miR-145抑制结肠癌的实验研究
摘要
结肠癌是一种常见的消化道恶性肿瘤,发病率呈逐年上升趋势。2010年美国癌症协会发布的统计报告指出,在美国男性和女性肿瘤患者中,发病率和病死率均上升,分别位列第2位和第3位。我国原本是结肠癌发病率比较低的国家,随着我国经济的发展、人们生活水平的提高及膳食结构的改变,近年来我国结肠癌发病率逐年上升。统计资料显示,我国男性结肠癌的发病率仅次于肺癌和胃癌,女性仅次于乳腺癌和子宫颈癌,均位列第三位,其术后5年生存率仅50%左右。目前研究表明,结肠癌可能起因于结肠上皮原癌基因突变、抑癌基因失活、基因组表观遗传学修饰改变和环境等多因素共同作用,但其确切机制仍不十分明了。深入研究结肠癌的发病机制,对结肠癌的早期诊断、治疗和改善患者生活质量有着十分重要的意义。
miRNA (microRNA)是生物体内一类长度为21.25个核苷酸,内源性的非编码小分子单链RNA,主要通过转录后水平调节,抑制靶基因的表达。miRNA广泛参与细胞增殖、分化、凋亡、组织器官发育等生理过程和肿瘤发生、发展、侵袭、转移等病理过程的调控。近年来,miRNA涉及到多种人类肿瘤,通过一系列不同的机制,miRNA功能的增强或减弱都影响了肿瘤的进程。正因为miRNA能调控肿瘤细胞的分化、增殖、存活及转移,所以无论正性还是负性的肿瘤相关miRNA,都可以为干预肿瘤的发生及发展提供强大的治疗策略。
为了深入研究miRNA在结肠癌中的作用机制,首先,本课题组采用miRNA表达芯片筛选结肠癌组织和癌旁组织中差异表达miRNA分子,获得了一组结肠癌相关候选miRNA分子,其中miR-145差异表达最明显,相对于癌旁组织,结肠癌组织中miR-145表达水平显著下调,并在实时定量RT-PCR实验中得到了证实；其次,选取miR-145作为进一步研究对象,通过体内、体外实验研究miR-145在结肠癌中的生物学功能；最后在结肠癌细胞HCT116中通过荧光素酶活性分析和Western blot初步证明miR-145可以靶向调控转录因子SOX9。本研究首次提出“miR-145可能通过靶向调控转录因子SOX9参与结肠癌恶性表型发生”的假说,将为全面阐明miR-145在结肠癌中的作用机制奠定理论基础,更为结肠癌候选诊断分子和治疗药物的研发提供理论依据。
第一部分结肠癌相关候选miRNA的筛选与验证
目的：
探讨结肠癌组织和癌旁组织中miRNA的表达差异,获得结肠癌相关的候选miRNA分子。
方法：
对6例结肠癌组织和癌旁组织进行miRNA表达芯片检测,对感兴趣的差异表达miRNA分子采用实时定量RT-PCR进行验证。
结果：
1. miRNA差异表达谱分析发现,51个miRNA在结肠癌组织和癌旁组织表达有差异,差异均在2倍变化以上,具有统计学意义。其中,24个miRNA在结肠癌组织中表达下调,27个miRNA表达上调。相对于癌旁组织,结肠癌组织中miR-145和miR-143表达下调3.492和2.899倍,结肠癌组织中miR-96表达上调3.083倍。
2.实时定量RT-PCR检测显示结肠癌组织中miR-145和miR-143表达水平相对癌旁组织下调3.533倍和2.425倍,miR-96表达水平相对癌旁组织上调2.363倍。
结论：
相对于癌旁组织,结肠癌中miRNA具有差异表达谱,miR-145表达水平变化与结肠癌密切相关,可供进一步研究。
第二部分miR-145体外抑制结肠癌细胞HCT116增殖的实验研究
目的：
体外实验研究miR-145在结肠癌细胞HCT116中的生物学功能。
方法：
1.采用PCR扩增miR-145基因片段,将其克隆入真核表达载体pCMV-myc中,构建重组质粒pCMV-miR-145。
2.酶切及DNA测序鉴定重组质粒后,脂质体法转染结肠癌细胞HCT116,运用实时定量RT-PCR检测转染后细胞中miR-145的表达情况。
3.分别运用CCK8、流式细胞术和细胞划痕试验检测过表达miR-145对HCT116细胞增殖、细胞周期和迁移能力的影响。
结果：
1.成功构建了miR-145的真核表达载体pCMV-miR-145。实时定量RT-PCR证明转染该载体后,相对于空载体转染组,HCT116细胞中miR-145表达水平上升754倍。
2.相对于对照组,真核表达载体pCMV-miR-145转染组G1期百分比升高(62.80±1.22%vs51.61±0.91%),吸光度值降低(0.345±0.032vs0.632±0.033),划痕中央区细胞数目减少(176±24vs276±26)。
结论：
体外过表达miR-145,可导致结肠癌细胞HCT116细胞周期发生G1期阻滞、细胞增殖速度变慢、细胞迁移能力降低。
第三部分miR-145抑制裸鼠皮下移植瘤的实验研究
目的：
体内实验研究miR-145在结肠癌中的生物学功能。
方法：
