程中miR-135a的表达水平降低了约3倍。miR-135a处理组细胞的甘油三酯含量低于对照组,同时脂肪细胞分化标记基因(PPARγ、C/EBPa.ap2和FAS)的表达水平也降低。
采用多种方法对预测的靶基因APC进行验证：与对照组相比,miR-135a显著抑制APC3'UTR载体的荧光活性(p<0.01)。miR-135a抑制3T3-L1细胞内源APCmRNA及蛋白质的表达水平。反之,抑制miR-135a的表达,3T3-L1细胞中的APC蛋白的表达量升高。miR-135a提高经典Wnt/β-catenin信号通路CCNDI和c-myc的mRNA及细胞核中β-catenin蛋白质的表达水平。因此,miR-135a通过靶向作用APC基因,激活经典的Wnt/β-catenin信号通路,从而抑制3T3-L1前脂肪细胞的分化和脂肪形成。
4.miR-183对3T3-L1前脂肪细胞分化过程中脂肪形成的影响
诱导3T3-L1前脂肪细胞分化后的第2天和第4天,转染miR-183组的脂肪细胞分化标记基因(C/EBPa.PPARy.adiponectin和FAS)的mRNA表达量高于对照组。诱导5天的3T3-L1脂肪细胞的分析结果显示,miR-183组细胞较对照组的脂滴多、甘油三酯含量高(p<0.05)。另外,miR-183inhibitor抑制脂肪细胞分化标记基因PPARγ和C/EBPa的表达及甘油三酯的积累。
对预测的靶基因LRP6进行验证：与对照相比,miR-183显著抑制LR尸63'UTR载体的荧光活性(p<0.01)。miR-183抑制3T3-L1细胞内源LRP6mRNA及蛋白质的表达水平。miR-183抑制经典Wnt/β-catenin信号通路c-myc的mRNA及细胞核中β-catenin的表达水平。因此,miR-183通过靶向结合LRP6,抑制经典的Wnt/β-catenin信号通路的激活,从而促进脂肪形成。
5.小鼠mir-183启动子的转录活性研究
多种在线网站和软件对小鼠mir-183的预测结果表明,mir-183上游存在多个CpG岛、有可能的转录起始位点和众多的转录因子结合位点,miR-183侧翼序列存在多个EST数据报告。据此推测mir-183很有可能存在独立的转录单元。
分两段扩增miR-183上游5kb的区域,即无CpG岛的promoter1及其上游含多个CpG岛的promoter2片段。结果表明,pGL3-mir-183promoter1无荧光活性,而pGL3-mir-183promoter2的荧光活性则极显著增强(p<0.01)。进一步分析结果显示promoter2片段5端的176bp区域能够极显著增强细胞荧光活性(p<0.01),其可能是]mir-183的核心启动子区域。TFSEARCH和TESS软件预测发现核心启动子中与脂肪形成相关的转录因子sp1和GATA3。试验结果表明,GATA3转录因子极显著降低核心启动子的荧光活性(p<0.01)。与正常核心启动子相比,GATA3结合位点突变的核心启动子活性显著增强(p<0.05)。另外,GATA3转录因子抑制成熟miR-183的表达量,ChIP-PCR试验证实转录因子GATA3与mir-183核心启动子的结合。以上试验结果表明转录因子GATA3与小鼠mir-183核心启动子的结合可能抑制pri-miR-183的转录,从而导致成熟miR-183的表达量下降。
结论：①鉴定了更多的猪miRNAs,极大地丰富了猪的miRNA数据库；试验验证结果与Solexa测序结果基本一致,说明高通量Solexa测序方法的结果精确、真实可靠。②miR-135a通过直接靶向作用APC基因,促进细胞质中β-catenin蛋白的转位入核,激活经典的Wnt/β-catenin信号通路,抑制甘油三酯的积累和脂肪细胞分化标记基因的表达,从而阻碍3T3-L1前脂肪细胞的分化和脂肪形成。③miR-183通过靶向结合细胞表面受体基因LRP6,抑制经典的Wnt/β-catenin信号通路的激活,促进脂肪细胞分化和甘油三酯的积累,从而促进3T3-L1前脂肪细胞的脂肪形成。④转录因子GATA3与小鼠mir-183核心启动子区的结合成熟miR-183的表达。
This study aimed to investigate the effect of miRNA on adipocyte differentiation, and clarify the molecular mechanisms of miRNA regulating adipocyte differentiation and adipogenesis. Firstly, we chose the backfat tissues of150-day-old Large White and Meishan pigs as samples, and then constructed two small RNA libraries. High-throuput Solexa sequencing method and bio informatics softwares were used to identify miRNAs, analyze miRNAs with differentional expressions and predict novel miRNAs in both libraries. Then, these miRNAs with differentional expressions were subjected to validation, target prediction and function analysis, and miRNAs potentially correlated with preadipocyte differentiation, adipogenesis and lipid metabolism would be selected. Afterwards the analysis results found that miR-135a and miR-183may affect adipogenesis by regulating the canonical Wnt/β-catenin singnaling pathway. We hereby synthesized oligonucleotide miRNA mimics and inhibitor which simulate "gain-of-function" and "loss-of-function" to investigate the effects of miRNAs on triglyceride content and adipogenic marker genes expression levels of adipocyte. Meanwhile, the dual luciferase reporter gene analysis, QPCR assay and Western blotting examination were all adopted to validate the predicted targets. Finally, the flanking sequences of mmu-miR-183were experienced to predict by several bio informatics softwares. The core promoter of mir-183was subsequently isolated and cloned. ChlP-PCR experiment verified that GATA3is a transcription factor of mmu-mir-183, binding of GATA3with mir-183core promoter negatively regulated the transcriptional activity of pri-miR-183and maturity of miR-183. The main results as follows:
1. Screening and identification of miRNAs in porcine backfat
The analyzed results by high-throughput Solexa sequencing showed that:①21,911,830and22,384,940small RNA raw reads were generated in backfat tissues of Large White and Meishan libraries, respectively, of which19,510,268and19,952,323were high-quality reads, accounting for89.04%and89.13%of raw reads.19,271,999(accounted for98.78%of high-quality reads) and19,613,350(accounted for98.30%of high-quality reads) clean reads were obtained.②121mature miRNAs were detected in both libraries comprising72known porcine miRNAs and49miRNA*were first identified in pigs which had homologs in other species.9conserved miRNAs with significantly differential expressions were found, of which the expressions of ssc-miR-135, ssc-miR-224, ssc-miR-146b and ssc-miR-215were higher in Large White library, opposite to the patterns shown by ssc-miR-la, ssc-miR-122, ssc-miR-133a, ssc-miR-183and ssc-miR-204.③140novelly predicted miRNAs (corresponding to156independent genomic loci) were identified in both libraries, of which87miRNAs were found in Large White library and102in Meishan library. Almost all novel miRNAs could be considered pig-specific except ssc-miR-1343, miR-2320, miR-2326, miR-2411and miR-2483which had homologs in Bos taurus.
2. Expression pattern analysis and function prediction of miRNAs in procine backfat
The stem-loop QPCR method was utilized to analyzed6conserved miRNAs with significantly differential expressions and4novelly predicted miRNAs. The QPCR results validated the sequencing results and they were consistent but miR-la was an exception.
Several bioinformatics target prediction softwares were used to predict targets of mouse miR-135a and miR-183. The results showed that3'UTR of APC gene contained two potential binding sites with miR-135a, GSK3β and LRP6may also be the separate target of miR-135a and miR-183. KEGG software analysis results showed that APC, GSK3β and LRP6are all important members of and participate in the canonical Wnt/β-catenin singaling pathway. We hereby speculated that miR-135a and miR-183may involve in the canonical Wnt/β-catenin singaling pathway by separately targeting APC, GSK3β and LRP6, and then regulate adipogenesis.
3. Effect of miR-135a on3T3-L1preadipocyte differentiation and adipogenesis
The expression level of miR-135a decreased almost3times during3T3-L1preadipocyte adipogenic differentitation. The content of triglyceride was lower in miR-135a group, and meanwhile the expression levels of adipogenic maker genes (PPARy, C/EBPa, ap2and FAS) were also decreased.
Validation of predicted target APC by several methods:miR-135a significantly inhibited the luciferase activity of APC3'UTR plasmid when compared to control groups (p<0.01). The mRNA and protein expression levels of endogenous APC were both suppressed by miR-135a, and viceversa, miR-135a inhibitor increased the expression of APC protein. Futhermore, mRNA expression levels of CCND1, c-myc and protein expression levels of nuclear P-catenin downstream canonical Wnt/β-catenin singaling pathway were increased in miR-135a group. Therefore, miR-135a activated the canonical Wnt/β-catenin signaling pathway by directly targeting APC, and then impaired3T3-L1preadipocyte differentiation and adipogenesis.
4. Influence of miR-183on adipogenesis of3T3-L1preadipocyte differentiation
The mRNA expression levels of adipogenic marker genes (C/EBPa, PPARy, adiponectin and FAS) were higher in miR-183group when compared to control groups after3T3-L1cells experienced to adipogenic differentiation for2days and4days. The amount of lip id droplets and triglyceride content were both increased in miR-183group at5days. Moreover, miR-183inhibitor also decreased the expressions of PPARy and C/EBPa and accumulation of triglyceride during3T3-L1adipogenic induction.
Validation of predicted target LRP6:miR-183significantly inhibited the luciferase activity of LRP63'UTR plasmid when compared to control groups (p<0.01). The mRNA and protein expression levels of endogenous LRP6were both suppressed by miR-183. Futhermore, mRNA expression levels of c-myc and protein expression levels of nuclear P-catenin were impaired in miR-183group. Therefore, miR-183inhibited canonical Wnt/β-catenin signaling pathway by targeting LRP6, and then facilitated adipogenesis.
5. Study of transcriptional activity of mmu-mir-183promoter
The sequences of mouse mir-183were predicted and analyzed by several online websites and softwares. The results displayed that the upstream sequences of miR-183have potential transcriptional strat sites, numerous transcription factor binding sites, CpG islands (about1200bp) and many EST data reports. We thus speculated that mir-183may have its own transcription unite.
The promoter1(2309bp) and upstream fragment promoter2(1927bp) of miR-183were amplified, respectively. The results of dual luciferase reporter gene analysis showed that pGL3-mir-183promoter1has no luciferase activity, and pGL3-mir-183promoter2could significantly enhance the luciferase activity in3T3-L1, C2C12and BHK cells (p<0.01). Further experiments demonstrated that the176bp fragment in the proximal5'end of mir-183promoter2significantly enhanced the luciferase activity (p<0.01) and this176bp area may be the core promoter of mir-183. The transcription factors sp1and GATA3, which are related to adipogenesis, were found may bind with the core promoter predicted by online TFSEARCH and TESS softwares. The results of dual luciferase reporter gene analysis demonstrated that GATA3significantly decreased the luciferase activity of core promoter (p<0.01). Furthermore, the luciferase activity of core promoter with mutated GATA3binding sites was significantly increased when compared to wild core promoter (p<0.05). QPCR analysis results indicated that the expression level of mature miR-183was significantly inhibited by transcription factor GATA3. ChIP-PCR experiment verified binding of transcription factor GATA3with mir-183core promoter. These outcomes confirmed that binding of transcription factor GATA3with mmu-mir-183core promoter may inhibit the transcriptional activity of pri-miR-183and generation of mature miR-183and thus decreased its expression.
Conclusion:①Identificaiton of more porcine miRNAs, and enrich the repertoire of miRNAs in pigs; Our experimental results displayed a high level of concordance with that of Solexa sequencing, showing that the results of high-throughput Solexa sequencing strategy are accurate, true and reliable.②miR-135a promoted the translocation of β-catenin protein from cytoplasm to nuclear by directly targeting APC, activated the canonical Wnt/β-catenin singaling pathway, inhibited the accumulation of triglyceride and expressions of adipogenic marker genes, and then impaired the3T3-L1adipogenic differentiation and adipogenesis.③miR-183inhibited canonical Wnt/β-catenin singaling pathway by directly targeting cellular surface receptor gene LRP6, acclerated adipocyte differentiation and accumulation of triglyceride, and then supported the adipogenesis of3T3-L1cells.④Binding of transcription factor GATA3with mmu-mir-183core promoter inhibited the transcriptional activity of pri-miR-183and generation of mature miR-183, and then led to the final decreased miR-183expression.
