模拟失重条件下microRNA-494抑制成骨细胞分化作用机制的研究
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摘要
空间长期重力环境的改变会引起机体多个系统的变化,包括骨质疏松、肌肉萎缩、免疫系统功能不全、心血管系统适应性减弱等。通过物理训练和营养补充等措施的实施可以缓解肌肉萎缩、提高心血管系统的适应性,然而,目前尚没有办法对抗微重力引起的宇航员骨质流失,这是威胁长期进行太空工作宇航员身体健康的最主要因素之一。微重力导致骨质流失速度大约为每月减少2 %的骨矿物质密度,相当于绝经后妇女一年流失的骨量。研究表明,重力和机械负荷是保持骨骼完整性的重要因素,然而微重力环境引起骨质丢失的机制尚不完全清楚。已有的研究显示,失重引起的骨量减少是因为骨形成与重吸收之间的平衡被破坏,骨质形成减少,而重吸收保持正常或增加,最终导致了骨量丢失。由于成骨细胞分化是骨骼形成和维持骨量的关键步骤,所以目前被普遍接受的观点认为微重力导致的骨丢失的主要原因是成骨的细胞分化障碍,但是导致成骨细胞分化障碍的分子机制并不完全清楚。
miRNA是一类长度约18-25nt的小分子RNA,存在于包括哺乳动物在内的多种有机生命内。miRNA通过完全或不完全互补的方式结合于靶基因的mRNA的3’UTR,负向调控基因表达。大约超过30 %的基因表达都受到miRNA的调控,表明miRNA调控基因表达这一现象是普遍存在的。miRNA在细胞增殖、分化、死亡等过程中发挥着至关重要的作用。许多研究认为,miRNA参与了对成骨细胞成骨过程的表达调控,是成骨细胞分化的重要的调节分子。
本研究中我们以BMP-2诱导小鼠间充质多能前体细胞C2C12成骨分化为研究对象,探讨模拟失重状态下,成骨细胞分化过程中miRNA表达谱的改变,进而研究miRNA在失重性骨丢失中的作用及其分子机制。本研究的主要发现和结果如下:
1.微重力抑制成骨细胞分化和成熟。我们首先在细胞水平上检测微重力对成骨细胞分化的影响。我们将C2C12细胞置于回转器培养,同时加入300 ng/ml BMP-2诱导分化,对照组不加BMP-2刺激。72小时后,Real-time PCR检测成骨细胞特异基因碱性磷酸酶(Alkaline phosphatase,ALP),骨钙素(osteocalcin,OC),骨保护素(osteoprotegerin,OPG)和Runx2,结果显示,在模拟失重环境下,成骨细胞分化受到显著抑制。成骨细胞分化过程有多条通路参与,我们检测了微重力对部分通路相关分子表达的影响。我们按上述方法处理C2C12细胞,72小时后收集细胞裂解液,Western blot结果显示,微重力引起BMPR2、FGFR2、Runx2的蛋白表达降低。尾部悬吊能够消除大鼠后肢机械负荷,是研究模拟失重的良好动物模型。本研究中我们采用核素骨扫描检测了悬吊大鼠骨质形成。我们发现,与对照组大鼠相比悬吊大鼠骨和关节中99mTc-MDP的聚集明显减少,而且差异随悬吊时间延长而增大。该结果说明,微重力引起骨代谢减弱,骨生成减少。
2.微重力环境可以引起成骨细胞miRNA表达谱的改变。我们将C2C12细胞置于回转器中模拟失重培养,模拟失重培养细胞与对照细胞均加入BMP-2诱导其向成骨方向分化。72小时后,收集细胞提取总RNA并进行miRNA芯片检测。miRNA芯片筛选结果发现了7个显著差异表达的miRNA,其中表达上调的miRNA有2个,表达下调的miRNA有5个。Real-time PCR验证结果与芯片结果基本一致,其中mmu-miR-494模拟失重后表达水平明显升高,mmu-miR-18*, mmu-miR-122a, mmu-miR-301, and mmu-miR-340表达水平则显著降低,但mmu-miR-143的表达与对照组相比无显著差别。生物信息学方法预测表达上调的miR-494的靶基因参与成骨细胞分化,因此我们将关注的焦点放在对miR-494的研究上。给予BMP-2处理的C2C12细胞在模拟失重0、2、4、8、12、24、48和72小时后分别收集细胞检测miR-494表达水平的变化。我们发现,在模拟失重2小时,miR-494表达开始上升,并且随着失重时间的延长,miR-494表达持续升高。此外,我们分离了悬吊大鼠股骨近侧干垢端成骨细胞,检测其中miR-494表达水平,发现悬吊2周和4周的大鼠其成骨细胞内miR-494表达均明显上调,并随悬吊时间的延长而持续升高。该实验表明微重力环境能引起成骨细胞miRNA表达谱的改变,其中mmu-miR-494的升高十分显著,且其表达水平与模拟失重的时间正相关。
3. miR-494抑制成骨细胞分化。为了验证miR-494对骨形成的作用,我们首先检测miR-494对细胞增殖和细胞周期的影响。我们将miR-494 mimics及其相应阴性对照(N.C.)转染C2C12细胞和MC3T3-E1细胞,MTT实验和流式细胞术分析结果表明,miR-494对细胞增殖和周期无明显影响。进一步的研究显示,在BMP-2存在时,miR-494能明显抑细胞ALP活性,但未给予BMP-2刺激时,这种抑制效果不显著,表明miR-494可能参与了对C2C12细胞成骨分化的抑制。随后,我们用Reai-time PCR、ELISA和Western Blot分别在mRNA水平和蛋白水平检测了miR-494对成骨细胞特异基因ALP、OC、OPG、Runx2表达的影响。与ALP染色和活性测定结果一致,转染miR-494后细胞中ALP mRNA表达降低,而且无论是否存在BMP-2,OC、OPG和Runx2表达也有所下降。ELIAS分析结果显示,过表达miR-494减少了细胞分泌OC和OPG。在BMP-2诱导下,Runx2蛋白表达上调,但是转染miR-494后Runx2的上调被抑制。Osx是Runx2下游基因,我们发现在BMP-2诱导分化的C2C12细胞中过表达miR-494抑制了Osx的表达。另外,我们还发现在C2C12细胞中过表达miR-494后,其成肌分化的相关基因表达发生了上调,而miR-494对C2C12细胞成脂分化无明显作用。我们的研究表明,miR-494能抑制细胞的成骨分化。
