miR-29c调控MMP2在胰腺癌转移中的作用

发布时间：2018-06-06 10:02

本文选题：miR-29c + MMP2　；参考：《第三军医大学》2015年硕士论文

【摘要】：目的:研究miR-29c在胰腺癌侵袭转移中的作用方法:1.RT-PCR检测5株不同分化能力胰腺癌细胞株(PANC-1,Bx Pc-3,Hs766t,cFPAN,Capan1)的miR-29s的表达,Western Blot及明胶酶谱法检测5株不同分化能力胰腺癌细胞MMP2的表达和活性,利用Spearman等级相关统计方法分析在胰腺癌细胞中miR-29s表达与MMP2表达的相关性。2.通过转染miRNA mimic和miRNA inhibitor分别构建mi R-29c的过表达和干扰表达;CCK-8检测mi R-29c表达对胰腺癌细胞增殖能力影响;Transwell迁移实验和Transwell侵袭实验分别检测mi R-29c表达对胰腺癌细胞迁移和侵袭能力的影响。3.结合生物信息学预测和荧光素酶活性报告基因证实mi R-29c调控MMP2表达的分子机制。4.通过裸鼠原位胰腺癌种植模型筛选出自发性高肝转移胰腺癌细胞亚群(Hs766t、Hs766t-L1和Hs766t-L2),分别检测mi R-29c和MMP2的表达,分析其表达与转移能力的相关性;利用所获得高肝转移胰腺癌细胞活体验证mi R-29s对胰腺癌转移能力的影响。5.RT-PCR检测胰腺癌组织及其癌旁组织mi R-29c的表达,免疫组化检测胰腺癌组织及其癌旁组织MMP2蛋白表达,分别分析其表达与临床病理参数相关性;Kaplan Meier统计方法分析mi R-29c表达对胰腺癌预后的影响。结果:1.在胰腺癌细胞中,mi R-29c表达与MMP2蛋白表达呈负相关。2.过表达miR-29c对胰腺癌细胞的增殖能力无影响,但可明显抑制胰腺癌细胞的迁移和侵袭能力;干扰mi R-29c表达后可显著增强胰腺癌细胞的迁移和侵袭能力。3.生物信息学预测提示:MMP2 3'UTR存在一个mi R-29c结合位点;过表达mi R-29c*2011年国家自然科学基金面上项目(81071975)可显著降低MMP2 mRNA和蛋白表达水平,干扰miR-29c表达后可显著上调MMP2mRNA和蛋白表达水平;miR-29c可显著抑制野生型报告基因荧光素酶活性,而对突变型报告基因荧光素酶活性无影响。4.通过裸鼠原位胰腺癌种植模型,体外分离并培养出自发性高肝转移胰腺癌细胞亚群(Hs766t、Hs766t-L1和Hs766t-L2),mi R-29c表达与胰腺癌细胞转移潜能呈负相关,而MMP2蛋白表达与胰腺癌细胞的转移潜能呈正相关;利用高转移潜能胰腺癌细胞Hs766t-L2构建裸鼠原位胰腺癌动物模型,过表达miR-29c明显抑制裸鼠原位胰腺癌自发性肝转移。5.在胰腺癌临床样本中,miR-29c的表达明显低于其对应的癌旁组织,且与MMP2蛋白的表达呈负相关;miR-29c的表达与胰腺癌的分化程度、淋巴结转移和TNM期相关;同时miR-29c的表达是影响胰腺癌预后的独立因素。结论:1.在细胞水平和活体水平证实mi R-29c通过靶向调控MMP2表达抑制胰腺癌侵袭转移。2.mi R-29c调控MMP2是调控胰腺癌转移的一个重要机制,但存在其它机制调控胰腺癌侵袭转移;在胰腺癌肝转移过程中,miR-29c是调控MMP2的一个重要因素,但并不是唯一的。3.mi R-29c靶向调控MMP2表达为望为胰腺癌肝转移的诊断和治疗提供理论基础。
[Abstract]:Objective: to study the role of miR-29c in the invasion and metastasis of pancreatic carcinoma. Methods: 1. RT-PCR was used to detect the expression of miR-29s in 5 pancreatic cancer cell lines (PANC-1BX Pc-3Hs766tFPANCAN-1). Western Blot and gelatinase spectrum were used to detect the expression and activity of MMP2 in 5 pancreatic cancer cell lines with different differentiation ability. The correlation between miR-29s expression and MMP2 expression in pancreatic cancer cells was analyzed by Spearman rank correlation method. 2. 2. Overexpression and interference expression of mi R-29c were constructed by transfection of miRNA mimic and miRNA inhibitor respectively. CCK-8 was used to detect the effect of mi R-29c expression on the proliferation of pancreatic cancer cells. Transwell migration assay and Transwell invasion assay were used to detect the effect of mi R-29c expression on the migration of pancreatic cancer cells. And the influence of invasiveness. Combined with bioinformatics prediction and luciferase activity reporter gene, the molecular mechanism of MMP2 expression regulated by mi R-29c was confirmed. Hs766t-L1 and Hs766t-L2 cells were isolated from nude mice with high hepatic metastases. The expression of miR-29c and MMP2 were detected, and the correlation between the expression and metastasis ability was analyzed. The effect of mi R-29s on the metastatic ability of pancreatic carcinoma was tested in vivo by using the obtained high hepatic metastasis pancreatic cancer cells. 