1.猪背部脂肪组织miRNAs的筛选和鉴定
Solexa高通量测序分析结果发现：①在大白猪和梅山猪的背部脂肪组织样品中分别获得21,911,830和22,384,940条小RNA原始序列,其中高质量序列各为19,510,268和19,952,323条,分别占小RNA序列的89.04%和89.13%。得到的纯净小RNA读数(clean reads)分别为19,271,999条(占高质量序列的98.78%)和19,613,350条(占高质量序列的98.30%)。②在两样品库中共鉴定出121个成熟miRNAs,包括72个已知的猪miRNAs和49个同源的猪中新的miRNA*.在9个差异表达显著的保守miRNAs, ssc-miR-135\ssc-miR-224、ssc-miR-146b和ssc-miR-215在大白猪中高表达,而ssc-miR-1a、ssc-miR-122、ssc-miR-133a、ssc-miR-183和ssc-miR-204则在梅山猪中高表达。③在两样品库中共发现了140个新的候选miRNAs(对应156个基因组位点),大白猪样品中87个,梅山猪样品中102个。其中miR-1343、miR-2320、miR-2326、miR-2411和miR-2483只与牛同源保守,其他都是猪特异的miRNAs。
2.猪背部脂肪组织niRNAs的表达分析和功能预测
对6个差异表达显著的保守miRNAs及4个新的候选miRNAs进行stem-loop QPCR试验验证。分析结果显示,除miR-la以外,其余miRNAs的表达趋势与Solexa测序结果一致。
多种靶基因软件预测结果显示,小鼠的miR-135a与APC基因的3'UTR具有两个潜在的结合位点；GSK3β也可能是miR-135a的靶标；LRP6是miR-183的潜在靶基因。KEGG软件分析结果表明APC、GSK3fl和LRP6是经典Wnt/β-catenin信号通路中的重要成员。据此推测,miR-135a和miR-183有可能分别通过靶向作用APC、GSK3β和LRP6参与经典的Wnt/β-catenin信号通路来调控脂肪的形成。
3.miR-135a对3T3-L1前脂肪细胞的分化及脂肪形成的影响
与未分化的3T3-L1前脂肪细胞相比,成脂诱导分化过程中miR-135a的表达水平降低了约3倍。miR-135a处理组细胞的甘油三酯含量低于对照组,同时脂肪细胞分化标记基因(PPARγ、C/EBPa.ap2和FAS)的表达水平也降低。
采用多种方法对预测的靶基因APC进行验证：与对照组相比,miR-135a显著抑制APC3'UTR载体的荧光活性(p<0.01)。miR-135a抑制3T3-L1细胞内源APCmRNA及蛋白质的表达水平。反之,抑制miR-135a的表达,3T3-L1细胞中的APC蛋白的表达量升高。miR-135a提高经典Wnt/β-catenin信号通路CCNDI和c-myc的mRNA及细胞核中β-catenin蛋白质的表达水平。因此,miR-135a通过靶向作用APC基因,激活经典的Wnt/β-catenin信号通路,从而抑制3T3-L1前脂肪细胞的分化和脂肪形成。
4.miR-183对3T3-L1前脂肪细胞分化过程中脂肪形成的影响
诱导3T3-L1前脂肪细胞分化后的第2天和第4天,转染miR-183组的脂肪细胞分化标记基因(C/EBPa.PPARy.adiponectin和FAS)的mRNA表达量高于对照组。诱导5天的3T3-L1脂肪细胞的分析结果显示,miR-183组细胞较对照组的脂滴多、甘油三酯含量高(p<0.05)。另外,miR-183inhibitor抑制脂肪细胞分化标记基因PPARγ和C/EBPa的表达及甘油三酯的积累。
对预测的靶基因LRP6进行验证：与对照相比,miR-183显著抑制LR尸63'UTR载体的荧光活性(p<0.01)。miR-183抑制3T3-L1细胞内源LRP6mRNA及蛋白质的表达水平。miR-183抑制经典Wnt/β-catenin信号通路c-myc的mRNA及细胞核中β-catenin的表达水平。因此,miR-183通过靶向结合LRP6,抑制经典的Wnt/β-catenin信号通路的激活,从而促进脂肪形成。
5.小鼠mir-183启动子的转录活性研究
多种在线网站和软件对小鼠mir-183的预测结果表明,mir-183上游存在多个CpG岛、有可能的转录起始位点和众多的转录因子结合位点,miR-183侧翼序列存在多个EST数据报告。据此推测mir-183很有可能存在独立的转录单元。
分两段扩增miR-183上游5kb的区域,即无CpG岛的promoter1及其上游含多个CpG岛的promoter2片段。结果表明,pGL3-mir-183promoter1无荧光活性,而pGL3-mir-183promoter2的荧光活性则极显著增强(p<0.01)。进一步分析结果显示promoter2片段5端的176bp区域能够极显著增强细胞荧光活性(p<0.01),其可能是]mir-183的核心启动子区域。TFSEARCH和TESS软件预测发现核心启动子中与脂肪形成相关的转录因子sp1和GATA3。试验结果表明,GATA3转录因子极显著降低核心启动子的荧光活性(p<0.01)。与正常核心启动子相比,GATA3结合位点突变的核心启动子活性显著增强(p<0.05)。另外,GATA3转录因子抑制成熟miR-183的表达量,ChIP-PCR试验证实转录因子GATA3与mir-183核心启动子的结合。以上试验结果表明转录因子GATA3与小鼠mir-183核心启动子的结合可能抑制pri-miR-183的转录,从而导致成熟miR-183的表达量下降。
结论：①鉴定了更多的猪miRNAs,极大地丰富了猪的miRNA数据库；试验验证结果与Solexa测序结果基本一致,说明高通量Solexa测序方法的结果精确、真实可靠。②miR-135a通过直接靶向作用APC基因,促进细胞质中β-catenin蛋白的转位入核,激活经典的Wnt/β-catenin信号通路,抑制甘油三酯的积累和脂肪细胞分化标记基因的表达,从而阻碍3T3-L1前脂肪细胞的分化和脂肪形成。③miR-183通过靶向结合细胞表面受体基因LRP6,抑制经典的Wnt/β-catenin信号通路的激活,促进脂肪细胞分化和甘油三酯的积累,从而促进3T3-L1前脂肪细胞的脂肪形成。④转录因子GATA3与小鼠mir-183核心启动子区的结合成熟miR-183的表达。
This study aimed to investigate the effect of miRNA on adipocyte differentiation, and clarify the molecular mechanisms of miRNA regulating adipocyte differentiation and adipogenesis. Firstly, we chose the backfat tissues of150-day-old Large White and Meishan pigs as samples, and then constructed two small RNA libraries. High-throuput Solexa sequencing method and bio informatics softwares were used to identify miRNAs, analyze miRNAs with differentional expressions and predict novel miRNAs in both libraries. Then, these miRNAs with differentional expressions were subjected to validation, target prediction and function analysis, and miRNAs potentially correlated with preadipocyte differentiation, adipogenesis and lipid metabolism would be selected. Afterwards the analysis results found that miR-135a and miR-183may affect adipogenesis by regulating the canonical Wnt/β-catenin singnaling pathway. We hereby synthesized oligonucleotide miRNA mimics and inhibitor which simulate "gain-of-function" and "loss-of-function" to investigate the effects of miRNAs on triglyceride content and adipogenic marker genes expression levels of adipocyte. Meanwhile, the dual luciferase reporter gene analysis, QPCR assay and Western blotting examination were all adopted to validate the predicted targets. Finally, the flanking sequences of mmu-miR-183were experienced to predict by several bio informatics softwares. The core promoter of mir-183was subsequently isolated and cloned. ChlP-PCR experiment verified that GATA3is a transcription factor of mmu-mir-183, binding of GATA3with mir-183core promoter negatively regulated the transcriptional activity of pri-miR-183and maturity of miR-183. The main results as follows:
1. Screening and identification of miRNAs in porcine backfat
The analyzed results by high-throughput Solexa sequencing showed that:①21,911,830and22,384,940small RNA raw reads were generated in backfat tissues of Large White and Meishan libraries, respectively, of which19,510,268and19,952,323were high-quality reads, accounting for89.04%and89.13%of raw reads.19,271,999(accounted for98.78%of high-quality reads) and19,613,350(accounted for98.30%of high-quality reads) clean reads were obtained.②121mature miRNAs were detected in both libraries comprising72known porcine miRNAs and49miRNA*were first identified in pigs which had homologs in other species.9conserved miRNAs with significantly differential expressions were found, of which the expressions of ssc-miR-135, ssc-miR-224, ssc-miR-146b and ssc-miR-215were higher in Large White library, opposite to the patterns shown by ssc-miR-la, ssc-miR-122, ssc-miR-133a, ssc-miR-183and ssc-miR-204.③140novelly predicted miRNAs (corresponding to156independent genomic loci) were identified in both libraries, of which87miRNAs were found in Large White library and102in Meishan library. Almost all novel miRNAs could be considered pig-specific except ssc-miR-1343, miR-2320, miR-2326, miR-2411and miR-2483which had homologs in Bos taurus.
2. Expression pattern analysis and function prediction of miRNAs in procine backfat
The stem-loop QPCR method was utilized to analyzed6conserved miRNAs with significantly differential expressions and4novelly predicted miRNAs. The QPCR results validated the sequencing results and they were consistent but miR-la was an exception.
Several bioinformatics target prediction softwares were used to predict targets of mouse miR-135a and miR-183. The results showed that3'UTR of APC gene contained two potential binding sites with miR-135a, GSK3β and LRP6may also be the separate target of miR-135a and miR-183. KEGG software analysis results showed that APC, GSK3β and LRP6are all important members of and participate in the canonical Wnt/β-catenin singaling pathway. We hereby speculated that miR-135a and miR-183may involve in the canonical Wnt/β-catenin singaling pathway by separately targeting APC, GSK3β and LRP6, and then regulate adipogenesis.
3. Effect of miR-135a on3T3-L1preadipocyte differentiation and adipogenesis
The expression level of miR-135a decreased almost3times during3T3-L1preadipocyte adipogenic differentitation. The content of triglyceride was lower in miR-135a group, and meanwhile the expression levels of adipogenic maker genes (PPARy, C/EBPa, ap2and FAS) were also decreased.
Validation of predicted target APC by several methods:miR-135a significantly inhibited the luciferase activity of APC3'UTR plasmid when compared to control groups (p<0.01). The mRNA and protein expression levels of endogenous APC were both suppressed by miR-135a, and viceversa, miR-135a inhibitor increased the expression of APC protein. Futhermore, mRNA expression levels of CCND1, c-myc and protein expression levels of nuclear P-catenin downstream canonical Wnt/β-catenin singaling pathway were increased in miR-135a group. Therefore, miR-135a activated the canonical Wnt/β-catenin signaling pathway by directly targeting APC, and then impaired3T3-L1preadipocyte differentiation and adipogenesis.
4. Influence of miR-183on adipogenesis of3T3-L1preadipocyte differentiation
The mRNA expression levels of adipogenic marker genes (C/EBPa, PPARy, adiponectin and FAS) were higher in miR-183group when compared to control groups after3T3-L1cells experienced to adipogenic differentiation for2days and4days. The amount of lip id droplets and triglyceride content were both increased in miR-183group at5days. Moreover, miR-183inhibitor also decreased the expressions of PPARy and C/EBPa and accumulation of triglyceride during3T3-L1adipogenic induction.
Validation of predicted target LRP6:miR-183significantly inhibited the luciferase activity of LRP63'UTR plasmid when compared to control groups (p<0.01). The mRNA and protein expression levels of endogenous LRP6were both suppressed by miR-183. Futhermore, mRNA expression levels of c-myc and protein expression levels of nuclear P-catenin were impaired in miR-183group. Therefore, miR-183inhibited canonical Wnt/β-catenin signaling pathway by targeting LRP6, and then facilitated adipogenesis.
5. Study of transcriptional activity of mmu-mir-183promoter
The sequences of mouse mir-183were predicted and analyzed by several online websites and softwares. The results displayed that the upstream sequences of miR-183have potential transcriptional strat sites, numerous transcription factor binding sites, CpG islands (about1200bp) and many EST data reports. We thus speculated that mir-183may have its own transcription unite.
The promoter1(2309bp) and upstream fragment promoter2(1927bp) of miR-183were amplified, respectively. The results of dual luciferase reporter gene analysis showed that pGL3-mir-183promoter1has no luciferase activity, and pGL3-mir-183promoter2could significantly enhance the luciferase activity in3T3-L1, C2C12and BHK cells (p<0.01). Further experiments demonstrated that the176bp fragment in the proximal5'end of mir-183promoter2significantly enhanced the luciferase activity (p<0.01) and this176bp area may be the core promoter of mir-183. The transcription factors sp1and GATA3, which are related to adipogenesis, were found may bind with the core promoter predicted by online TFSEARCH and TESS softwares. The results of dual luciferase reporter gene analysis demonstrated that GATA3significantly decreased the luciferase activity of core promoter (p<0.01). Furthermore, the luciferase activity of core promoter with mutated GATA3binding sites was significantly increased when compared to wild core promoter (p<0.05). QPCR analysis results indicated that the expression level of mature miR-183was significantly inhibited by transcription factor GATA3. ChIP-PCR experiment verified binding of transcription factor GATA3with mir-183core promoter. These outcomes confirmed that binding of transcription factor GATA3with mmu-mir-183core promoter may inhibit the transcriptional activity of pri-miR-183and generation of mature miR-183and thus decreased its expression.