4. miR-494通过调节Runx2、BMPR2和FGFR2的表达抑制成骨细胞分化。我们用生物信息学方法在包括miRanda、TargetScan、pictar和RNAhybrid databases等在内的多个网站进行miR-494靶基因预测,得到可能的靶基因超过1,000个。我们进一步从中筛选出参与成骨细胞分化的靶基因30个,并将这些基因3’UTR克隆入荧光素酶报告载体,与miR-494 mimics或N.C.共转染293A细胞,检测其相对荧光强度。结果发现,miR-494能显著抑制BMPR2、FGFR2和Runx2 3’UTR报告基因的荧光素酶活性,且抑制效率达到40-60 %。相反地,突变掉这些基因上miR-494的结合位点后,miR-494对荧光素酶活性的影响消失,表明miR-494可以结合并作用于上述基因的3’UTR。Western Blot和Real-time PCR结果显示,过表达miR-494明显抑制内源性BMPR2、FGFR2和Runx2的蛋白和mRNA表达。为了进一步确定miR-494是通过抑制BMPR2、FGFR2和Runx2的表达影响成骨细胞分化,我们合成了针对这3个基因的siRNA。将3种siRNA分别转染C2C12细胞72小时,成骨细胞特异基因表达下调,骨分化受抑制,其趋势与转染miR-494一致。我们的研究证明,miR-494通过直接下调和Runx2、BMPR2和FGFR2抑制成骨细胞的分化。
5.转录因子MyoD可能参与了对miR-494的表达调控。为了探讨miR-494转录调控机制,我们对miR-494上游序列进行了生物信息学分析。分析结果显示,距离pre-miR-494 5’端2-3kb的上游序列在不同种属中有高度的保守性,提示这段序列可能参与了对miR-494的转录调控。进一步的分析表明该区域有多个Myod的结合位点,提示转录因子Myod可能参与了miR-494的转录调节。我们首先分析了模拟失重时,BMP-2诱导分化的C2C12细胞中Myod表达情况,发现随着失重时间的延长,Myod表达发生了上调,这与miR-494表达变化相一致。随后,我们在正常重力条件下培养的C2C12细胞中加入BMP-2,研究细胞分化过程中mi-494与Myod表达变化情况,Real-time PCR结果显示两者表达水平随诱导时间的延长同时下降。此外,我们还发现转染miR-494后,MyoD的表达水平也发生了上调。我们的研究初步显示了转录因子MyoD可能参与了对miR-494的表达调控。为了证实我们的推测,进一步的的相关实验还在深入进行中。
6. miR-494 inhibitor能够部分缓解失重引起的成骨分化障碍。模拟失重时成骨细胞分化受抑制,miR-494表达升高。我们将针对miR-494的反义寡核苷酸,即miR-494 inhibitor及其相应对照N.C. inhibitor分别转染C2C12细胞,同时加入300 ng/ml BMP-2诱导分化,置于回转器模拟失重培养72小时,用Real-time PCR方法检测细胞成骨相关基因表达,发现抑制miR-494的表达能够使ALP染色增强,说明miR-494 inhibitor能够增加骨形成能力,部分缓解失重引起的成骨分化障碍。
总之,我们发现miRNA参与失重性成骨细胞功能异常的发生。其中,我们对表达上调的miR-494功能和作用机制进行了较为全面的研究,发现在模拟失重时miR-494表达升高,而且与失重时间正相关。体外实验证明,miR-494通过降低其靶基因BMPR2、FGFR2和Runx2的表达参与成骨细胞分化。过表达miR-494抑制成骨相关基因的表达,从而抑制骨形成;降低内源性miR-494表达则促进成骨分化,并且能够部分缓解由于失重引起的成骨分化障碍。该研究将对预防和治疗失重性骨质疏松提供理论依据。
Many serious adverse physiological changes occur during space?ight. Some of these include bone deterioration, muscle loss, immune system dysfunction, cardiovascular deconditioning and so on. Some of these pathophysiological adjustments can be counteracted adequately with physical exercise or nutritional supplementation. But many of the changes cannot be overcome. In particular, microgravity(MG)-induced osteoporosis is one of the most severe harms and the mechanism is not clear, which have always been the critical issue in this field. In the most severe forms of MG-induced bone loss, there is an approximately 2 % decrease in bone mineral density in only 1 month, equal to that of a postmenopausal woman in 1 year. Gravity and mechanical loading are known to be essential for the maintenance of skeletal integrity. It has been reported that the change induce by microgravity in bone mass is a result of the uncoupling of bone remodeling between decreased formation and normal (or increased) resorption. Osteoblast differentiation is a key step in proper skeletal development and acquisition of bone mass, and previous syudy proved that altered osteoblast function and development play an important role in MG-induced bone loss. But it is unclear that what mechanisms result in the dysfunction of osteoblast and what moleculars are involved in it.