建立结肠癌细胞HCT116裸鼠皮下移植瘤模型,观察过表达miR-145对肿瘤体积大小、倍增时间的影响。
结果：
过表达miR-145后,肿瘤体积明显减少(368.18±78mm3vs914.14±204mm3)、倍增时间延长(6.5±1.5天vs3.8±0.9天)。
结论：
miR-145可在体内抑制结肠癌细胞HCT116增殖。
第四部分miR-145抑制结肠癌细胞HCT116增殖机制的研究
目的：
探讨miR-145抑制结肠癌细胞HCT116增殖功能的可能机制。
方法：
1.采用生物信息学分析miR-145的下游靶基因及其与靶基因的结合位点。
2.构建基于靶基因SOX9含miR-145结合位点的荧光素酶报告载体pMIR-Report, DNA测序鉴定重组质粒。
3.将重组质粒转染入结肠癌细胞HCT116,检测荧光素酶活性的变化。
4. Western-blot实验检测过表达miR-145和降低内源性miR-145水平时靶基因SOX9的表达变化。
5.实时定量RT-PCR和Western-blot检测癌组织与癌旁组织中miR-145和SOX9及其下游分子MUC2的mRNA表达水平和蛋白表达水平。
结果：
1.生物信息学结合文献检索,确定转录因子SOX9为miR-145的潜在下游靶基因,SOX9的mRNA3'UTR区-282和-1402有两个可能结合位点。
2.含有miR-145结合位点的pMIR-282和pMIR-1402,在过表达miR-145时,其荧光素酶活性相对空载体分别下降50.29%和59.83%；在对核心结合位点突变后,荧光素酶活性恢复,与野生型水平接近。
3.过表达miR-145后,Western-blot检测显示SOX9蛋白显著下降,MUC2蛋白水平相应增加；通过化学修饰的反义寡核苷酸降低内源性miR-145表达水平后,Western-blot检测发现SOX9蛋白显著增加,MUC2蛋白表达减少。
4.相对于癌旁组织,miR-145的mRNA水平在癌组织中下调,SOX9的mRNA水平相应增加,MUC2的mRNA水平相应下降,同时,癌组织中SOX9的蛋白水平上调,其下游MUC2因为受到SOX9抑制其蛋白水平下降。
结论：
miR-145可能通过调节SOX9及下游分子MUC2抑制结肠癌细胞增殖。
Colon cancer is one of the most common gastrointestinal malignant tumors, with the incidence increasing each year. Statistical reports of the American Cancer Society in2010shows that the incidence and case fatality rate of colon cancer in male and female cancer patients was ranked No.3and ranked2in the tumor in the U.S. respectively. China was in countries with low incidence of colon cancer, but in recent years as China's rapid economic development and improved living standards, lifestyle and dietary pattern of change, there is an increasing trend in the incidence of colon cancer which was ranked NO.3both in male and female. The five-year survival rate after surgery is only about fifty percent. The previous study showed that the colon cancer caused by gain of function of oncogene and lose of function of tumor suppressor gene in colonic epithelial cells, epigenetic modification of genomic changes and environment factors. But its pathogenesis is not yet very clear, in-depth study of the pathogenesis of colon cancer is greatly benefits for early diagnosis, treatment and improved quality of life of patients.