Conclusion:①Identificaiton of more porcine miRNAs, and enrich the repertoire of miRNAs in pigs; Our experimental results displayed a high level of concordance with that of Solexa sequencing, showing that the results of high-throughput Solexa sequencing strategy are accurate, true and reliable.②miR-135a promoted the translocation of β-catenin protein from cytoplasm to nuclear by directly targeting APC, activated the canonical Wnt/β-catenin singaling pathway, inhibited the accumulation of triglyceride and expressions of adipogenic marker genes, and then impaired the3T3-L1adipogenic differentiation and adipogenesis.③miR-183inhibited canonical Wnt/β-catenin singaling pathway by directly targeting cellular surface receptor gene LRP6, acclerated adipocyte differentiation and accumulation of triglyceride, and then supported the adipogenesis of3T3-L1cells.④Binding of transcription factor GATA3with mmu-mir-183core promoter inhibited the transcriptional activity of pri-miR-183and generation of mature miR-183, and then led to the final decreased miR-183expression.
引文
1. 柴进.宰前应激对猪肉质的影响及其机制研究.[博士学位论文].武汉：华中农业大学图书馆,2010
2. 李国喜.猪脂肪组织发育相关miRNA的鉴定及miR-103生物学功能的初步研究.[博士学位论文].武汉：华中农业大学图书馆,2011
3. 李鹏昊.miR-27a、miR-143对猪脂肪沉积和miR-1、miR-206对肌肉肥大的调控研究.[硕士学位论文].武汉：华中农业大学图书馆,2011
4. 宁小敏.miR-196a对猪脂肪细胞增殖分化的影响.[硕士学位论文].武汉：华中农业大学图书馆,2010
5. 屈长青,张国华,陈粉粉,赵丽丽,杨公社.猪前体脂肪细胞的原代培养.农业生物技术学报,2005,13：649-653
6. 覃丽梅.脂肪细胞分化的WNT信号通路中microRNA表达谱分析及其功能研究.[博士学位论文].武汉：华中农业大学图书馆,2010
7. 徐露莲,史春梅,陈玲,季晨博,郭锡熔,徐美玉.脂肪细胞分化相关miRNA-hsa-miR-378生物信息学分析.中国儿童保健杂志,2012,20：1001-1004
8. 张国华,杨公社,屈长青,孙世铎.猪前体脂肪细胞的分离培养.细胞生物学杂志,2005,27：693-696
9. 朱璐,史春梅,崔焱,徐广峰,季晨博,倪毓辉,郭锡熔.hsa-miR-335靶基因预测及生物信息学分析.西部医学,2012,24：1851-1855
10. AguilO F, Camarero N, Relat J, Marrero PF, Haro D. Transcriptional regulation of the human acetoacetyl-CoA synthetase gene by PPARy. Biochem J,2010,427: 255-264
11. Altuvia Y, Landgraf P, Lithwick G, Elefant N, Pfeffer S, Aravin A, Brownstein MJ, Tuschl T, Margalit H. Clustering and conservation patterns of human microRNAs. Nucleic Acids Res,2005,33:2697-2706
12. Ambros V. The functions of animal microRNAs. Nature,2004,431:350-355
13. Andersen DC, Jensen CH, Schneider M, Nossent AY, Eskildsen T, Hansen JL, Teisner B, Sheikh SP. MicroRNA-15a fine-tunes the level of Delta-like 1 homolg (DLK1) in proliferating 3T3-L1 preadipocytes. Exp Cell Res,2010,316:1681-1691
14. Antonson P, Pray MG, Jacobsson A, Xanthopoulos KG Myc inhibits CCAAT/enhancer-binding protein alpha-gene expression in HIB-1B hibernoma cells through interactions with the core promoter region. Eur JBiochem,1995,232: 397-403
15. Aravin A, Tuschl T. Identification and characterization of small RNAs involved in RNA silencing. FEBS Lett,2005,579:5830-5840
16. Bartel DP. MicroRNA:genomics, biogenesis, mechanism, and function. Cell,2004, 116:281-297
17. Barth N, Langmann T, SchOlmerich J, Schmitz G, Schaffler A. Identification of regulatory elements in the human adipose most abundant gene transcript-1 (apM-1) promoter:role of SP1/SP3 and TNF-a as regulatory pathways. Diabetologia,2002, 45:1425-1433
18. Bejerano G, Pheasant M, Makunin I, Stephen S, Kent WJ, Mattick JS, Haussler D. Ultraconserved elements in human genome. Science,2004,304:1321-1325
19. Bennett CN, Ross SE, Longo KA, Bajnok L, Hemati N, Johnson KW, Harrison SD, MacDougald OA. Regulation of Wnt signaling during adipogenesis. J Biol Chem, 2002,277:30998-31004
20. Bhat RA, Stauffer B, Komm BS, Bodine PVN. Structure-function analysis of secreted frizzled-related protein-1 for its Wnt antagonist function. J Cell Biochem, 2007,102:1519-1528
21. Bolw MJ, Grocock RJ, Dongen SV, Enright AJ, Dicks E, Futreal PA, Wooster R, Stratton MR. RNA editing of human microRNAs. Genome Biol,2006,7:R27
22. Card DA, Hebbar PB, Li LP, Trotter KW, Komatsu Y, Mishina Y, Archer TK. Oct4/Sox2-regulated miR-302 targets cyclin D1 in human embryonic stem cells. Mol Cell Biol,2008,28:6426-6438
23. Chen CF, Ridzon DA, Broomer AJ, Zhou ZH, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvaker VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res,2005,33: e179
24. Chen JF, Mandel EM, Thomson JM, Wu QL, Callis TE, Hammond SM, Conlon FL, Wang DZ. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet,2005,38:228-233
25. Chendrimada TP, Finn KJ, Ji XJ, Baillat D, Gregory RI, Liebhaber SA, Pasquinelli AE, Shikhattar R. MicroRNA silencing through RISC recruitment of eIF6. Nature, 2007,447:823-828
26. Cho IS, Kim J, Seo HY, Lim DH, Hong JS, Park YH, Park DC, Hong KC, Whang KY, Lee YS. Cloning and characterization of microRNAs from porcine skeletal muscle and adipose tissue. Mol Biol Rep,2010,37:3567-3574
27. Christodoulides C, Lagathu C, Sethi JK, Vidal-Puig A. Adipogenesis and WNT signaling. Trends Endocrinol Metab,2009,20:16-24
28. Cirera S, Birck M, Busk PK, Fredholm M. Expression profiles of miRNA-122 and its target CAT1 in minipigs(Sus scrofa) fed a high-cholesterol diet. Comp Med, 2010,60:136-141
29. Cullen BR. Viruses and microRNAs. Nat Genet,2006,38:S25-S30
30. Davalos A, Goedeke L, Smibert P, Ramirez CM, Warrier NP, Andreo U, Cirera-Salinas D, Rayner K, Suresh U, Pastor-Pareja JC, Esplugues E, Fisher EA, Penalva LO, Moore KJ, Suarez Y, Lai EC, Fernadez-Hernando C. miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling. Proc Natl Acad Sci USA,2011,108:9232-9237
31. Doench JG, Petersen CP, Sharp PA. siRNAs can function as miRNAs. Gene Dev, 2003,17:438-442
32. Ebhardt HA, Tsang HH, Dai DC, Liu YF, Bostan B, Fahlman RP. Meta-analysis of small RNA-sequencing errors reveals ubiquitous post-transcriptional RNA modifications. Nucleic Acids Res,2009,37:2461-2470
33. Esau C, Davis S, Murray SF, Yu XX, Pandey SK, Pear M, Watts L, Booten SL, Graham M, McKay R, Subramaniam A, Propp S, Lollo BA, Freier SS, Bennett CF, Bhanot S, Monia BP. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab,2006,3:87-98
34. Kennell JA, Gerin I, MacDougald OA, Cadigan KM. The microRNA miR-8 is a conserved negative regulator of Wnt signaling. Proc Natl Acad Sci USA,2008,105: 15417-15422
35. Esau C, Kang XL, Peralta E, Hanson E, Marcusson EG, Ravichandran LV, Sun YQ, Koo S, Perera RJ, Jain R, Dean NM, Freier SM, Bennett F, Lollo B, Griffey R. MicroRNA-143 regulates adipocyte differentiation.J Biol Chem,2004,279: 52361-52365
36. Feederle R, Linstaedt SD, Bannert H, Lips H, Bryan M, Cullen BR, Delecluse HJ. A viral microRNA cluster strongly potentiates the transforming properties of a human herpesvirus. PLoS Pathog,2011,7:e10001294
37. Freytag SO, Geddes TJ. Reciprocal regulation of adipogenesis by Myc and C/EBP alpha. Science,1992,256:379-382
38. Fu MF, Rao M, Bouras T, Wang CG, Wu KM, Zhang XP, Li ZP, Yao TP, Pestell RG Cyclin D1 inhibits peroxisome proliferator-activated receptor y-mediated adipogenesis through histone deacetylase recruitment. J Biol Chem,2005,280: 16934-16941
39. Gerin I, Bommer GT, McCoin CS, Sousa KM, Krishnan V, MacDougald OA. Roles for miRNA-378/378* in adipocyte gene expression and lipogenesis. Am J Physiol Endocrinol Metab,2010,299:E198-E206
40. Glazov EA, Cottee PA, Barris WC, Moore RJ, Dalrymple BP, Tizard ML. A microRNA catalog of the developing chicken embryo identified by a deep sequencing approach. Genome Res,2008:957-964
41. Glazov EA, Kongsuwan K, Assavalapsakul W, Horwood PF, Mitter N, Mahony TJ. Repertorie of bovine miRNA and miRNA-like small regulatory RNAs expressed upon viral infection. PLoS One,2009,4:e6349
42. Gorodkin J, Cirera S, Hedegaard J, Gilchrist MJ, Panitz F, J(?)rgensen C, Scheibye-Knudsen K, Arvin T, Lumholdt S, Sawera M, Green T, Nielsen BJ, Havgaard JH, Rosenkilde C, Wang J, Li H, Li RQ, Liu B, Hu SN, Dong W et al. Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags. Genome Biol,2007,8:R45
43. Gu J, He T, Pei YF, Li F, Wang XW, Zhang J, Zhang XG, Li YD. Primary transcripts and expressions of mammal intergenic microRNAs detected by mapping ESTs to their flanking sequences. Mamm Genome,2006,17:1033-1041
44. Guo YX, Mo DL, Zhang Y, Zhang Y, Cong PQ, Xiao SQ, He ZY, Liu XH, Chen YS. MicroRNAome comparison between intramuscular and subcutaneous vascular stem cell adipogenesis. PLoS One,2012,7:e45410
45. Gupta RK, Arany Z, Seale P, Mepani RJ, Ye L, Conroe HM, Roby YA, Kulaga H, Reed RR, Spiegelman BM. Transcriptional control of preadipocyte determination by Zfp423. Nature,2010,464:619-623
46. He A, Zhu LL, Gupta N, Chang YS, Fang FD. Overexpression of micro ribonucleic acid 29, highly up-regulated in diabetic rats, leads to insulin resistance in 3T3-L1 adipocytes. Mol Endocrinol,2007,21:2785-2794
47. He TC, Sparks AB, Rago C, Hermeking H, Zawel L, Da Costa LT, Morin PJ, Vogelstein B, Kinzler KW. Identification of c-MYC as a target of the APC pathway. Science,1998,281:1509-1512
48. Heneghan HM, Miller N, McAnena OJ, O'Brien T, Kerin MJ. Differential miRNA expression in omental adipose tissue and in the circulation of obese patients identifies novel metabolic biomarkers. J Clin Endocrinol Metab,2011,96: E846-E850
49. Herrera BM, Lockstone HE, Taylor JM, Ria M, Barrett A, Collins S, Kaisaki P, Argoud K, Fernandez C, Travers ME, Grew JP, Randall JC, Gloyn AL, Gaufuier D, McCarthy MI, Lindgren CM. Global microRNA expression profiles in insulin target tissues in a spontaneous rat model of type 2 diabetes. Diabetologia,2010,53: 1099-1109
50. Herrera BM, Lockstone HE, Taylor JM, Wills QF, Kaisaki PJ, Barrett A, Camps C, Fernandez C, Ragoussis J, Gauguier D, McCarthy MI, Lindgren CM. MicroRNA-125a is over-expressed in insulin target tissues in a spontaneous rat model of type 2 diabetes. BMC Med Genomics,2009,2:54
51. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease RL. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene,1989,77:51-59
52. Huang J, Zhao L, Xing LP, Chen D. MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation. Stem Cells,2010,28: 357-364
53. Huang K, Zhang JX, Han L, You YP, Jiang T, Pu PY, Kang CS. MicroRNA roles in beta-catenin pathway. Mol Cancer,2010,9:252
54. Huang TH, Zhu MJ, Li XY, Zhao SH. Discovery of porcine microRNAs and profiling from skeletal muscle tissues during development. PLoS One,2008,3: e3225
55. Huelsken J, Brichmeier W. New aspects of Wnt signaling pathways in higher vertebrates. Curr Opin Genet Dev,2001,11:547-553