MiRNA are endogenously expressed single-stranded noncoding RNAs of 18-25 nucleotides in length which have been identified in diverse organisms, including mammalian cells. MiRNA negatively regulate the translations of specific target genes by binding to the 3’untranslated regions (3’UTR) of the genes with partially or fully complementary sequences. It has been reported that over 30% of protein coding genes in humans are regulated by miRNA, which are involved in the regulation of development and homeostatic events. MiRNA play critical roles in cell proliferation, cell differentiation, and cell death. Many studies have identified that miRNA attenuation of gene expression posttranscripted has emerged as an important regulator of mesenchymal cell differentiation into the osteoblast lineage.
In this study, we determined the changes of miRNA expression partten in the BMP-2-induced differertiation of the pluripotent mesenchymal precursor cell line C2C12 simulated by microgravity exposure and further explored roles and mechanisms of miRNA in MG-induced bone loss. The main results and findings of this work are as follows:
1. Osteoblast differentiation and maturation are suppressed in microgravity. To identify the alterations of osteoblast differentiation induced by microgravity at the cellular level, C2C12 cells were cultured in clinorotation condition for 72 h without or with 300ng/ml BMP2. Real-time PCR for mRNA of the typical osteoblastic markers Alkaline phosphatase(ALP), osteocalcin(OC), osteoprotegerin(OPG), and runt-related transcription factor 2(Runx2)results showed the inhibition of osteogenesis either the BMP-2 absence or not. There are many pathways involved in osteogenesis. We investigated the expression levels of the critical moleculars related to osteoblast differentiation and maturation. We treated C2C12 cells under the same conditions for 72 hr and collected cell lysis. Western blot analysis revealed that the expressions of FGFR2, BMPR2, and Runx2 were reduced in microgrativity with or without BMP-2. The rat model of tail-suspended to make hind limb unloading has been used for detection the effects of microgrativity. The bone formation of tail-suspended rats was investigated by radionuclide bone scintigraphy, which has always been used to examine bone metabolism. We found that compard with the control group the accumulation of 99mTc-MDP was significantly lower either in bones or joints of the tail-suspended rats. And the di?erences became more and more significcant along with the time prolongation. These findings suggested that microgrativity resulted in decrease of bone metabolism and osteoblastogenesis.
2. Microarray analysis reveals the differential expression partten of microRNAs in microgravity. To detect the potential involvement of miRNA in osteoblast differentiation program in microgravity, microarray was performed by using total RNA from C2C12 cells cultured in clinorotation condition for 72h. The expression levels of 2 miRs (ratio≥2.0) increased and 5 miRNA decreased between stationary control and microgravity group. To validate the results of microarray profiling, Real-time PCR was performed to analyze miRNA in C2C12 cells cultured in microgravity condition for 72hr. Precursor of miR-494 (mmu-miR-494) was robustly up-regulated (>2-fold), and precursors of miR-18*, miR-122a, miR-301, and miR-340 (mmu-miR-18*, mmu-miR-122a, mmu-miR-301, and mmu-miR-340) were significantly down-regulated (>2-fold). However, precursor of miR-143 (mmu-miR-143) did not show significant change between stationary control and microgrativity group, which was not coincidence with the data from microarray analysis. Among the differentially expressed miRNA screened, we focused on the up-regulation of miR-494, because the potential target genes of miR-494 predicted by bioinformatic method play positive roles in osteoblast differentiation. To clarify the time-dependent expression of miR-494, we collected RNA from stationary control and microgrativity-treated samples at 2, 4, 8, 12, 24, 48 and72 h. Real-time PCR was performed to further analyze the expression pattern of miR-494 during microgravity of C2C12 cells. We found that miR-494 began to upregulate at 2h and kept increasing along with the time of microgravity. In addition, we isolated osteoblasts from the proximal femurs of tail-suspended rats to detecte the expression levels of miR-494. Real-time PCR analysis showed the expression of miR-494 was dramatically increased after 2 weeks of tail-suspension, and kept increasing with the time of unloading. These results indicited that miRNA may play important roles in abnomal osteblastogenesis induced by microgrativity.