miRNA (microRNA) is a endogenous single-stranded non-coding small RNA with its length of21-25nucleotide in organisms, mainly through the inhibition of target gene expression at post-transcriptional level. miRNA are widely involved in cell proliferation, differentiation, apoptosis, tissue and organ development, tumor genesis, and tumor metastasis. In recent years, many studies show that miRNAs involved in a variety of human tumors through a series of different mechanisms. Enhancing or diminishing miRNA function will affect the process of tumor. Precisely because of the miRNA regulation of cancer cell differentiation, proliferation, survival and metastasis, positive or negative tumor-associated of miRNA can be thought to provide a powerful treatment strategy for tumor occurrence and development of intervention.
For further research on the mechanism of miRNAs in colon cancer, firstly we carried out the expression profiling analysis both in the tumor tissues and adjacent tissues from clinical patients respectively and gain a group of colon cancer-related candidate miRNAs. The expression level of miR-145in colon cancer is significantly lower than adjacent tissue by using Chip array and the real time reverse transcription polymerase chain reaction experiments.Secondly this study intends to verify and analyze the biological role of miR-145in colon cancer. Finally, we want to initially verify the hypothesis that miR-145may lead to the occurrence of colon cancer malignant phenotype through the regulation of target gene SOX9and related downstream signaling pathways by fluciferase assay and western blot. This research put forward the hypothesis that miR-145may cause development of colon cancer by target regulating transcription factors SOX9for the first time, lay base for a comprehensive understanding of the roles of miR-145in colon cancer, provide the theory basis for the diagnosis and drug treatment of colon cancer drug.
PartⅠ The screening and validation of the colon cancer-related candidate miRNAs
Objective
To explore the miRNA expression pattern difference between colon cancer tissues and adjacent tissues and gain a group of colon cancer-related candidate miRNAs.
Methods
miRNA expression microarray analysis was carried out in tumor tissue and adjacent tissue from six clinical patients. We further validated the expression of interesting candidate miRNA using real time reverse transcription polymerase chain reaction (real-time RT-PCR) method.
Results
1. The Genechip found51miRNAs, including that there are24miRNAs downregulated and27miRNA upregulated in tumor tissue comparing with adjacent tissues, the differences were statistically significant in more than two times. Expression levels of miR-145and miR-143were down-regualted3.492fold and2.899fold respectively, while expression level of miR-96was up-regualted 3.083fold in colon cancer.
2. The expression levels of miR-145and miR-143in tumor tissue were down-regualted3.533fold and2.425fold comparing to adjacent tissues through real-time RT-PCR validation, the expression level of miR-96in tumor tissues was up-regualted2.363fold comparing to adjacent tissues through real-time RT-PCR validation.
Conclusion
The expression profile of miRNA is different between tumor tissues and adjacent tissues. miR-145may play roles in colon cancer and available for further study.
Partll The study of miR-145suppressing the proliferation of HCT116cells in vitro
Objective
To investigate the biological function of miR-145involved in HCT116cells in vitro.
Methods
1. miR-145genes fragment was obtained by PCR from genomic DNA extracted from normal tissue and inserted into eukaryotic expression plasmid pCMV-myc.
2. The constructed plasmids containing right sequence of target miR-145were transfected into HCT116cells via lipofectamine. The expression level of miR-145was detected by real-time RT-PCR.
3. The proliferation of HCT116over-expressed with miR-145was detected using CCK8kit and the cell cycle was detected by Flow Cytometer, and the transfer ability change was detected by scratch test.
Results
1. The pCMV-miR-145plasmid which over expressing miR-145was successfully constructed, the plasmid could increase expression level of miR-145in HCT116cells by754times comparing to empty vector.