5. The expression of mi R-29c in pancreatic carcinoma and its adjacent tissues was detected by RT-PCR, and the expression of MMP2 protein in pancreatic carcinoma and its adjacent tissues was detected by immunohistochemistry. The correlation between the expression of miR-29c and the clinicopathologic parameters was analyzed. The expression of mi R-29c was analyzed by Kaplan Meier method. The result is 1: 1. There was a negative correlation between the expression of MMP2 protein and mRNA expression in pancreatic cancer cells. Overexpression of miR-29c had no effect on the proliferation of pancreatic cancer cells, but significantly inhibited the migration and invasion of pancreatic cancer cells, and interfering with the expression of R-mi-29c could significantly enhance the migration and invasion ability of pancreatic cancer cells. Bioinformatics prediction suggested that there was a miR-29c binding site in the 3'UTR, and that overexpression of mi R-29c * in 2011 NSFC project 8107 1975) could significantly reduce the expression level of MMP2 mRNA and protein. Interfering with miR-29c expression significantly up-regulated MMP2mRNA and protein expression level. MiR-29c significantly inhibited luciferase activity of wild type reporter gene, but had no effect on luciferase activity of mutant reporter gene. The expression of Hs766tHs766t-L1 and Hs76t-L2OMI-29c was negatively correlated with the metastatic potential of pancreatic cancer cells. The expression of MMP2 protein was positively correlated with the metastatic potential of pancreatic cancer cells, and high metastatic potential pancreatic cancer cell line Hs766t-L2 was used to construct nude mice in situ pancreatic carcinoma animal model, overexpression of miR-29c significantly inhibited spontaneous liver metastasis of nude mice. The expression of miR-29c in clinical samples of pancreatic cancer was significantly lower than that in the corresponding adjacent tissues, and the expression of miR-29c was negatively correlated with the expression of MMP2 protein and the degree of differentiation, lymph node metastasis and TNM stage of pancreatic cancer. At the same time, the expression of miR-29c is an independent factor affecting the prognosis of pancreatic cancer. Conclusion 1. It was confirmed at cell level and in vivo level that mi R-29c inhibits invasion and metastasis of pancreatic carcinoma by targeting MMP2 expression. Regulating MMP2 is an important mechanism of regulating pancreatic cancer metastasis, but there are other mechanisms to regulate invasion and metastasis of pancreatic carcinoma. MiR-29c is an important factor in the regulation of MMP2 in the process of hepatic metastasis of pancreatic cancer, but it is not the only .3.miR-29c targeted to regulate the expression of MMP2, which is expected to provide a theoretical basis for the diagnosis and treatment of hepatic metastasis of pancreatic cancer.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.9

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