3. MiR-494 inhibits the differentition of osteoblast in normal grativity. To elucidate the role of miR-494 in osteogenesis, firstly we investigated the effects of miR-494 on proliferation and cell cycles. We transfected C2C12 cells and MC3T3-E1 cells separately with miR-494 mimics and negative contro(lN.C.), and MTT assay and flow cytometry analysis suggested miR-494 had no significant effects on cell proliferation and cycles either in C2C12 cells or MC3T3-E1 cells. Then we measured ALP activities in C2C12 cells transfected with N.C. or miR-494 mimic with and without BMP-2 treatment for 72 h. The ALP activities in the transfected with miR-494 cells were significantly suppressed in the presence of BMP-2. There was no significant difference between transfection with negative control and miR-494, because mesenchymal stem cell C2C12 did not osteogenicly differentiate without BMP-2. To further investigated whether miR-494 are directly coupled to BMP-2-induced C2C12 osteoblastogenesis, C2C12 cells were transfected with N.C. or miR-494 and treated with BMP-2 simultaneously for 72 h. Real-time PCR, ELISA, and Western Blot were performed to determine the mRNA levels ALP, OC, OPG, and Runx2. Consistent with the ALP staining analysis, the mRNA expression of ALP was reduced, and the gene expressionsof OC, OPG, Runx2 were decreased whether BMP-2 presence or not. ELISA analysis showed miR-494 reduced the secretions of OC and OPG. Runx2 protein level was enhanced by BMP-2, but the enhancement was deducted in cells transfected with miR-494 compared with cells transfected with negative control. Thus, for osteogenesis of C2C12 cells to proceed, there is a requirement for BMP-2. But the introduction of miR-494 inhibits the osteoblast differentiation whether BMP-2 exist or not. Osx acts the downstreamof Runx2, and we found miR-494 inhibited Osx mRNA expression in C2C12 cell induced by BMP-2. In addition, miR-494 could promote myogenesis and have no effect on adipogenesis.
4. MiR-494 targeting Runx2, BMPR2, and FGFR2 inhibits the osteoblast differentition. More than 1,000 genes were predicted by miRanda, TargetScan, pictar or RNAhybrid databases to be potential target genes for miR-494. We selected more than 30 putative miR-494 target genes involved in osteoblastic differentiation from all the predicted genes and used a luciferase reporter assay system and transfection of miR-494 mimics to determine which targets were for miR-494. We cotransfected the luciferase reporter plasmids cloned the 3’-untranslated region (3’-UTR) sequences of the selected genes and negative control or miR-494 into 293A cells, the relative luciferase activity demonstrated whether miR-494 had effect on the genes. In 293A cells cotransfected with luciferase reporter genes carrying 3’-UTRs of BMPR2, FGFR2, and Runx2 with a putative miR-494 binding site, the level of suppression was about 40-60%. In contrast, the suppressive activity was lost when the seed sequence of miR-494 on 3’-UTRs of these genes were mutant. MiR-494 was able to down-regulated endogenous proteins of BMPR2, FGFR2, and Runx2, and able to suppress the mRNA levels of the three genes, consistent with the mechanism of miRNA regulation. To further address the hypothesis that miR-494 negatively regulated osteoblast differentiation by targeting key signal transduction factors as BMPR2, FGFR2, and Runx2, we introduced siRNAs targeted these genes which repression effects were confirmed by western blot analysis into C2C12 cells for 72 h. Real-time PCR analysis suggested knockdown of the three genes suppress the osteoblast differerntiation. Together, these results suggest that BMPR2, FGFR2, and Runx2 are targets for miR-494. On the other hand, miR-494 may indirectly downregulate Runx2 through suppressing the expressions of BMPR2 and FGFR2 which are the upstream genes of Runx2.
5. Myod enhances the expression of miR-494. To investigate the transcriptional regulation mechanism of miR-494, we analyzed the conservatism of 5’terminal of miR-494 by bioinformatics method. The region about 2-3 kb upstream of 5’terminal of pre-miR-494 is highly conserved, and there are several binding sites of Myod in this sequence. We identified the expression pattern of Myod during microgravity and BMP-2 induction in C2C12 cells. Real-time PCR result suggested the expression of Myod was increasd along with the time of MG and decreased with BMP-2 induction. So we presumed that Myod promote the expression of miR-494, and miR-494 upregulate Myod mRNA.
6. MiR-494 inhibitor can partly rescue the dysdifferentiation of osteoblast in microgrativity. Osteoblastic differentiation is suppressed and the expression of miR-494 is upregulated in microgrativity condition. C2C12 cells were transfected with miR-494 inhibitor or N.C.inhibitor and cultured in clinorotation condition for 72 h without or with 300ng/ml BMP-2. Real-time PCR analysis for the osteoblastic markers suggested that the inhibition of miR-494 partly enhanced ostogenesis with the presence of BMP-2 or not. Consistent with Real-time PCR array, ALP staining was increased following transfection of miR-494 inhibitor compared with transfection of N.C. inhibitor.