2. Comparing to control, after transferred by pCMV-miR-145plasmid, percentage of G1phase increased (62.80±1.22%vs51.61±0.91%); OD value decresed (0.345±0.032vs0.632±0.033); cells number of scramble decresed (176±24vs276±26)
Conclusion
Over-expression of exogenous miR-145can suppress the proliferation of HCT116cells by cuasing G1cell cycle block and suppressing proliferation and transfer ability in vitro.
PartⅢ The experiment of miR-145restraining growth of subcutaneous transplant tumors in nude mice
Objective
To investigate the biological function of miR-145involved in HCT116cells in vivo.
Methods
Animal model of subcutaneous transplant tumor induced by colon cancer HCT116cells in nude mice was established. Tumor size and doubling time were observed after overexpression of miR-145.
Results
Tumor size significantly reduced (368.18±78mm3vs914.14±204mm3), as well as doubling time extended after overexpression of miR-145(6.5±1.5day vs3.8±0.9day).
Conclusion
Over-expression of exogenous miR-145can suppress the proliferation of HCT116cells in vivo.
PartⅣ The mechan ism of miR-145suppressing the proliferation of HCT116cells Objective
To study possible mechanisms of miR-145inhibiting proliferation of HCT116cells.
Methods
1. Identification of the potential target of miR-145using bioinformatics.
2. miR-145binding site in SOX9gene locus was obtained by PCR from genomic DNA extracted from normal tissue and inserted into luciferase reporter plasmid pMIR-reporter.
3. The constructed plasmids containing right sequence of target were transfected into HCT116cells via lipofectamine for the detection of luciferase activity. The expression change of SOX9was detected by western blotting, when over expressed or low expressed miR-145.
4. Detect the mRNA level of miR-145, SOX9and MUC2in tumor and adjacent tissues from clinical patients. Furthermore, the protein level of SOX9and MUC2was also detected by western blotting.
Results
1. SOX9was selected as the potential target of miR-145by bioinformatics. There are two miR-145binding site in the3'UTR of SOX9, which located at the-282bp and-1402bp downstream of stop codon respectively.
2. The luciferase activity of pMIR-282and pMIR-1402was decreased significantly50.29%and59.83%respectively comparing with the control. After the'seed sequence'was mutated, the luciferase activity can back to the normal level.
3. Over-expressed miR-145can decrease the protein level of SOX9and elevate the protein level of MUC2. Knock down of miR-145using antisense inhibitor can increase the protein level of SOX9, vice versa.
4. The expression level of miR-145is significantly down-regulated in tumor tissues. Meanwhile, the expression level of SOX9increased and MUC2decreased on the contrary.
Conclusion
miR-145may suppress the proliferation of colon cancer by suppressing target gene SOX9and downstream MUC2.
miRNA (microRNA)是生物体内一类长度为21.25个核苷酸,内源性的非编码小分子单链RNA,主要通过转录后水平调节,抑制靶基因的表达。miRNA广泛参与细胞增殖、分化、凋亡、组织器官发育等生理过程和肿瘤发生、发展、侵袭、转移等病理过程的调控。近年来,miRNA涉及到多种人类肿瘤,通过一系列不同的机制,miRNA功能的增强或减弱都影响了肿瘤的进程。正因为miRNA能调控肿瘤细胞的分化、增殖、存活及转移,所以无论正性还是负性的肿瘤相关miRNA,都可以为干预肿瘤的发生及发展提供强大的治疗策略。