56. Iliopoulos D, Drosatos K, Hiyama Y, Goldberg IJ, Zannis VI. MicroRNA-370 controls the expression of MicroRNA-122 and Cptla and affects lipid metabolism. JLipid Res,2010,51:1513-1523
57. John E, Wienecke-Baldacchino A, Liivrand M, Heinaniemi M, Carlberg C, Sinkkonen L. Dataset integration identifies transcriptional regulation of microRNA genes by PPARy in differentiating mouse 3T3-L1 adipocytes. Nucleic Acids Res, 2012,40:4446-4460
58. Jopling CL, Norman KL, Sarnow P. Positive and negative modulation of viral and cellular mRNAs by liver-specific microRNA miR-22. Cold Spring Harb Symp Quant Biol,2006,71:369-376
59. Kajimoto K, Naraba H, Iwai N. MicroRNA and 3T3-L1 pre-adipocyte differentiation. RNA,2006,12:1626-1632
60. Kanazawa A, Tsukada S, Kamiyama M, Yanagimoto T, Nakajima M, Maeda S. Wnt5b partially inhibits canonical Wnt/β-catenin signaling pathway and promotes adipogenesis in 3T3-L1 preadipocytes. Biochem Biophys Res Commun,2005,330: 505-510
61. Karbiener M, Fischer C, Nowitsch S, Opriessnig P, Papak C, Ailhaud G, Dani C, Amri EZ, Scheideler M. microRNA miR-27b impairs human adipocyte differentiation and targets PPARy. Biochem Biophys Res Commun,2009,390: 247-251
62. Karbiener M, Neuhold C, Opriessnig P, Prokesch A, Bogner-Strauss JG, Scheideler M. MicroRNA-30c promotes human adipocyte differentiation and co-represses PAI-1 and ALK2. RNA Biol,2011,8:850-860
63. Kawai M, Mushiake S, Bessho K, Murakami M, Namba N, Kokubu C, Michigami T, Ozono K. Wnt/Lrp/β-catenin signaling suppress adipogenesis by inhibiting mutual activation of PPRAy and C/EBPa. Biochem Biophys Res Commun,2007,363: 276-282
64. Keller P, Gburcik V, Petrovic N, Gallagher IJ, Nedergaard J, Cannon B, Timmons JA. Gene-chip studies of adipogenesis-regualted microRNAs in mouse primary adipocytes and human obesity. BMC Endocr Disord,2011,11:7
65. Kershwa EE, Flier JS. Adipose tissue as an endocrine organ. J Clin Endocrinol Metab.2004,89:2548-2556
66. Kim HJ, Cui XS, Kim EJ, Kim WJ, Kim NH. New porcine microRNA genes found by homo logy search. Genome,2006,49:1283-1286
67. Kim J, Cho IS, Hong JS, Choi YK, Kim H, Lee YS. Identification and characterization of new microRNAs from pig. Mamm Genome,2008,19:570-580
68. Kim SY, Kim AY, Lee HY, Son HY, Lee GY, Lee JW, Lee YS, Kim JB. miR-27a is a negative regulator of adipocyte differentiation via suppressing PPARy expression. Biochem Biophys Res Commun,2010,392:323-328
69. Kim YJ, Hwang SH, Cho HH, Shin KK, Bae YC, Jung JS. MicroRNA 21 regulates the proliferation of human adipose tissue-derived mesenchymal stem cells and high-fat diet-induced obesity alters microRNA 21 expression in white adipose tissues. J Cell Physiol,2012,227:183-193
70. Kim YJ, Hwang SJ, Bae YC, Jung JS. MiR-21 regulates adipogenic differentiation through the modulation of TGF-beta signaling in mesenchymal stem cells derived from human adipose tissue. Stem Cells,2009,27:3093-3102
71. Kinoshita M, Ono K, Horie T, Nagao K, Nishi H, Kuwabara Y, Takanabe-Mori R, Hasegawa K, Kita T, Kimura T. Regulation of adipocyte differentiation by actibation of serotonin (5-HT) receptors 5-HT2AR and 5-HT2cR and involvement of microRNA-448-mediated repression of KLF5. Mol Endocrinol,2010,24: 1978-1987
72. KrUtzfeldt J, Rajewsky N, Braich R, Rajeevv KG, Tuschl T, Manoharan M, Stoffel M. Silencing of microRNA in vivo with'antagomirs'. Nature,2005,438:685-689
73. Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T. Identification of tissue-specific microRNAs from mouse. Curr Biol,2002,12: 735-739
74. Lee EK, Lee MJ, Abdelmohsen K, Kim W, Kim MM, Srikantan S, Martindale JL, Hutchison ER, Kim HH, Marasa BS, Selimyan R, Egan JM, Smith SR, Fried SK, Gorospe M. miR-130 suppress adipogenesis by inhibiting'peroxisome proliferator-activated receptor y expression. Mol Cell Biol,2010,31:626-638
75. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell,1993,75:843-854
76. Lefterova MI, Lazar MA. New developments in adipogenesis. Trends Endocrinol Metab,2009,20:107-114
77. Li GX, Li YJ, Li XJ, Ning XM, Li MH, Yang GS. MicroRNA identity and abundance in developing swine adipose tissue as determined by Solexa sequencing. J Cell Biochem,2011,112:1318-1328
78. Li HX, Luo X, Liu RX, Yang YJ, Yang GS. Roles of Wnt/β-catenin signaling in adipogenic differentiation potential of adipose-derived mesenchymal stem cells. Mol Cell Endocrinol,2008,291:116-124
79. Li HY, Xi QY, Xiong YY, Liu XL, Cheng X, Shu G, Wang SB, Wang LN, Gao P, Zhu XT, Jiang QY, Yuan L, Zhang YL. Identification and comparison of microRNAs from skeletal muscle and adipose tissues from two porcine breeds. Anim Genet,2012,43:704-713
80. Li MZ, Xia YL, Gu YR, Zhang K, Lang QL, Chen L, Guan JQ, Luo ZG, Chen HS, Li Y, Li QH, Li X, Jiang AA, Shuai SR, Wang JR, Zhu Q, Zhou XC, Gao XL, Li XW. MicroRNAome of porcine pre-and postnatal development. PLoS One,2010,5; e11541
81. Li ZY, Hassan MQ, Volinia S, Van Wijnen AJ, Stein JL, Croce CM, Lian JB, Stein GS. A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci USA,2008,105:13906-13911
82. Lian CJ, Sun BX, Niu SL, Yang RJ, Liu BY, Lu CY, Meng JL, Qiu ZY, Zhang LY, Zhao ZH. A comparative profile of the microRNA transcriptome in immature and mature porcine testes using Solexa deep sequencing. FEBSJ,2012,279:964-975
83. Liao BJ, Bao XC, Liu LQ, Feng SP, Zovoilis A, Liu WB, Xue YT, Cai J, Guo XP, Qin BM, Zhang RS, Wu JY, Lai LX, Teng MK, Niu LW, Zhang BL, Esteban MA, Pei DQ. MicroRNA cluster 302-367 enhances somatic cell reprogramming by accelerating a mesenchymal-to-epithelial transition. J Biol Chem,2011,286: 17359-17364
84. Lin KY, Zhang XJ, Feng DD, Zhang H, Zeng CW, Han BW, Zhou AD, Ou LH, Xu L, Chen YQ. miR-125b, a target of CDX2, regulates cell differentiation through repression of the core binding factor in hematopoietic malignancies. J Biol Chem, 2011,286:38253-38263
85. Lin Q, Gao Z, Alarcon RM, Ye J, Yun Z. A role of miR-27 in the regulation of adipogenesis. FEBS Lett,2009,276:2348-2358
86. Ling HY, Ou HS, Feng SD, Zhang XY, Tuo QH, Chen LX, Zhu BY, Gao ZP, Tang CK, Yin WD, Zhang L, Liao DF. Changes in microRNA (miR) profile and effects of miR-320 in insulin-resistant 3T3-L1 adipocytes. Clin Exp Pharmacol Physiol,2009, 36:e32-e39
87. Ling HY, Wen GB, Feng SD, Tuo QH, Ou HS, Yao CH, Zhu YB, Gao ZP, Zhang L, Liao DF. MicroRNA-375 promotes 3T3-L1 adipocyte differentiation through modulation of extracellular signal-regulated kinase signalling. Clin Exp Pharmacol Physiol,2011,38:239-246
88. Liu H, Lin H, Zhang L, Sun Q, Yuan GH, Zhang L, Chen S, Chen Z. miR-145 and miR-143 regulate odontoblast differentiation through targeting Klf4 and Osx in a feedback loop. JBiol Chem,2013,288:9261-9271
89. Liu SP, Li D, Li QB, Zhao P, Xiang ZH, Xia QY. MicroRNAs of Bombyx mori identified by Solexa sequencing. BMC Genomics,2010,11:148
90. Lizuka K, Bruick RK, Liang GS, Horton JD, Uyeda K. Deficiency of carbohydrate response element-binding protein (ChREBP) reduces lipogenesis as well as glycolysis. Proc Natl Acad Sci USA,2004,101:7281-7286
91. Longo KA, Wright WS, Kang S, Gerin I, Chiang SH, Lucas PC, Opp MR, MacDougald OA. WntlOb inhibits development of white and brown adipose tissues. JBiol Chem,2004,279:35503-35509
92. Lowe CE, O'Rahilly S, Rochford JJ. Adipogenesis at a glance. J Cell Sci,2011,124: 2681-2686
93. Luo LF, Ye LZ, Liu G, Shao GC, Zheng R, Ren ZQ, Zuo B, Xu DQ, Lei MG, Jiang SW, Deng CY, Xiong YZ, Li FE. Microarry-based approach identifies differentially expressed microRNAs in porcine sexually immature and mature testes. PLoS One, 2010,5:e11744
94. Mann B, Gelos M, Siedow A, Hanski ML, Gratchev A, Ilyas M, Bodmer WF, Moyer MP, Riecken EO, Buhr HJ, Hanski C. Target genes of beta-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas. Proc Natl Acad Sci USA,1999,96:1603-1608
95. Martinelli R, Nardelli C, Pilone V, Buonomo T, Liguori R, Castano I, Buono P, Masone S, Persico G, Forestieri P, Pastore L, Sacchetti L. miR-519d overexpression is associated with human obesity. Obesity,2010,18:2170-2176
96. McDaneld TG, Smith TPL, Doumit ME, Miles JR, Coutinho LL, Sonstegard TS, Matukumalli LK, Nonneman DJ, Wiedmann RT. MicroRNA transcriptome profiles during swine skeletal muscle development. BMC Genomics,2009,10:77
97. McGregor RA, Choi MS. microRNAs in the regulation of adipogenesis and obesity. Curr Mol Med,2011,11:304-316
98. Meng YJ, Chen DJ, Jin YF, Mao CZ, Wu P, Chen M. RNA editing of nuclear transcripts in Arabidopsis thaliana. BMC Genomics,2010,11:S12
99. Miclea RL, Van der Horst G, Robanus-Maandag EC, L□wik CW, Oostdijk W, Wit JM, Karperien M. Apc bridges Wnt/β-catenin and BMP singaling during osteoblast differentiation of KS483 cells. Exp Cell Res,2011,317:1411-1421
100. Miller JR. The Wnts. Genome Biol,2001,3:1-15
101. Morin RD, O'Connor MD, Griffith M, Kuchenbauer F, Delaney A, Prabhu AL, Zhao YJ, McDonald H, Zeng T, Hirst M, Eaves CJ, Marra MA. Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. Genome Res,2008,18:610-621
102. Nagel R, Sage CL, Diosdado B, Van der Waal M, Oude Vrielink JAF, Bolijin A, Meijer GA, Agami R. Regulation of the adenomatous polyposis coli gene by the miR-135 family in colorectal cancer. Cancer Res,2008,68:5795-5802
103. Nakanishi N, Nakagawa Y, Tokushige N, Aoki N, Matsuzaka T, Ishii K, Yahagi N, Kobayashi K, Yatoh S, Takahashi A, Suzuki H, Urayama O, Yamada N, Shimano H. The up-regulation of microRNA-335 is associated with lipid metabolism in liver and white adipose tissue of genetically obese mice. Biochem Biophys Res Commun, 2009,385:492-496
104. Nascimento CM, Ribeiro EB, Oyama LM. Metabolism and secretory function of white adipose tissue:effect of dietary fat. An Acad Bras Cienc,2009,81:453-466
105. Neville MJ, Collins JM, Gloyn AL, McCarthy MI, Karpe F. Comprehensive human adipose tissue mRNA and microRNA endogenous control selection for quantitative real-time-PCR normalization. Obesity,2011,19:888-892
106. Nishizuka M, Koyanagi A, Osada S, Imagawa M. Wnt4 and Wnt5a promote adipocyte differentiation. FEBS Lett,2008,582:3201-3205
107. Nusse R, Varmus HE. Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome. Cell,1982,31: 99-109