In conclusion, we found miRNA involved in dysfunction of osteoblast during MG. Overexpression of miR-494 inhibits osteogenesis and inhibition of endogenous miR-494 promotes ostenblast differentiation. Our finding might help to provide some theoretical basis to the protection and treatment of MG-induced Osteoporosis.
miRNA是一类长度约18-25nt的小分子RNA,存在于包括哺乳动物在内的多种有机生命内。miRNA通过完全或不完全互补的方式结合于靶基因的mRNA的3’UTR,负向调控基因表达。大约超过30 %的基因表达都受到miRNA的调控,表明miRNA调控基因表达这一现象是普遍存在的。miRNA在细胞增殖、分化、死亡等过程中发挥着至关重要的作用。许多研究认为,miRNA参与了对成骨细胞成骨过程的表达调控,是成骨细胞分化的重要的调节分子。
本研究中我们以BMP-2诱导小鼠间充质多能前体细胞C2C12成骨分化为研究对象,探讨模拟失重状态下,成骨细胞分化过程中miRNA表达谱的改变,进而研究miRNA在失重性骨丢失中的作用及其分子机制。本研究的主要发现和结果如下:
1.微重力抑制成骨细胞分化和成熟。我们首先在细胞水平上检测微重力对成骨细胞分化的影响。我们将C2C12细胞置于回转器培养,同时加入300 ng/ml BMP-2诱导分化,对照组不加BMP-2刺激。72小时后,Real-time PCR检测成骨细胞特异基因碱性磷酸酶(Alkaline phosphatase,ALP),骨钙素(osteocalcin,OC),骨保护素(osteoprotegerin,OPG)和Runx2,结果显示,在模拟失重环境下,成骨细胞分化受到显著抑制。成骨细胞分化过程有多条通路参与,我们检测了微重力对部分通路相关分子表达的影响。我们按上述方法处理C2C12细胞,72小时后收集细胞裂解液,Western blot结果显示,微重力引起BMPR2、FGFR2、Runx2的蛋白表达降低。尾部悬吊能够消除大鼠后肢机械负荷,是研究模拟失重的良好动物模型。本研究中我们采用核素骨扫描检测了悬吊大鼠骨质形成。我们发现,与对照组大鼠相比悬吊大鼠骨和关节中99mTc-MDP的聚集明显减少,而且差异随悬吊时间延长而增大。该结果说明,微重力引起骨代谢减弱,骨生成减少。
2.微重力环境可以引起成骨细胞miRNA表达谱的改变。我们将C2C12细胞置于回转器中模拟失重培养,模拟失重培养细胞与对照细胞均加入BMP-2诱导其向成骨方向分化。72小时后,收集细胞提取总RNA并进行miRNA芯片检测。miRNA芯片筛选结果发现了7个显著差异表达的miRNA,其中表达上调的miRNA有2个,表达下调的miRNA有5个。Real-time PCR验证结果与芯片结果基本一致,其中mmu-miR-494模拟失重后表达水平明显升高,mmu-miR-18*, mmu-miR-122a, mmu-miR-301, and mmu-miR-340表达水平则显著降低,但mmu-miR-143的表达与对照组相比无显著差别。生物信息学方法预测表达上调的miR-494的靶基因参与成骨细胞分化,因此我们将关注的焦点放在对miR-494的研究上。给予BMP-2处理的C2C12细胞在模拟失重0、2、4、8、12、24、48和72小时后分别收集细胞检测miR-494表达水平的变化。我们发现,在模拟失重2小时,miR-494表达开始上升,并且随着失重时间的延长,miR-494表达持续升高。此外,我们分离了悬吊大鼠股骨近侧干垢端成骨细胞,检测其中miR-494表达水平,发现悬吊2周和4周的大鼠其成骨细胞内miR-494表达均明显上调,并随悬吊时间的延长而持续升高。该实验表明微重力环境能引起成骨细胞miRNA表达谱的改变,其中mmu-miR-494的升高十分显著,且其表达水平与模拟失重的时间正相关。
3. miR-494抑制成骨细胞分化。为了验证miR-494对骨形成的作用,我们首先检测miR-494对细胞增殖和细胞周期的影响。我们将miR-494 mimics及其相应阴性对照(N.C.)转染C2C12细胞和MC3T3-E1细胞,MTT实验和流式细胞术分析结果表明,miR-494对细胞增殖和周期无明显影响。进一步的研究显示,在BMP-2存在时,miR-494能明显抑细胞ALP活性,但未给予BMP-2刺激时,这种抑制效果不显著,表明miR-494可能参与了对C2C12细胞成骨分化的抑制。随后,我们用Reai-time PCR、ELISA和Western Blot分别在mRNA水平和蛋白水平检测了miR-494对成骨细胞特异基因ALP、OC、OPG、Runx2表达的影响。与ALP染色和活性测定结果一致,转染miR-494后细胞中ALP mRNA表达降低,而且无论是否存在BMP-2,OC、OPG和Runx2表达也有所下降。ELIAS分析结果显示,过表达miR-494减少了细胞分泌OC和OPG。在BMP-2诱导下,Runx2蛋白表达上调,但是转染miR-494后Runx2的上调被抑制。Osx是Runx2下游基因,我们发现在BMP-2诱导分化的C2C12细胞中过表达miR-494抑制了Osx的表达。另外,我们还发现在C2C12细胞中过表达miR-494后,其成肌分化的相关基因表达发生了上调,而miR-494对C2C12细胞成脂分化无明显作用。我们的研究表明,miR-494能抑制细胞的成骨分化。