为了深入研究miRNA在结肠癌中的作用机制,首先,本课题组采用miRNA表达芯片筛选结肠癌组织和癌旁组织中差异表达miRNA分子,获得了一组结肠癌相关候选miRNA分子,其中miR-145差异表达最明显,相对于癌旁组织,结肠癌组织中miR-145表达水平显著下调,并在实时定量RT-PCR实验中得到了证实；其次,选取miR-145作为进一步研究对象,通过体内、体外实验研究miR-145在结肠癌中的生物学功能；最后在结肠癌细胞HCT116中通过荧光素酶活性分析和Western blot初步证明miR-145可以靶向调控转录因子SOX9。本研究首次提出“miR-145可能通过靶向调控转录因子SOX9参与结肠癌恶性表型发生”的假说,将为全面阐明miR-145在结肠癌中的作用机制奠定理论基础,更为结肠癌候选诊断分子和治疗药物的研发提供理论依据。
第一部分结肠癌相关候选miRNA的筛选与验证
目的：
探讨结肠癌组织和癌旁组织中miRNA的表达差异,获得结肠癌相关的候选miRNA分子。
方法：
对6例结肠癌组织和癌旁组织进行miRNA表达芯片检测,对感兴趣的差异表达miRNA分子采用实时定量RT-PCR进行验证。
结果：
1. miRNA差异表达谱分析发现,51个miRNA在结肠癌组织和癌旁组织表达有差异,差异均在2倍变化以上,具有统计学意义。其中,24个miRNA在结肠癌组织中表达下调,27个miRNA表达上调。相对于癌旁组织,结肠癌组织中miR-145和miR-143表达下调3.492和2.899倍,结肠癌组织中miR-96表达上调3.083倍。
2.实时定量RT-PCR检测显示结肠癌组织中miR-145和miR-143表达水平相对癌旁组织下调3.533倍和2.425倍,miR-96表达水平相对癌旁组织上调2.363倍。
结论：
相对于癌旁组织,结肠癌中miRNA具有差异表达谱,miR-145表达水平变化与结肠癌密切相关,可供进一步研究。
第二部分miR-145体外抑制结肠癌细胞HCT116增殖的实验研究
目的：
体外实验研究miR-145在结肠癌细胞HCT116中的生物学功能。
方法：
1.采用PCR扩增miR-145基因片段,将其克隆入真核表达载体pCMV-myc中,构建重组质粒pCMV-miR-145。
2.酶切及DNA测序鉴定重组质粒后,脂质体法转染结肠癌细胞HCT116,运用实时定量RT-PCR检测转染后细胞中miR-145的表达情况。
3.分别运用CCK8、流式细胞术和细胞划痕试验检测过表达miR-145对HCT116细胞增殖、细胞周期和迁移能力的影响。
结果：
1.成功构建了miR-145的真核表达载体pCMV-miR-145。实时定量RT-PCR证明转染该载体后,相对于空载体转染组,HCT116细胞中miR-145表达水平上升754倍。
2.相对于对照组,真核表达载体pCMV-miR-145转染组G1期百分比升高(62.80±1.22%vs51.61±0.91%),吸光度值降低(0.345±0.032vs0.632±0.033),划痕中央区细胞数目减少(176±24vs276±26)。
结论：
体外过表达miR-145,可导致结肠癌细胞HCT116细胞周期发生G1期阻滞、细胞增殖速度变慢、细胞迁移能力降低。
第三部分miR-145抑制裸鼠皮下移植瘤的实验研究
目的：
体内实验研究miR-145在结肠癌中的生物学功能。
方法：
建立结肠癌细胞HCT116裸鼠皮下移植瘤模型,观察过表达miR-145对肿瘤体积大小、倍增时间的影响。
结果：
过表达miR-145后,肿瘤体积明显减少(368.18±78mm3vs914.14±204mm3)、倍增时间延长(6.5±1.5天vs3.8±0.9天)。
结论：
miR-145可在体内抑制结肠癌细胞HCT116增殖。
第四部分miR-145抑制结肠癌细胞HCT116增殖机制的研究
目的：
探讨miR-145抑制结肠癌细胞HCT116增殖功能的可能机制。
方法：
1.采用生物信息学分析miR-145的下游靶基因及其与靶基因的结合位点。
2.构建基于靶基因SOX9含miR-145结合位点的荧光素酶报告载体pMIR-Report, DNA测序鉴定重组质粒。
3.将重组质粒转染入结肠癌细胞HCT116,检测荧光素酶活性的变化。
4. Western-blot实验检测过表达miR-145和降低内源性miR-145水平时靶基因SOX9的表达变化。
5.实时定量RT-PCR和Western-blot检测癌组织与癌旁组织中miR-145和SOX9及其下游分子MUC2的mRNA表达水平和蛋白表达水平。
结果：
1.生物信息学结合文献检索,确定转录因子SOX9为miR-145的潜在下游靶基因,SOX9的mRNA3'UTR区-282和-1402有两个可能结合位点。
2.含有miR-145结合位点的pMIR-282和pMIR-1402,在过表达miR-145时,其荧光素酶活性相对空载体分别下降50.29%和59.83%；在对核心结合位点突变后,荧光素酶活性恢复,与野生型水平接近。
3.过表达miR-145后,Western-blot检测显示SOX9蛋白显著下降,MUC2蛋白水平相应增加；通过化学修饰的反义寡核苷酸降低内源性miR-145表达水平后,Western-blot检测发现SOX9蛋白显著增加,MUC2蛋白表达减少。
4.相对于癌旁组织,miR-145的mRNA水平在癌组织中下调,SOX9的mRNA水平相应增加,MUC2的mRNA水平相应下降,同时,癌组织中SOX9的蛋白水平上调,其下游MUC2因为受到SOX9抑制其蛋白水平下降。
结论：
miR-145可能通过调节SOX9及下游分子MUC2抑制结肠癌细胞增殖。
Colon cancer is one of the most common gastrointestinal malignant tumors, with the incidence increasing each year. Statistical reports of the American Cancer Society in2010shows that the incidence and case fatality rate of colon cancer in male and female cancer patients was ranked No.3and ranked2in the tumor in the U.S. respectively. China was in countries with low incidence of colon cancer, but in recent years as China's rapid economic development and improved living standards, lifestyle and dietary pattern of change, there is an increasing trend in the incidence of colon cancer which was ranked NO.3both in male and female. The five-year survival rate after surgery is only about fifty percent. The previous study showed that the colon cancer caused by gain of function of oncogene and lose of function of tumor suppressor gene in colonic epithelial cells, epigenetic modification of genomic changes and environment factors. But its pathogenesis is not yet very clear, in-depth study of the pathogenesis of colon cancer is greatly benefits for early diagnosis, treatment and improved quality of life of patients.