108. O'Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c-Myc-regulated microRNAs modulate E2F1 expression. Nature,2005,435:839-843
109. Ortega FJ, Moreno-Navarrete JM, Pardo G, Sabater M, Hummel M, Ferrer A, Rodriguez-Hernosa JI, Ruiz B, Ricart W, Peral B, Fernandez-Real JM. MiRNA expression profile of human subcutaneous adipose and during adipocyte differentiation. PLoS One,2010,5:e9022
110. Oskowitz AZ, Lu J, Penfornis P, Ylostalo J, McBride J, Flemington EK, Prockop DJ, Pochampally R. Human multipotent stromal cells from bone marrow and microRNA:regulation of differentiation and leukemia inhibitory factor expression. Proc Natl Acad Sci USA,2008,105:18372-18377
111. Per□bner I, Karow M, Jochum M, Neth P. LRP6 mediates Wnt/β-catenin signaling and regulates adipogenic differentiation in human mesenchymal stem cells. Int J Biochem Cell Biol,2012,44:1970-1982
112. Piriyapongsa J, Jordan IK, Conley AB, Ronan T, Smalheiser NR. Transcription factor binding sites are highly enriched within microRNA precursor sequences. Biol Direct,2011,6:61
113. Romao JM, Jin WW, Dodson MV, Hausman GJ, Moore SS, Guan LL. MicroRNA regulation in mammalian adipogenesis. Exp Biol Med,2011,236:997-1004
114. Prestwich TC, MacDougald OA. Wnt/β-catenin signaling in adipogenesis and metabolism. Curr Opin Cell Biol,2007,19:612-617
115. Qin LM, Chen YS, Niu YN, Chen WQ, Wang QW, Xiao SQ, Li AN, Xie Y, Li J, Zhao X, He ZU, Mo DL. A deep investigation into the adipogenesis mechanism: profile of microRNAs regulating adipogenesis by modulating the canonical Wnt/β-catenin signaling pathway. BMC Genomics,2010,11:320
116. Ramirez CM, Davalos A, Goedeke L, Salerno AG, Warrier N, Cirera-Salinas D, Suarez Y, Fernandez-Hernando C. MicroRNA-758 regulates cholesterol efflux through posttranscriptional repression of ATP-binding cassette transporter A1. Arterioscler Thromb Vasc Biol,2011,31:2707-2714
117. Rao PK, Kumar RM, Farkhondeh M, Baskerville S, Lodish HF. Myogenic factors that regulate expression of muscle-specific microRNAs. Proc Natl Acad Sci USA, 2006,103:8721-8726
118. Rathjen T, Pais H, Sweetman D, Moulton V, Munsterberg A, Dalmay T. High throughput sequencing of microRNAs in chicken somites. FEBS Lett,2009,583: 1422-1426
119. Rayner KJ, Esau CC, Hussain FN, McDaniel AL, Marshall SM, Van Gils JM, Ray TD, Sheedy FJ, Goedeke L, Liu XQ, Khatsenko OG, Kaimal V, Lees CJ, Fernandez-Hernando C, Fisher EA, Temel RE, Moore KJ. Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides. Nature, 2011,478:404-407
120. Rayner KJ, Sheedy FJ, Esau CC, Hussain FN, Temel RE, Parathath S, Van Gils JM, Rayner AJ, Chang AN, Suarez Y, Fernandez-Hernando C, Fisher EA, Moore KJ. Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis. J Clin Invest,2011,121:2921-2931
121. Rayner KJ, Suarez Y, Davalos A, Parathath S, Fitzgerald ML, Tamehiro N, Fisher EA, Moore KJ, Fernandez-Hernando C. MiR-33 contributes to the regulation of cholesterol homeostasis. Science,2010,328:1570-1573
122. Reddy AM, Zheng Y, Jagadeeswaran G, Macmil SL, Graham WB, Roe BA, Desilva U, Zhang WX, Sunkar R. Cloning, characterization and expression analysis of porcine microRNAs. BMC Genomics,2009,10:65
123. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Robert Hhrvitz H, Ruvkun G. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature,2000,403:901-906
124. Rippe V, Dittberner L, Lorenz VN, Drieschner N, Nimzyk R, Sendt W, Junker K, Belge G, Bullerdiek J. The two stem cell microRNA gene clusters C19MC and miR-371-3 are activated by specific chromosomal rearrangements in a subgroup of thyroid adenomas. PLoS One,2010,5:e9485
125. Robin Lytle J, Yario TA, Steitz JA. Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5'UTR as in the 3'UTR. Proc Natl Acad Sci USA, 2007,104:9667-9672
126. Rodriguez A, Griffiths-Jones S, Ashurst JL, Bradley A. Identification of mammalian microRNA host genes and transcription units. Genome Res,2004,14:1902-1910
127. Ross SE, Erickson RL, Hemati N, MacDouglad OA. Glycogen synthase kinase 3 is an insulin-regulated C/EBPa kinase. Mol Cell Biol,1999,19:8433-8441
128. Ross SE, Hemati N, Longo KA, Bennett CN, Lucas PC, Erickson RL, MacDougald OA. Inhibition of adipogenesis by Wnt signaling. Science,2000,289:950-953
129. Ruan MB, Zhao YT, Meng ZH, Wang XJ, Yang WC. Conserved miRNA analysis in Gossypium hirsutum through small RNA sequencing. Genomics,2009,94:263-268
130. Ruby JG, Jan C, Player C, Axtell MJ, Lee W, Nusbaum C, Ge H, Bartel DP. Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell,2006,126:1193-1207
131. Saini HK, Enright AJ, Griffiths-Jones S. Annotation of mammalian primary of microRNAs. BMC Genomics,2008,9:564
132. Sempere LF, Freemantle S, Pitha-Rowe I, Moss E, Dmitrovsky E, Ambros V. Expression profiling of mammalian microRNAs uncovers a subsets of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation. Genome Biol,2004,5:R13
133. Sethi JK, Vidal-Puig A. Wnt signaling and the control of cellular metabolism. Biochem J,2010,427:1-17
134. Shtutman M, Zhurinsky J, Simcha I, Albanese C, D'Amico M, Pestell R, Ben-Ze'ev A. The cyclin Dl gene is a target of the beta-catenin/LEF-1 pathway. Proc Natl Acad Sci USA,1999,96:5522-5527
135. Smalheiser NR. EST analyses predict the existence of a population of chimeric microRNA precursor-mRNA transcripts expressed in normal human and mouse tissues. Genome Biol,2003,4:403
136. Sober S, Laan M, Annilo T. MicroRNAs miR-124 and miR-135a are potential regulators of the mineralocorticoid receptor gene (NR3C2) expression. Biochem Biophys Res Commun,2010,391:727-732
137. Sun TW, Fu MG, Bookout AL, Kliewer SA, Mangelsdorf DJ. MicroRNA let-7 regulates 3T3-L1 adipogenesis. Mol Endocrinol,2009,23:925-931
138. Sweetman D, Goljanek K, Rathjen T, Oustanina S, Braun T, Dalmay T, Munsterberg A. Specific requirements of MRFs for the expression of muscle specific microRNAs, miR-1, miR-206 and miR-133. Dev Biol,2008,321:491-499
139. Takanabe R, Ono K, Abe Y, Takaya T, Horie T, Wada H, Kita T, Satoh N, Shimatsu A, Hasegawa K. Up-regulated expression of microRNA-143 in association with obesity in adipose tissue of mice fed high-fat diet. Biochem Biophys Res Commun, 2008,376:728-732
140. Tanzer A, Stadler PF. Molecular evolution of a microRNA cluster. J Mol Biol,2004, 339:327-335
141. Tay Y, Zhang JQ, Thomson AM, Lim B, Rigoutsos I. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature, 2008,455:1124-1128
142. Tazawa H, Tsuchiya N, Izumiya M, Nakagama H, Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells. Proc Natl Acad Sci USA,2007,104:15472-15477
143. Tetsu O, McCormick F. (3-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature,1999,398:422-426
144. Tong Q, Tsai J, Tan G, Dalgin G, Hotamisligil GS. Interaction between GATA and the C/EBP family of transcription fctors is critical in GATA-mediated suppression of adipose differentiation. Mol Cell Biol,2005,25:p706-p715
145. Trajkovski M, Hausser J, Soutschek J, Bhat B, Akin A, Zavolan M, Heim MH, Stoffel M. MicroRNAs 103 and 107 regulate insulin sensitivity. Nature,2011,474: 649-653
146. Ugarte F, Ryser M, Thieme S, Fierro FA, Navratiel K, Bornhauser M, Brenner S. Notch signaling enhances osteogenic differentiation while inhibiting adipogenesis in primary human bone marrow stromal cells. Exp Hematol,2009,37:867-875
147. Van Tienen FHJ, Laeremans H, Van der Kallen, Smeets HJM. Wnt5b stimulates adipogenesis by activating PPARy, and inhibiting the β-catenin dependent Wnt signaling pathway together with Wnt5a. Biochem Biophys Res Commun,2009,387: 207-211
148. Wada Taira, Shimba S, Tezuka M. Transcriptional regulation of the hypoxia inducible factor-2a (HIF-2a) gene during adipose differentiation in 3T3-L1 Cells. Biol Pharm Bull,2006,29:49-54
149. Wade JT, Struhl K, Busby SJW, Grainger DC. Genomic analysis of protein-DNA interactions in bacteria:insights into transcription and chromosome organization. Mol Microbiol,2007,65:21-26
150. Wang Q, Li YC, Wang JH, Kong J, Qi YC, Quigg RJ, Li XM. miR-17-92 cluster accelerates adipocyte differentiation by negatively regulating tumor-suppressor Rb2/p130. Proc Natl Acad Sci USA,2008,105:2889-2894
151. Wernersson R, Schierup MH, J(?)rgensen FG, Gorodkin J, Panitz F, Staerfeldt HH, Christensen OF, Mailund T, Hornsh0j H, Klein A, Wang J, Liu B, Hu SN, Dong W, Li W, Wong GKS, Yu J, Wang J, Bendixen C, Fredholm M et al. Pigs in sequence space:a 0.66 X coverage pig genome survey based on shotgun sequencing. BMC Genomics,2005,6:70
152. White UA, Stephens JM. Transcriptional factors that promote formation of white adipose tissue. Mol Cell Endocrinol,2010,328:10-14
153. Wodarz A, Nusse R. Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol,1998,14:59-88
154. Wright WS, Longo KA, Dolinsky VW, Gerin I, Kang S, Bennett CN, Chiang SH, Prestwich TC, Gress C, Burant CF, Susulic VS, MacDougald OA. WntlOb inhibits obesity in ob/ob and agouti mice. Diabetes,2007,56:295-303
155. Wu D, Hu L. MicroRNA:a new kind of gene regulators. Agr Sci China,2006,5: 77-80
156. Xie H, Lim B, Lodish HR MicroRNAs induced during adipogenesis that accelerate fat cell development are downregulated in obesity. Diabetes,2009,58:1050-1057
157. Xie SS, Huang TH, Shen Y, Li XY, Zhang XX, Zhu MJ, Qin HY, Zhao SH. Identification and characterization of microRNAs from porcine skeletal muscle. Anim Genet,2010,41:179-190
158. Xu PZ, Vernooy SY, Guo M. The Drosophila microRNA mir-14 suppresses cell death and is required for normal fat metabolism. Curr Biol,2003,13:790-795
159. Yamakuchi M, Lowenstein CJ. MiR-34, SIRT1, and p53:the feedback loop. Cell Cycle,2009,8:712-715
160. Yang Z, Bian CJ, Zhou H, Huang S, Wang SH, Liao LM, Zhao RC. MicroRNA hsa-miR-138 inhibits adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells through adenovirus EID-1. Stem Cells Dev,2011,20: 259-267
161. Zaragosi LE, Wdziekonski B, Brigand KL, Villageois P, Mari B, Waldmann R, Dani C, Barbry P. Small RNA sequencing reveals miR-642a-3p. as a novel adipocyte-specific microRNA and miR-30 as a key regulator of human adipogenesis. Genome Biol,2011,12:R64
162. Zaragosi LE, Wdziekonski B, Fontaine C, Villageois P, Peraldi P, Dani C. Effect of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes. BMC Cell Biol,2008,9:11