4. miR-494通过调节Runx2、BMPR2和FGFR2的表达抑制成骨细胞分化。我们用生物信息学方法在包括miRanda、TargetScan、pictar和RNAhybrid databases等在内的多个网站进行miR-494靶基因预测,得到可能的靶基因超过1,000个。我们进一步从中筛选出参与成骨细胞分化的靶基因30个,并将这些基因3’UTR克隆入荧光素酶报告载体,与miR-494 mimics或N.C.共转染293A细胞,检测其相对荧光强度。结果发现,miR-494能显著抑制BMPR2、FGFR2和Runx2 3’UTR报告基因的荧光素酶活性,且抑制效率达到40-60 %。相反地,突变掉这些基因上miR-494的结合位点后,miR-494对荧光素酶活性的影响消失,表明miR-494可以结合并作用于上述基因的3’UTR。Western Blot和Real-time PCR结果显示,过表达miR-494明显抑制内源性BMPR2、FGFR2和Runx2的蛋白和mRNA表达。为了进一步确定miR-494是通过抑制BMPR2、FGFR2和Runx2的表达影响成骨细胞分化,我们合成了针对这3个基因的siRNA。将3种siRNA分别转染C2C12细胞72小时,成骨细胞特异基因表达下调,骨分化受抑制,其趋势与转染miR-494一致。我们的研究证明,miR-494通过直接下调和Runx2、BMPR2和FGFR2抑制成骨细胞的分化。
5.转录因子MyoD可能参与了对miR-494的表达调控。为了探讨miR-494转录调控机制,我们对miR-494上游序列进行了生物信息学分析。分析结果显示,距离pre-miR-494 5’端2-3kb的上游序列在不同种属中有高度的保守性,提示这段序列可能参与了对miR-494的转录调控。进一步的分析表明该区域有多个Myod的结合位点,提示转录因子Myod可能参与了miR-494的转录调节。我们首先分析了模拟失重时,BMP-2诱导分化的C2C12细胞中Myod表达情况,发现随着失重时间的延长,Myod表达发生了上调,这与miR-494表达变化相一致。随后,我们在正常重力条件下培养的C2C12细胞中加入BMP-2,研究细胞分化过程中mi-494与Myod表达变化情况,Real-time PCR结果显示两者表达水平随诱导时间的延长同时下降。此外,我们还发现转染miR-494后,MyoD的表达水平也发生了上调。我们的研究初步显示了转录因子MyoD可能参与了对miR-494的表达调控。为了证实我们的推测,进一步的的相关实验还在深入进行中。
6. miR-494 inhibitor能够部分缓解失重引起的成骨分化障碍。模拟失重时成骨细胞分化受抑制,miR-494表达升高。我们将针对miR-494的反义寡核苷酸,即miR-494 inhibitor及其相应对照N.C. inhibitor分别转染C2C12细胞,同时加入300 ng/ml BMP-2诱导分化,置于回转器模拟失重培养72小时,用Real-time PCR方法检测细胞成骨相关基因表达,发现抑制miR-494的表达能够使ALP染色增强,说明miR-494 inhibitor能够增加骨形成能力,部分缓解失重引起的成骨分化障碍。
总之,我们发现miRNA参与失重性成骨细胞功能异常的发生。其中,我们对表达上调的miR-494功能和作用机制进行了较为全面的研究,发现在模拟失重时miR-494表达升高,而且与失重时间正相关。体外实验证明,miR-494通过降低其靶基因BMPR2、FGFR2和Runx2的表达参与成骨细胞分化。过表达miR-494抑制成骨相关基因的表达,从而抑制骨形成;降低内源性miR-494表达则促进成骨分化,并且能够部分缓解由于失重引起的成骨分化障碍。该研究将对预防和治疗失重性骨质疏松提供理论依据。
Many serious adverse physiological changes occur during space?ight. Some of these include bone deterioration, muscle loss, immune system dysfunction, cardiovascular deconditioning and so on. Some of these pathophysiological adjustments can be counteracted adequately with physical exercise or nutritional supplementation. But many of the changes cannot be overcome. In particular, microgravity(MG)-induced osteoporosis is one of the most severe harms and the mechanism is not clear, which have always been the critical issue in this field. In the most severe forms of MG-induced bone loss, there is an approximately 2 % decrease in bone mineral density in only 1 month, equal to that of a postmenopausal woman in 1 year. Gravity and mechanical loading are known to be essential for the maintenance of skeletal integrity. It has been reported that the change induce by microgravity in bone mass is a result of the uncoupling of bone remodeling between decreased formation and normal (or increased) resorption. Osteoblast differentiation is a key step in proper skeletal development and acquisition of bone mass, and previous syudy proved that altered osteoblast function and development play an important role in MG-induced bone loss. But it is unclear that what mechanisms result in the dysfunction of osteoblast and what moleculars are involved in it.