miRNA (microRNA) is a endogenous single-stranded non-coding small RNA with its length of21-25nucleotide in organisms, mainly through the inhibition of target gene expression at post-transcriptional level. miRNA are widely involved in cell proliferation, differentiation, apoptosis, tissue and organ development, tumor genesis, and tumor metastasis. In recent years, many studies show that miRNAs involved in a variety of human tumors through a series of different mechanisms. Enhancing or diminishing miRNA function will affect the process of tumor. Precisely because of the miRNA regulation of cancer cell differentiation, proliferation, survival and metastasis, positive or negative tumor-associated of miRNA can be thought to provide a powerful treatment strategy for tumor occurrence and development of intervention.
For further research on the mechanism of miRNAs in colon cancer, firstly we carried out the expression profiling analysis both in the tumor tissues and adjacent tissues from clinical patients respectively and gain a group of colon cancer-related candidate miRNAs. The expression level of miR-145in colon cancer is significantly lower than adjacent tissue by using Chip array and the real time reverse transcription polymerase chain reaction experiments.Secondly this study intends to verify and analyze the biological role of miR-145in colon cancer. Finally, we want to initially verify the hypothesis that miR-145may lead to the occurrence of colon cancer malignant phenotype through the regulation of target gene SOX9and related downstream signaling pathways by fluciferase assay and western blot. This research put forward the hypothesis that miR-145may cause development of colon cancer by target regulating transcription factors SOX9for the first time, lay base for a comprehensive understanding of the roles of miR-145in colon cancer, provide the theory basis for the diagnosis and drug treatment of colon cancer drug.
PartⅠ The screening and validation of the colon cancer-related candidate miRNAs
Objective
To explore the miRNA expression pattern difference between colon cancer tissues and adjacent tissues and gain a group of colon cancer-related candidate miRNAs.
Methods
miRNA expression microarray analysis was carried out in tumor tissue and adjacent tissue from six clinical patients. We further validated the expression of interesting candidate miRNA using real time reverse transcription polymerase chain reaction (real-time RT-PCR) method.
Results
1. The Genechip found51miRNAs, including that there are24miRNAs downregulated and27miRNA upregulated in tumor tissue comparing with adjacent tissues, the differences were statistically significant in more than two times. Expression levels of miR-145and miR-143were down-regualted3.492fold and2.899fold respectively, while expression level of miR-96was up-regualted 3.083fold in colon cancer.