163. Zhang BH, Pan XP, Cobb GP, Anderson TA. Plant microRNA:a small regulatory molecule with big impact. Dev Biol,2006,289:3-16
164. Zhao CZ, Xia H, Frazier TP, Yao YY, Bi YP, Li AQ, Li MJ, Li CS, Zhagn BH, Wang XJ. Deep sequencing identifies novel and conserved microRNAs in peanuts (Arachis Hypogaea L.). BMC Plant Biol,2010,10:3
165. Zhao Y, Samal E, Srivastava D. Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature,2005,436:214-220
166. Zhou B, Liu HL, Shi FX, Wang JY. MicroRNA expression profiles of porcine skeletal muscle. Anim Genet,2010,41:499-508
2. 李国喜.猪脂肪组织发育相关miRNA的鉴定及miR-103生物学功能的初步研究.[博士学位论文].武汉：华中农业大学图书馆,2011
3. 李鹏昊.miR-27a、miR-143对猪脂肪沉积和miR-1、miR-206对肌肉肥大的调控研究.[硕士学位论文].武汉：华中农业大学图书馆,2011
4. 宁小敏.miR-196a对猪脂肪细胞增殖分化的影响.[硕士学位论文].武汉：华中农业大学图书馆,2010
5. 屈长青,张国华,陈粉粉,赵丽丽,杨公社.猪前体脂肪细胞的原代培养.农业生物技术学报,2005,13：649-653
6. 覃丽梅.脂肪细胞分化的WNT信号通路中microRNA表达谱分析及其功能研究.[博士学位论文].武汉：华中农业大学图书馆,2010
7. 徐露莲,史春梅,陈玲,季晨博,郭锡熔,徐美玉.脂肪细胞分化相关miRNA-hsa-miR-378生物信息学分析.中国儿童保健杂志,2012,20：1001-1004
8. 张国华,杨公社,屈长青,孙世铎.猪前体脂肪细胞的分离培养.细胞生物学杂志,2005,27：693-696
9. 朱璐,史春梅,崔焱,徐广峰,季晨博,倪毓辉,郭锡熔.hsa-miR-335靶基因预测及生物信息学分析.西部医学,2012,24：1851-1855
10. AguilO F, Camarero N, Relat J, Marrero PF, Haro D. Transcriptional regulation of the human acetoacetyl-CoA synthetase gene by PPARy. Biochem J,2010,427: 255-264
11. Altuvia Y, Landgraf P, Lithwick G, Elefant N, Pfeffer S, Aravin A, Brownstein MJ, Tuschl T, Margalit H. Clustering and conservation patterns of human microRNAs. Nucleic Acids Res,2005,33:2697-2706
12. Ambros V. The functions of animal microRNAs. Nature,2004,431:350-355
13. Andersen DC, Jensen CH, Schneider M, Nossent AY, Eskildsen T, Hansen JL, Teisner B, Sheikh SP. MicroRNA-15a fine-tunes the level of Delta-like 1 homolg (DLK1) in proliferating 3T3-L1 preadipocytes. Exp Cell Res,2010,316:1681-1691
14. Antonson P, Pray MG, Jacobsson A, Xanthopoulos KG Myc inhibits CCAAT/enhancer-binding protein alpha-gene expression in HIB-1B hibernoma cells through interactions with the core promoter region. Eur JBiochem,1995,232: 397-403
15. Aravin A, Tuschl T. Identification and characterization of small RNAs involved in RNA silencing. FEBS Lett,2005,579:5830-5840
16. Bartel DP. MicroRNA:genomics, biogenesis, mechanism, and function. Cell,2004, 116:281-297
17. Barth N, Langmann T, SchOlmerich J, Schmitz G, Schaffler A. Identification of regulatory elements in the human adipose most abundant gene transcript-1 (apM-1) promoter:role of SP1/SP3 and TNF-a as regulatory pathways. Diabetologia,2002, 45:1425-1433
18. Bejerano G, Pheasant M, Makunin I, Stephen S, Kent WJ, Mattick JS, Haussler D. Ultraconserved elements in human genome. Science,2004,304:1321-1325
19. Bennett CN, Ross SE, Longo KA, Bajnok L, Hemati N, Johnson KW, Harrison SD, MacDougald OA. Regulation of Wnt signaling during adipogenesis. J Biol Chem, 2002,277:30998-31004
20. Bhat RA, Stauffer B, Komm BS, Bodine PVN. Structure-function analysis of secreted frizzled-related protein-1 for its Wnt antagonist function. J Cell Biochem, 2007,102:1519-1528
21. Bolw MJ, Grocock RJ, Dongen SV, Enright AJ, Dicks E, Futreal PA, Wooster R, Stratton MR. RNA editing of human microRNAs. Genome Biol,2006,7:R27
22. Card DA, Hebbar PB, Li LP, Trotter KW, Komatsu Y, Mishina Y, Archer TK. Oct4/Sox2-regulated miR-302 targets cyclin D1 in human embryonic stem cells. Mol Cell Biol,2008,28:6426-6438
23. Chen CF, Ridzon DA, Broomer AJ, Zhou ZH, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvaker VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res,2005,33: e179
24. Chen JF, Mandel EM, Thomson JM, Wu QL, Callis TE, Hammond SM, Conlon FL, Wang DZ. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet,2005,38:228-233
25. Chendrimada TP, Finn KJ, Ji XJ, Baillat D, Gregory RI, Liebhaber SA, Pasquinelli AE, Shikhattar R. MicroRNA silencing through RISC recruitment of eIF6. Nature, 2007,447:823-828
26. Cho IS, Kim J, Seo HY, Lim DH, Hong JS, Park YH, Park DC, Hong KC, Whang KY, Lee YS. Cloning and characterization of microRNAs from porcine skeletal muscle and adipose tissue. Mol Biol Rep,2010,37:3567-3574
27. Christodoulides C, Lagathu C, Sethi JK, Vidal-Puig A. Adipogenesis and WNT signaling. Trends Endocrinol Metab,2009,20:16-24
28. Cirera S, Birck M, Busk PK, Fredholm M. Expression profiles of miRNA-122 and its target CAT1 in minipigs(Sus scrofa) fed a high-cholesterol diet. Comp Med, 2010,60:136-141
29. Cullen BR. Viruses and microRNAs. Nat Genet,2006,38:S25-S30
30. Davalos A, Goedeke L, Smibert P, Ramirez CM, Warrier NP, Andreo U, Cirera-Salinas D, Rayner K, Suresh U, Pastor-Pareja JC, Esplugues E, Fisher EA, Penalva LO, Moore KJ, Suarez Y, Lai EC, Fernadez-Hernando C. miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling. Proc Natl Acad Sci USA,2011,108:9232-9237
31. Doench JG, Petersen CP, Sharp PA. siRNAs can function as miRNAs. Gene Dev, 2003,17:438-442
32. Ebhardt HA, Tsang HH, Dai DC, Liu YF, Bostan B, Fahlman RP. Meta-analysis of small RNA-sequencing errors reveals ubiquitous post-transcriptional RNA modifications. Nucleic Acids Res,2009,37:2461-2470
33. Esau C, Davis S, Murray SF, Yu XX, Pandey SK, Pear M, Watts L, Booten SL, Graham M, McKay R, Subramaniam A, Propp S, Lollo BA, Freier SS, Bennett CF, Bhanot S, Monia BP. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab,2006,3:87-98
34. Kennell JA, Gerin I, MacDougald OA, Cadigan KM. The microRNA miR-8 is a conserved negative regulator of Wnt signaling. Proc Natl Acad Sci USA,2008,105: 15417-15422
35. Esau C, Kang XL, Peralta E, Hanson E, Marcusson EG, Ravichandran LV, Sun YQ, Koo S, Perera RJ, Jain R, Dean NM, Freier SM, Bennett F, Lollo B, Griffey R. MicroRNA-143 regulates adipocyte differentiation.J Biol Chem,2004,279: 52361-52365
36. Feederle R, Linstaedt SD, Bannert H, Lips H, Bryan M, Cullen BR, Delecluse HJ. A viral microRNA cluster strongly potentiates the transforming properties of a human herpesvirus. PLoS Pathog,2011,7:e10001294
37. Freytag SO, Geddes TJ. Reciprocal regulation of adipogenesis by Myc and C/EBP alpha. Science,1992,256:379-382
38. Fu MF, Rao M, Bouras T, Wang CG, Wu KM, Zhang XP, Li ZP, Yao TP, Pestell RG Cyclin D1 inhibits peroxisome proliferator-activated receptor y-mediated adipogenesis through histone deacetylase recruitment. J Biol Chem,2005,280: 16934-16941
39. Gerin I, Bommer GT, McCoin CS, Sousa KM, Krishnan V, MacDougald OA. Roles for miRNA-378/378* in adipocyte gene expression and lipogenesis. Am J Physiol Endocrinol Metab,2010,299:E198-E206
40. Glazov EA, Cottee PA, Barris WC, Moore RJ, Dalrymple BP, Tizard ML. A microRNA catalog of the developing chicken embryo identified by a deep sequencing approach. Genome Res,2008:957-964
41. Glazov EA, Kongsuwan K, Assavalapsakul W, Horwood PF, Mitter N, Mahony TJ. Repertorie of bovine miRNA and miRNA-like small regulatory RNAs expressed upon viral infection. PLoS One,2009,4:e6349
42. Gorodkin J, Cirera S, Hedegaard J, Gilchrist MJ, Panitz F, J(?)rgensen C, Scheibye-Knudsen K, Arvin T, Lumholdt S, Sawera M, Green T, Nielsen BJ, Havgaard JH, Rosenkilde C, Wang J, Li H, Li RQ, Liu B, Hu SN, Dong W et al. Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags. Genome Biol,2007,8:R45
43. Gu J, He T, Pei YF, Li F, Wang XW, Zhang J, Zhang XG, Li YD. Primary transcripts and expressions of mammal intergenic microRNAs detected by mapping ESTs to their flanking sequences. Mamm Genome,2006,17:1033-1041
44. Guo YX, Mo DL, Zhang Y, Zhang Y, Cong PQ, Xiao SQ, He ZY, Liu XH, Chen YS. MicroRNAome comparison between intramuscular and subcutaneous vascular stem cell adipogenesis. PLoS One,2012,7:e45410
45. Gupta RK, Arany Z, Seale P, Mepani RJ, Ye L, Conroe HM, Roby YA, Kulaga H, Reed RR, Spiegelman BM. Transcriptional control of preadipocyte determination by Zfp423. Nature,2010,464:619-623
46. He A, Zhu LL, Gupta N, Chang YS, Fang FD. Overexpression of micro ribonucleic acid 29, highly up-regulated in diabetic rats, leads to insulin resistance in 3T3-L1 adipocytes. Mol Endocrinol,2007,21:2785-2794
47. He TC, Sparks AB, Rago C, Hermeking H, Zawel L, Da Costa LT, Morin PJ, Vogelstein B, Kinzler KW. Identification of c-MYC as a target of the APC pathway. Science,1998,281:1509-1512
48. Heneghan HM, Miller N, McAnena OJ, O'Brien T, Kerin MJ. Differential miRNA expression in omental adipose tissue and in the circulation of obese patients identifies novel metabolic biomarkers. J Clin Endocrinol Metab,2011,96: E846-E850
49. Herrera BM, Lockstone HE, Taylor JM, Ria M, Barrett A, Collins S, Kaisaki P, Argoud K, Fernandez C, Travers ME, Grew JP, Randall JC, Gloyn AL, Gaufuier D, McCarthy MI, Lindgren CM. Global microRNA expression profiles in insulin target tissues in a spontaneous rat model of type 2 diabetes. Diabetologia,2010,53: 1099-1109
50. Herrera BM, Lockstone HE, Taylor JM, Wills QF, Kaisaki PJ, Barrett A, Camps C, Fernandez C, Ragoussis J, Gauguier D, McCarthy MI, Lindgren CM. MicroRNA-125a is over-expressed in insulin target tissues in a spontaneous rat model of type 2 diabetes. BMC Med Genomics,2009,2:54
51. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease RL. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene,1989,77:51-59
52. Huang J, Zhao L, Xing LP, Chen D. MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation. Stem Cells,2010,28: 357-364
53. Huang K, Zhang JX, Han L, You YP, Jiang T, Pu PY, Kang CS. MicroRNA roles in beta-catenin pathway. Mol Cancer,2010,9:252
54. Huang TH, Zhu MJ, Li XY, Zhao SH. Discovery of porcine microRNAs and profiling from skeletal muscle tissues during development. PLoS One,2008,3: e3225
55. Huelsken J, Brichmeier W. New aspects of Wnt signaling pathways in higher vertebrates. Curr Opin Genet Dev,2001,11:547-553
56. Iliopoulos D, Drosatos K, Hiyama Y, Goldberg IJ, Zannis VI. MicroRNA-370 controls the expression of MicroRNA-122 and Cptla and affects lipid metabolism. JLipid Res,2010,51:1513-1523
57. John E, Wienecke-Baldacchino A, Liivrand M, Heinaniemi M, Carlberg C, Sinkkonen L. Dataset integration identifies transcriptional regulation of microRNA genes by PPARy in differentiating mouse 3T3-L1 adipocytes. Nucleic Acids Res, 2012,40:4446-4460