MiRNA are endogenously expressed single-stranded noncoding RNAs of 18-25 nucleotides in length which have been identified in diverse organisms, including mammalian cells. MiRNA negatively regulate the translations of specific target genes by binding to the 3’untranslated regions (3’UTR) of the genes with partially or fully complementary sequences. It has been reported that over 30% of protein coding genes in humans are regulated by miRNA, which are involved in the regulation of development and homeostatic events. MiRNA play critical roles in cell proliferation, cell differentiation, and cell death. Many studies have identified that miRNA attenuation of gene expression posttranscripted has emerged as an important regulator of mesenchymal cell differentiation into the osteoblast lineage.
In this study, we determined the changes of miRNA expression partten in the BMP-2-induced differertiation of the pluripotent mesenchymal precursor cell line C2C12 simulated by microgravity exposure and further explored roles and mechanisms of miRNA in MG-induced bone loss. The main results and findings of this work are as follows:
1. Osteoblast differentiation and maturation are suppressed in microgravity. To identify the alterations of osteoblast differentiation induced by microgravity at the cellular level, C2C12 cells were cultured in clinorotation condition for 72 h without or with 300ng/ml BMP2. Real-time PCR for mRNA of the typical osteoblastic markers Alkaline phosphatase(ALP), osteocalcin(OC), osteoprotegerin(OPG), and runt-related transcription factor 2(Runx2)results showed the inhibition of osteogenesis either the BMP-2 absence or not. There are many pathways involved in osteogenesis. We investigated the expression levels of the critical moleculars related to osteoblast differentiation and maturation. We treated C2C12 cells under the same conditions for 72 hr and collected cell lysis. Western blot analysis revealed that the expressions of FGFR2, BMPR2, and Runx2 were reduced in microgrativity with or without BMP-2. The rat model of tail-suspended to make hind limb unloading has been used for detection the effects of microgrativity. The bone formation of tail-suspended rats was investigated by radionuclide bone scintigraphy, which has always been used to examine bone metabolism. We found that compard with the control group the accumulation of 99mTc-MDP was significantly lower either in bones or joints of the tail-suspended rats. And the di?erences became more and more significcant along with the time prolongation. These findings suggested that microgrativity resulted in decrease of bone metabolism and osteoblastogenesis.
2. Microarray analysis reveals the differential expression partten of microRNAs in microgravity. To detect the potential involvement of miRNA in osteoblast differentiation program in microgravity, microarray was performed by using total RNA from C2C12 cells cultured in clinorotation condition for 72h. The expression levels of 2 miRs (ratio≥2.0) increased and 5 miRNA decreased between stationary control and microgravity group. To validate the results of microarray profiling, Real-time PCR was performed to analyze miRNA in C2C12 cells cultured in microgravity condition for 72hr. Precursor of miR-494 (mmu-miR-494) was robustly up-regulated (>2-fold), and precursors of miR-18*, miR-122a, miR-301, and miR-340 (mmu-miR-18*, mmu-miR-122a, mmu-miR-301, and mmu-miR-340) were significantly down-regulated (>2-fold). However, precursor of miR-143 (mmu-miR-143) did not show significant change between stationary control and microgrativity group, which was not coincidence with the data from microarray analysis. Among the differentially expressed miRNA screened, we focused on the up-regulation of miR-494, because the potential target genes of miR-494 predicted by bioinformatic method play positive roles in osteoblast differentiation. To clarify the time-dependent expression of miR-494, we collected RNA from stationary control and microgrativity-treated samples at 2, 4, 8, 12, 24, 48 and72 h. Real-time PCR was performed to further analyze the expression pattern of miR-494 during microgravity of C2C12 cells. We found that miR-494 began to upregulate at 2h and kept increasing along with the time of microgravity. In addition, we isolated osteoblasts from the proximal femurs of tail-suspended rats to detecte the expression levels of miR-494. Real-time PCR analysis showed the expression of miR-494 was dramatically increased after 2 weeks of tail-suspension, and kept increasing with the time of unloading. These results indicited that miRNA may play important roles in abnomal osteblastogenesis induced by microgrativity.