2. The expression levels of miR-145and miR-143in tumor tissue were down-regualted3.533fold and2.425fold comparing to adjacent tissues through real-time RT-PCR validation, the expression level of miR-96in tumor tissues was up-regualted2.363fold comparing to adjacent tissues through real-time RT-PCR validation.
Conclusion
The expression profile of miRNA is different between tumor tissues and adjacent tissues. miR-145may play roles in colon cancer and available for further study.
Partll The study of miR-145suppressing the proliferation of HCT116cells in vitro
Objective
To investigate the biological function of miR-145involved in HCT116cells in vitro.
Methods
1. miR-145genes fragment was obtained by PCR from genomic DNA extracted from normal tissue and inserted into eukaryotic expression plasmid pCMV-myc.
2. The constructed plasmids containing right sequence of target miR-145were transfected into HCT116cells via lipofectamine. The expression level of miR-145was detected by real-time RT-PCR.
3. The proliferation of HCT116over-expressed with miR-145was detected using CCK8kit and the cell cycle was detected by Flow Cytometer, and the transfer ability change was detected by scratch test.
Results
1. The pCMV-miR-145plasmid which over expressing miR-145was successfully constructed, the plasmid could increase expression level of miR-145in HCT116cells by754times comparing to empty vector.
2. Comparing to control, after transferred by pCMV-miR-145plasmid, percentage of G1phase increased (62.80±1.22%vs51.61±0.91%); OD value decresed (0.345±0.032vs0.632±0.033); cells number of scramble decresed (176±24vs276±26)
Conclusion
Over-expression of exogenous miR-145can suppress the proliferation of HCT116cells by cuasing G1cell cycle block and suppressing proliferation and transfer ability in vitro.
PartⅢ The experiment of miR-145restraining growth of subcutaneous transplant tumors in nude mice
Objective
To investigate the biological function of miR-145involved in HCT116cells in vivo.
Methods
Animal model of subcutaneous transplant tumor induced by colon cancer HCT116cells in nude mice was established. Tumor size and doubling time were observed after overexpression of miR-145.
Results
Tumor size significantly reduced (368.18±78mm3vs914.14±204mm3), as well as doubling time extended after overexpression of miR-145(6.5±1.5day vs3.8±0.9day).
Conclusion
Over-expression of exogenous miR-145can suppress the proliferation of HCT116cells in vivo.
PartⅣ The mechan ism of miR-145suppressing the proliferation of HCT116cells Objective
To study possible mechanisms of miR-145inhibiting proliferation of HCT116cells.
Methods
1. Identification of the potential target of miR-145using bioinformatics.
2. miR-145binding site in SOX9gene locus was obtained by PCR from genomic DNA extracted from normal tissue and inserted into luciferase reporter plasmid pMIR-reporter.
3. The constructed plasmids containing right sequence of target were transfected into HCT116cells via lipofectamine for the detection of luciferase activity. The expression change of SOX9was detected by western blotting, when over expressed or low expressed miR-145.
4. Detect the mRNA level of miR-145, SOX9and MUC2in tumor and adjacent tissues from clinical patients. Furthermore, the protein level of SOX9and MUC2was also detected by western blotting.
Results
1. SOX9was selected as the potential target of miR-145by bioinformatics. There are two miR-145binding site in the3'UTR of SOX9, which located at the-282bp and-1402bp downstream of stop codon respectively.
2. The luciferase activity of pMIR-282and pMIR-1402was decreased significantly50.29%and59.83%respectively comparing with the control. After the'seed sequence'was mutated, the luciferase activity can back to the normal level.
3. Over-expressed miR-145can decrease the protein level of SOX9and elevate the protein level of MUC2. Knock down of miR-145using antisense inhibitor can increase the protein level of SOX9, vice versa.
4. The expression level of miR-145is significantly down-regulated in tumor tissues. Meanwhile, the expression level of SOX9increased and MUC2decreased on the contrary.
Conclusion
miR-145may suppress the proliferation of colon cancer by suppressing target gene SOX9and downstream MUC2.
引文
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