58. Jopling CL, Norman KL, Sarnow P. Positive and negative modulation of viral and cellular mRNAs by liver-specific microRNA miR-22. Cold Spring Harb Symp Quant Biol,2006,71:369-376
59. Kajimoto K, Naraba H, Iwai N. MicroRNA and 3T3-L1 pre-adipocyte differentiation. RNA,2006,12:1626-1632
60. Kanazawa A, Tsukada S, Kamiyama M, Yanagimoto T, Nakajima M, Maeda S. Wnt5b partially inhibits canonical Wnt/β-catenin signaling pathway and promotes adipogenesis in 3T3-L1 preadipocytes. Biochem Biophys Res Commun,2005,330: 505-510
61. Karbiener M, Fischer C, Nowitsch S, Opriessnig P, Papak C, Ailhaud G, Dani C, Amri EZ, Scheideler M. microRNA miR-27b impairs human adipocyte differentiation and targets PPARy. Biochem Biophys Res Commun,2009,390: 247-251
62. Karbiener M, Neuhold C, Opriessnig P, Prokesch A, Bogner-Strauss JG, Scheideler M. MicroRNA-30c promotes human adipocyte differentiation and co-represses PAI-1 and ALK2. RNA Biol,2011,8:850-860
63. Kawai M, Mushiake S, Bessho K, Murakami M, Namba N, Kokubu C, Michigami T, Ozono K. Wnt/Lrp/β-catenin signaling suppress adipogenesis by inhibiting mutual activation of PPRAy and C/EBPa. Biochem Biophys Res Commun,2007,363: 276-282
64. Keller P, Gburcik V, Petrovic N, Gallagher IJ, Nedergaard J, Cannon B, Timmons JA. Gene-chip studies of adipogenesis-regualted microRNAs in mouse primary adipocytes and human obesity. BMC Endocr Disord,2011,11:7
65. Kershwa EE, Flier JS. Adipose tissue as an endocrine organ. J Clin Endocrinol Metab.2004,89:2548-2556
66. Kim HJ, Cui XS, Kim EJ, Kim WJ, Kim NH. New porcine microRNA genes found by homo logy search. Genome,2006,49:1283-1286
67. Kim J, Cho IS, Hong JS, Choi YK, Kim H, Lee YS. Identification and characterization of new microRNAs from pig. Mamm Genome,2008,19:570-580
68. Kim SY, Kim AY, Lee HY, Son HY, Lee GY, Lee JW, Lee YS, Kim JB. miR-27a is a negative regulator of adipocyte differentiation via suppressing PPARy expression. Biochem Biophys Res Commun,2010,392:323-328
69. Kim YJ, Hwang SH, Cho HH, Shin KK, Bae YC, Jung JS. MicroRNA 21 regulates the proliferation of human adipose tissue-derived mesenchymal stem cells and high-fat diet-induced obesity alters microRNA 21 expression in white adipose tissues. J Cell Physiol,2012,227:183-193
70. Kim YJ, Hwang SJ, Bae YC, Jung JS. MiR-21 regulates adipogenic differentiation through the modulation of TGF-beta signaling in mesenchymal stem cells derived from human adipose tissue. Stem Cells,2009,27:3093-3102
71. Kinoshita M, Ono K, Horie T, Nagao K, Nishi H, Kuwabara Y, Takanabe-Mori R, Hasegawa K, Kita T, Kimura T. Regulation of adipocyte differentiation by actibation of serotonin (5-HT) receptors 5-HT2AR and 5-HT2cR and involvement of microRNA-448-mediated repression of KLF5. Mol Endocrinol,2010,24: 1978-1987
72. KrUtzfeldt J, Rajewsky N, Braich R, Rajeevv KG, Tuschl T, Manoharan M, Stoffel M. Silencing of microRNA in vivo with'antagomirs'. Nature,2005,438:685-689
73. Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T. Identification of tissue-specific microRNAs from mouse. Curr Biol,2002,12: 735-739
74. Lee EK, Lee MJ, Abdelmohsen K, Kim W, Kim MM, Srikantan S, Martindale JL, Hutchison ER, Kim HH, Marasa BS, Selimyan R, Egan JM, Smith SR, Fried SK, Gorospe M. miR-130 suppress adipogenesis by inhibiting'peroxisome proliferator-activated receptor y expression. Mol Cell Biol,2010,31:626-638
75. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell,1993,75:843-854
76. Lefterova MI, Lazar MA. New developments in adipogenesis. Trends Endocrinol Metab,2009,20:107-114
77. Li GX, Li YJ, Li XJ, Ning XM, Li MH, Yang GS. MicroRNA identity and abundance in developing swine adipose tissue as determined by Solexa sequencing. J Cell Biochem,2011,112:1318-1328
78. Li HX, Luo X, Liu RX, Yang YJ, Yang GS. Roles of Wnt/β-catenin signaling in adipogenic differentiation potential of adipose-derived mesenchymal stem cells. Mol Cell Endocrinol,2008,291:116-124
79. Li HY, Xi QY, Xiong YY, Liu XL, Cheng X, Shu G, Wang SB, Wang LN, Gao P, Zhu XT, Jiang QY, Yuan L, Zhang YL. Identification and comparison of microRNAs from skeletal muscle and adipose tissues from two porcine breeds. Anim Genet,2012,43:704-713
80. Li MZ, Xia YL, Gu YR, Zhang K, Lang QL, Chen L, Guan JQ, Luo ZG, Chen HS, Li Y, Li QH, Li X, Jiang AA, Shuai SR, Wang JR, Zhu Q, Zhou XC, Gao XL, Li XW. MicroRNAome of porcine pre-and postnatal development. PLoS One,2010,5; e11541
81. Li ZY, Hassan MQ, Volinia S, Van Wijnen AJ, Stein JL, Croce CM, Lian JB, Stein GS. A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci USA,2008,105:13906-13911
82. Lian CJ, Sun BX, Niu SL, Yang RJ, Liu BY, Lu CY, Meng JL, Qiu ZY, Zhang LY, Zhao ZH. A comparative profile of the microRNA transcriptome in immature and mature porcine testes using Solexa deep sequencing. FEBSJ,2012,279:964-975
83. Liao BJ, Bao XC, Liu LQ, Feng SP, Zovoilis A, Liu WB, Xue YT, Cai J, Guo XP, Qin BM, Zhang RS, Wu JY, Lai LX, Teng MK, Niu LW, Zhang BL, Esteban MA, Pei DQ. MicroRNA cluster 302-367 enhances somatic cell reprogramming by accelerating a mesenchymal-to-epithelial transition. J Biol Chem,2011,286: 17359-17364
84. Lin KY, Zhang XJ, Feng DD, Zhang H, Zeng CW, Han BW, Zhou AD, Ou LH, Xu L, Chen YQ. miR-125b, a target of CDX2, regulates cell differentiation through repression of the core binding factor in hematopoietic malignancies. J Biol Chem, 2011,286:38253-38263
85. Lin Q, Gao Z, Alarcon RM, Ye J, Yun Z. A role of miR-27 in the regulation of adipogenesis. FEBS Lett,2009,276:2348-2358
86. Ling HY, Ou HS, Feng SD, Zhang XY, Tuo QH, Chen LX, Zhu BY, Gao ZP, Tang CK, Yin WD, Zhang L, Liao DF. Changes in microRNA (miR) profile and effects of miR-320 in insulin-resistant 3T3-L1 adipocytes. Clin Exp Pharmacol Physiol,2009, 36:e32-e39
87. Ling HY, Wen GB, Feng SD, Tuo QH, Ou HS, Yao CH, Zhu YB, Gao ZP, Zhang L, Liao DF. MicroRNA-375 promotes 3T3-L1 adipocyte differentiation through modulation of extracellular signal-regulated kinase signalling. Clin Exp Pharmacol Physiol,2011,38:239-246
88. Liu H, Lin H, Zhang L, Sun Q, Yuan GH, Zhang L, Chen S, Chen Z. miR-145 and miR-143 regulate odontoblast differentiation through targeting Klf4 and Osx in a feedback loop. JBiol Chem,2013,288:9261-9271
89. Liu SP, Li D, Li QB, Zhao P, Xiang ZH, Xia QY. MicroRNAs of Bombyx mori identified by Solexa sequencing. BMC Genomics,2010,11:148
90. Lizuka K, Bruick RK, Liang GS, Horton JD, Uyeda K. Deficiency of carbohydrate response element-binding protein (ChREBP) reduces lipogenesis as well as glycolysis. Proc Natl Acad Sci USA,2004,101:7281-7286
91. Longo KA, Wright WS, Kang S, Gerin I, Chiang SH, Lucas PC, Opp MR, MacDougald OA. WntlOb inhibits development of white and brown adipose tissues. JBiol Chem,2004,279:35503-35509
92. Lowe CE, O'Rahilly S, Rochford JJ. Adipogenesis at a glance. J Cell Sci,2011,124: 2681-2686
93. Luo LF, Ye LZ, Liu G, Shao GC, Zheng R, Ren ZQ, Zuo B, Xu DQ, Lei MG, Jiang SW, Deng CY, Xiong YZ, Li FE. Microarry-based approach identifies differentially expressed microRNAs in porcine sexually immature and mature testes. PLoS One, 2010,5:e11744
94. Mann B, Gelos M, Siedow A, Hanski ML, Gratchev A, Ilyas M, Bodmer WF, Moyer MP, Riecken EO, Buhr HJ, Hanski C. Target genes of beta-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas. Proc Natl Acad Sci USA,1999,96:1603-1608
95. Martinelli R, Nardelli C, Pilone V, Buonomo T, Liguori R, Castano I, Buono P, Masone S, Persico G, Forestieri P, Pastore L, Sacchetti L. miR-519d overexpression is associated with human obesity. Obesity,2010,18:2170-2176
96. McDaneld TG, Smith TPL, Doumit ME, Miles JR, Coutinho LL, Sonstegard TS, Matukumalli LK, Nonneman DJ, Wiedmann RT. MicroRNA transcriptome profiles during swine skeletal muscle development. BMC Genomics,2009,10:77
97. McGregor RA, Choi MS. microRNAs in the regulation of adipogenesis and obesity. Curr Mol Med,2011,11:304-316
98. Meng YJ, Chen DJ, Jin YF, Mao CZ, Wu P, Chen M. RNA editing of nuclear transcripts in Arabidopsis thaliana. BMC Genomics,2010,11:S12
99. Miclea RL, Van der Horst G, Robanus-Maandag EC, L□wik CW, Oostdijk W, Wit JM, Karperien M. Apc bridges Wnt/β-catenin and BMP singaling during osteoblast differentiation of KS483 cells. Exp Cell Res,2011,317:1411-1421
100. Miller JR. The Wnts. Genome Biol,2001,3:1-15
101. Morin RD, O'Connor MD, Griffith M, Kuchenbauer F, Delaney A, Prabhu AL, Zhao YJ, McDonald H, Zeng T, Hirst M, Eaves CJ, Marra MA. Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. Genome Res,2008,18:610-621
102. Nagel R, Sage CL, Diosdado B, Van der Waal M, Oude Vrielink JAF, Bolijin A, Meijer GA, Agami R. Regulation of the adenomatous polyposis coli gene by the miR-135 family in colorectal cancer. Cancer Res,2008,68:5795-5802
103. Nakanishi N, Nakagawa Y, Tokushige N, Aoki N, Matsuzaka T, Ishii K, Yahagi N, Kobayashi K, Yatoh S, Takahashi A, Suzuki H, Urayama O, Yamada N, Shimano H. The up-regulation of microRNA-335 is associated with lipid metabolism in liver and white adipose tissue of genetically obese mice. Biochem Biophys Res Commun, 2009,385:492-496
104. Nascimento CM, Ribeiro EB, Oyama LM. Metabolism and secretory function of white adipose tissue:effect of dietary fat. An Acad Bras Cienc,2009,81:453-466
105. Neville MJ, Collins JM, Gloyn AL, McCarthy MI, Karpe F. Comprehensive human adipose tissue mRNA and microRNA endogenous control selection for quantitative real-time-PCR normalization. Obesity,2011,19:888-892
106. Nishizuka M, Koyanagi A, Osada S, Imagawa M. Wnt4 and Wnt5a promote adipocyte differentiation. FEBS Lett,2008,582:3201-3205
107. Nusse R, Varmus HE. Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome. Cell,1982,31: 99-109
108. O'Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c-Myc-regulated microRNAs modulate E2F1 expression. Nature,2005,435:839-843
109. Ortega FJ, Moreno-Navarrete JM, Pardo G, Sabater M, Hummel M, Ferrer A, Rodriguez-Hernosa JI, Ruiz B, Ricart W, Peral B, Fernandez-Real JM. MiRNA expression profile of human subcutaneous adipose and during adipocyte differentiation. PLoS One,2010,5:e9022
110. Oskowitz AZ, Lu J, Penfornis P, Ylostalo J, McBride J, Flemington EK, Prockop DJ, Pochampally R. Human multipotent stromal cells from bone marrow and microRNA:regulation of differentiation and leukemia inhibitory factor expression. Proc Natl Acad Sci USA,2008,105:18372-18377