3. MiR-494 inhibits the differentition of osteoblast in normal grativity. To elucidate the role of miR-494 in osteogenesis, firstly we investigated the effects of miR-494 on proliferation and cell cycles. We transfected C2C12 cells and MC3T3-E1 cells separately with miR-494 mimics and negative contro(lN.C.), and MTT assay and flow cytometry analysis suggested miR-494 had no significant effects on cell proliferation and cycles either in C2C12 cells or MC3T3-E1 cells. Then we measured ALP activities in C2C12 cells transfected with N.C. or miR-494 mimic with and without BMP-2 treatment for 72 h. The ALP activities in the transfected with miR-494 cells were significantly suppressed in the presence of BMP-2. There was no significant difference between transfection with negative control and miR-494, because mesenchymal stem cell C2C12 did not osteogenicly differentiate without BMP-2. To further investigated whether miR-494 are directly coupled to BMP-2-induced C2C12 osteoblastogenesis, C2C12 cells were transfected with N.C. or miR-494 and treated with BMP-2 simultaneously for 72 h. Real-time PCR, ELISA, and Western Blot were performed to determine the mRNA levels ALP, OC, OPG, and Runx2. Consistent with the ALP staining analysis, the mRNA expression of ALP was reduced, and the gene expressionsof OC, OPG, Runx2 were decreased whether BMP-2 presence or not. ELISA analysis showed miR-494 reduced the secretions of OC and OPG. Runx2 protein level was enhanced by BMP-2, but the enhancement was deducted in cells transfected with miR-494 compared with cells transfected with negative control. Thus, for osteogenesis of C2C12 cells to proceed, there is a requirement for BMP-2. But the introduction of miR-494 inhibits the osteoblast differentiation whether BMP-2 exist or not. Osx acts the downstreamof Runx2, and we found miR-494 inhibited Osx mRNA expression in C2C12 cell induced by BMP-2. In addition, miR-494 could promote myogenesis and have no effect on adipogenesis.
4. MiR-494 targeting Runx2, BMPR2, and FGFR2 inhibits the osteoblast differentition. More than 1,000 genes were predicted by miRanda, TargetScan, pictar or RNAhybrid databases to be potential target genes for miR-494. We selected more than 30 putative miR-494 target genes involved in osteoblastic differentiation from all the predicted genes and used a luciferase reporter assay system and transfection of miR-494 mimics to determine which targets were for miR-494. We cotransfected the luciferase reporter plasmids cloned the 3’-untranslated region (3’-UTR) sequences of the selected genes and negative control or miR-494 into 293A cells, the relative luciferase activity demonstrated whether miR-494 had effect on the genes. In 293A cells cotransfected with luciferase reporter genes carrying 3’-UTRs of BMPR2, FGFR2, and Runx2 with a putative miR-494 binding site, the level of suppression was about 40-60%. In contrast, the suppressive activity was lost when the seed sequence of miR-494 on 3’-UTRs of these genes were mutant. MiR-494 was able to down-regulated endogenous proteins of BMPR2, FGFR2, and Runx2, and able to suppress the mRNA levels of the three genes, consistent with the mechanism of miRNA regulation. To further address the hypothesis that miR-494 negatively regulated osteoblast differentiation by targeting key signal transduction factors as BMPR2, FGFR2, and Runx2, we introduced siRNAs targeted these genes which repression effects were confirmed by western blot analysis into C2C12 cells for 72 h. Real-time PCR analysis suggested knockdown of the three genes suppress the osteoblast differerntiation. Together, these results suggest that BMPR2, FGFR2, and Runx2 are targets for miR-494. On the other hand, miR-494 may indirectly downregulate Runx2 through suppressing the expressions of BMPR2 and FGFR2 which are the upstream genes of Runx2.
5. Myod enhances the expression of miR-494. To investigate the transcriptional regulation mechanism of miR-494, we analyzed the conservatism of 5’terminal of miR-494 by bioinformatics method. The region about 2-3 kb upstream of 5’terminal of pre-miR-494 is highly conserved, and there are several binding sites of Myod in this sequence. We identified the expression pattern of Myod during microgravity and BMP-2 induction in C2C12 cells. Real-time PCR result suggested the expression of Myod was increasd along with the time of MG and decreased with BMP-2 induction. So we presumed that Myod promote the expression of miR-494, and miR-494 upregulate Myod mRNA.
6. MiR-494 inhibitor can partly rescue the dysdifferentiation of osteoblast in microgrativity. Osteoblastic differentiation is suppressed and the expression of miR-494 is upregulated in microgrativity condition. C2C12 cells were transfected with miR-494 inhibitor or N.C.inhibitor and cultured in clinorotation condition for 72 h without or with 300ng/ml BMP-2. Real-time PCR analysis for the osteoblastic markers suggested that the inhibition of miR-494 partly enhanced ostogenesis with the presence of BMP-2 or not. Consistent with Real-time PCR array, ALP staining was increased following transfection of miR-494 inhibitor compared with transfection of N.C. inhibitor.
In conclusion, we found miRNA involved in dysfunction of osteoblast during MG. Overexpression of miR-494 inhibits osteogenesis and inhibition of endogenous miR-494 promotes ostenblast differentiation. Our finding might help to provide some theoretical basis to the protection and treatment of MG-induced Osteoporosis.
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