111. Per□bner I, Karow M, Jochum M, Neth P. LRP6 mediates Wnt/β-catenin signaling and regulates adipogenic differentiation in human mesenchymal stem cells. Int J Biochem Cell Biol,2012,44:1970-1982
112. Piriyapongsa J, Jordan IK, Conley AB, Ronan T, Smalheiser NR. Transcription factor binding sites are highly enriched within microRNA precursor sequences. Biol Direct,2011,6:61
113. Romao JM, Jin WW, Dodson MV, Hausman GJ, Moore SS, Guan LL. MicroRNA regulation in mammalian adipogenesis. Exp Biol Med,2011,236:997-1004
114. Prestwich TC, MacDougald OA. Wnt/β-catenin signaling in adipogenesis and metabolism. Curr Opin Cell Biol,2007,19:612-617
115. Qin LM, Chen YS, Niu YN, Chen WQ, Wang QW, Xiao SQ, Li AN, Xie Y, Li J, Zhao X, He ZU, Mo DL. A deep investigation into the adipogenesis mechanism: profile of microRNAs regulating adipogenesis by modulating the canonical Wnt/β-catenin signaling pathway. BMC Genomics,2010,11:320
116. Ramirez CM, Davalos A, Goedeke L, Salerno AG, Warrier N, Cirera-Salinas D, Suarez Y, Fernandez-Hernando C. MicroRNA-758 regulates cholesterol efflux through posttranscriptional repression of ATP-binding cassette transporter A1. Arterioscler Thromb Vasc Biol,2011,31:2707-2714
117. Rao PK, Kumar RM, Farkhondeh M, Baskerville S, Lodish HF. Myogenic factors that regulate expression of muscle-specific microRNAs. Proc Natl Acad Sci USA, 2006,103:8721-8726
118. Rathjen T, Pais H, Sweetman D, Moulton V, Munsterberg A, Dalmay T. High throughput sequencing of microRNAs in chicken somites. FEBS Lett,2009,583: 1422-1426
119. Rayner KJ, Esau CC, Hussain FN, McDaniel AL, Marshall SM, Van Gils JM, Ray TD, Sheedy FJ, Goedeke L, Liu XQ, Khatsenko OG, Kaimal V, Lees CJ, Fernandez-Hernando C, Fisher EA, Temel RE, Moore KJ. Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides. Nature, 2011,478:404-407
120. Rayner KJ, Sheedy FJ, Esau CC, Hussain FN, Temel RE, Parathath S, Van Gils JM, Rayner AJ, Chang AN, Suarez Y, Fernandez-Hernando C, Fisher EA, Moore KJ. Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis. J Clin Invest,2011,121:2921-2931
121. Rayner KJ, Suarez Y, Davalos A, Parathath S, Fitzgerald ML, Tamehiro N, Fisher EA, Moore KJ, Fernandez-Hernando C. MiR-33 contributes to the regulation of cholesterol homeostasis. Science,2010,328:1570-1573
122. Reddy AM, Zheng Y, Jagadeeswaran G, Macmil SL, Graham WB, Roe BA, Desilva U, Zhang WX, Sunkar R. Cloning, characterization and expression analysis of porcine microRNAs. BMC Genomics,2009,10:65
123. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Robert Hhrvitz H, Ruvkun G. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature,2000,403:901-906
124. Rippe V, Dittberner L, Lorenz VN, Drieschner N, Nimzyk R, Sendt W, Junker K, Belge G, Bullerdiek J. The two stem cell microRNA gene clusters C19MC and miR-371-3 are activated by specific chromosomal rearrangements in a subgroup of thyroid adenomas. PLoS One,2010,5:e9485
125. Robin Lytle J, Yario TA, Steitz JA. Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5'UTR as in the 3'UTR. Proc Natl Acad Sci USA, 2007,104:9667-9672
126. Rodriguez A, Griffiths-Jones S, Ashurst JL, Bradley A. Identification of mammalian microRNA host genes and transcription units. Genome Res,2004,14:1902-1910
127. Ross SE, Erickson RL, Hemati N, MacDouglad OA. Glycogen synthase kinase 3 is an insulin-regulated C/EBPa kinase. Mol Cell Biol,1999,19:8433-8441
128. Ross SE, Hemati N, Longo KA, Bennett CN, Lucas PC, Erickson RL, MacDougald OA. Inhibition of adipogenesis by Wnt signaling. Science,2000,289:950-953
129. Ruan MB, Zhao YT, Meng ZH, Wang XJ, Yang WC. Conserved miRNA analysis in Gossypium hirsutum through small RNA sequencing. Genomics,2009,94:263-268
130. Ruby JG, Jan C, Player C, Axtell MJ, Lee W, Nusbaum C, Ge H, Bartel DP. Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans. Cell,2006,126:1193-1207
131. Saini HK, Enright AJ, Griffiths-Jones S. Annotation of mammalian primary of microRNAs. BMC Genomics,2008,9:564
132. Sempere LF, Freemantle S, Pitha-Rowe I, Moss E, Dmitrovsky E, Ambros V. Expression profiling of mammalian microRNAs uncovers a subsets of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation. Genome Biol,2004,5:R13
133. Sethi JK, Vidal-Puig A. Wnt signaling and the control of cellular metabolism. Biochem J,2010,427:1-17
134. Shtutman M, Zhurinsky J, Simcha I, Albanese C, D'Amico M, Pestell R, Ben-Ze'ev A. The cyclin Dl gene is a target of the beta-catenin/LEF-1 pathway. Proc Natl Acad Sci USA,1999,96:5522-5527
135. Smalheiser NR. EST analyses predict the existence of a population of chimeric microRNA precursor-mRNA transcripts expressed in normal human and mouse tissues. Genome Biol,2003,4:403
136. Sober S, Laan M, Annilo T. MicroRNAs miR-124 and miR-135a are potential regulators of the mineralocorticoid receptor gene (NR3C2) expression. Biochem Biophys Res Commun,2010,391:727-732
137. Sun TW, Fu MG, Bookout AL, Kliewer SA, Mangelsdorf DJ. MicroRNA let-7 regulates 3T3-L1 adipogenesis. Mol Endocrinol,2009,23:925-931
138. Sweetman D, Goljanek K, Rathjen T, Oustanina S, Braun T, Dalmay T, Munsterberg A. Specific requirements of MRFs for the expression of muscle specific microRNAs, miR-1, miR-206 and miR-133. Dev Biol,2008,321:491-499
139. Takanabe R, Ono K, Abe Y, Takaya T, Horie T, Wada H, Kita T, Satoh N, Shimatsu A, Hasegawa K. Up-regulated expression of microRNA-143 in association with obesity in adipose tissue of mice fed high-fat diet. Biochem Biophys Res Commun, 2008,376:728-732
140. Tanzer A, Stadler PF. Molecular evolution of a microRNA cluster. J Mol Biol,2004, 339:327-335
141. Tay Y, Zhang JQ, Thomson AM, Lim B, Rigoutsos I. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature, 2008,455:1124-1128
142. Tazawa H, Tsuchiya N, Izumiya M, Nakagama H, Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells. Proc Natl Acad Sci USA,2007,104:15472-15477
143. Tetsu O, McCormick F. (3-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature,1999,398:422-426
144. Tong Q, Tsai J, Tan G, Dalgin G, Hotamisligil GS. Interaction between GATA and the C/EBP family of transcription fctors is critical in GATA-mediated suppression of adipose differentiation. Mol Cell Biol,2005,25:p706-p715
145. Trajkovski M, Hausser J, Soutschek J, Bhat B, Akin A, Zavolan M, Heim MH, Stoffel M. MicroRNAs 103 and 107 regulate insulin sensitivity. Nature,2011,474: 649-653
146. Ugarte F, Ryser M, Thieme S, Fierro FA, Navratiel K, Bornhauser M, Brenner S. Notch signaling enhances osteogenic differentiation while inhibiting adipogenesis in primary human bone marrow stromal cells. Exp Hematol,2009,37:867-875
147. Van Tienen FHJ, Laeremans H, Van der Kallen, Smeets HJM. Wnt5b stimulates adipogenesis by activating PPARy, and inhibiting the β-catenin dependent Wnt signaling pathway together with Wnt5a. Biochem Biophys Res Commun,2009,387: 207-211
148. Wada Taira, Shimba S, Tezuka M. Transcriptional regulation of the hypoxia inducible factor-2a (HIF-2a) gene during adipose differentiation in 3T3-L1 Cells. Biol Pharm Bull,2006,29:49-54
149. Wade JT, Struhl K, Busby SJW, Grainger DC. Genomic analysis of protein-DNA interactions in bacteria:insights into transcription and chromosome organization. Mol Microbiol,2007,65:21-26
150. Wang Q, Li YC, Wang JH, Kong J, Qi YC, Quigg RJ, Li XM. miR-17-92 cluster accelerates adipocyte differentiation by negatively regulating tumor-suppressor Rb2/p130. Proc Natl Acad Sci USA,2008,105:2889-2894
151. Wernersson R, Schierup MH, J(?)rgensen FG, Gorodkin J, Panitz F, Staerfeldt HH, Christensen OF, Mailund T, Hornsh0j H, Klein A, Wang J, Liu B, Hu SN, Dong W, Li W, Wong GKS, Yu J, Wang J, Bendixen C, Fredholm M et al. Pigs in sequence space:a 0.66 X coverage pig genome survey based on shotgun sequencing. BMC Genomics,2005,6:70
152. White UA, Stephens JM. Transcriptional factors that promote formation of white adipose tissue. Mol Cell Endocrinol,2010,328:10-14
153. Wodarz A, Nusse R. Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol,1998,14:59-88
154. Wright WS, Longo KA, Dolinsky VW, Gerin I, Kang S, Bennett CN, Chiang SH, Prestwich TC, Gress C, Burant CF, Susulic VS, MacDougald OA. WntlOb inhibits obesity in ob/ob and agouti mice. Diabetes,2007,56:295-303
155. Wu D, Hu L. MicroRNA:a new kind of gene regulators. Agr Sci China,2006,5: 77-80
156. Xie H, Lim B, Lodish HR MicroRNAs induced during adipogenesis that accelerate fat cell development are downregulated in obesity. Diabetes,2009,58:1050-1057
157. Xie SS, Huang TH, Shen Y, Li XY, Zhang XX, Zhu MJ, Qin HY, Zhao SH. Identification and characterization of microRNAs from porcine skeletal muscle. Anim Genet,2010,41:179-190
158. Xu PZ, Vernooy SY, Guo M. The Drosophila microRNA mir-14 suppresses cell death and is required for normal fat metabolism. Curr Biol,2003,13:790-795
159. Yamakuchi M, Lowenstein CJ. MiR-34, SIRT1, and p53:the feedback loop. Cell Cycle,2009,8:712-715
160. Yang Z, Bian CJ, Zhou H, Huang S, Wang SH, Liao LM, Zhao RC. MicroRNA hsa-miR-138 inhibits adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells through adenovirus EID-1. Stem Cells Dev,2011,20: 259-267
161. Zaragosi LE, Wdziekonski B, Brigand KL, Villageois P, Mari B, Waldmann R, Dani C, Barbry P. Small RNA sequencing reveals miR-642a-3p. as a novel adipocyte-specific microRNA and miR-30 as a key regulator of human adipogenesis. Genome Biol,2011,12:R64
162. Zaragosi LE, Wdziekonski B, Fontaine C, Villageois P, Peraldi P, Dani C. Effect of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes. BMC Cell Biol,2008,9:11
163. Zhang BH, Pan XP, Cobb GP, Anderson TA. Plant microRNA:a small regulatory molecule with big impact. Dev Biol,2006,289:3-16
164. Zhao CZ, Xia H, Frazier TP, Yao YY, Bi YP, Li AQ, Li MJ, Li CS, Zhagn BH, Wang XJ. Deep sequencing identifies novel and conserved microRNAs in peanuts (Arachis Hypogaea L.). BMC Plant Biol,2010,10:3
165. Zhao Y, Samal E, Srivastava D. Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature,2005,436:214-220
166. Zhou B, Liu HL, Shi FX, Wang JY. MicroRNA expression profiles of porcine skeletal muscle. Anim Genet,2010,41:499-508