本文選題：miR-29c + MMP2　； 參考：《第三軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：目的:研究miR-29c在胰腺癌侵襲轉(zhuǎn)移中的作用方法:1.RT-PCR檢測(cè)5株不同分化能力胰腺癌細(xì)胞株(PANC-1,Bx Pc-3,Hs766t,cFPAN,Capan1)的miR-29s的表達(dá),Western Blot及明膠酶譜法檢測(cè)5株不同分化能力胰腺癌細(xì)胞MMP2的表達(dá)和活性,利用Spearman等級(jí)相關(guān)統(tǒng)計(jì)方法分析在胰腺癌細(xì)胞中miR-29s表達(dá)與MMP2表達(dá)的相關(guān)性。2.通過(guò)轉(zhuǎn)染miRNA mimic和miRNA inhibitor分別構(gòu)建mi R-29c的過(guò)表達(dá)和干擾表達(dá);CCK-8檢測(cè)mi R-29c表達(dá)對(duì)胰腺癌細(xì)胞增殖能力影響;Transwell遷移實(shí)驗(yàn)和Transwell侵襲實(shí)驗(yàn)分別檢測(cè)mi R-29c表達(dá)對(duì)胰腺癌細(xì)胞遷移和侵襲能力的影響。3.結(jié)合生物信息學(xué)預(yù)測(cè)和熒光素酶活性報(bào)告基因證實(shí)mi R-29c調(diào)控MMP2表達(dá)的分子機(jī)制。4.通過(guò)裸鼠原位胰腺癌種植模型篩選出自發(fā)性高肝轉(zhuǎn)移胰腺癌細(xì)胞亞群(Hs766t、Hs766t-L1和Hs766t-L2),分別檢測(cè)mi R-29c和MMP2的表達(dá),分析其表達(dá)與轉(zhuǎn)移能力的相關(guān)性;利用所獲得高肝轉(zhuǎn)移胰腺癌細(xì)胞活體驗(yàn)證mi R-29s對(duì)胰腺癌轉(zhuǎn)移能力的影響。5.RT-PCR檢測(cè)胰腺癌組織及其癌旁組織mi R-29c的表達(dá),免疫組化檢測(cè)胰腺癌組織及其癌旁組織MMP2蛋白表達(dá),分別分析其表達(dá)與臨床病理參數(shù)相關(guān)性;Kaplan Meier統(tǒng)計(jì)方法分析mi R-29c表達(dá)對(duì)胰腺癌預(yù)后的影響。結(jié)果:1.在胰腺癌細(xì)胞中,mi R-29c表達(dá)與MMP2蛋白表達(dá)呈負(fù)相關(guān)。2.過(guò)表達(dá)miR-29c對(duì)胰腺癌細(xì)胞的增殖能力無(wú)影響,但可明顯抑制胰腺癌細(xì)胞的遷移和侵襲能力;干擾mi R-29c表達(dá)后可顯著增強(qiáng)胰腺癌細(xì)胞的遷移和侵襲能力。3.生物信息學(xué)預(yù)測(cè)提示:MMP2 3'UTR存在一個(gè)mi R-29c結(jié)合位點(diǎn);過(guò)表達(dá)mi R-29c*2011年國(guó)家自然科學(xué)基金面上項(xiàng)目(81071975)可顯著降低MMP2 mRNA和蛋白表達(dá)水平,干擾miR-29c表達(dá)后可顯著上調(diào)MMP2mRNA和蛋白表達(dá)水平;miR-29c可顯著抑制野生型報(bào)告基因熒光素酶活性,而對(duì)突變型報(bào)告基因熒光素酶活性無(wú)影響。4.通過(guò)裸鼠原位胰腺癌種植模型,體外分離并培養(yǎng)出自發(fā)性高肝轉(zhuǎn)移胰腺癌細(xì)胞亞群(Hs766t、Hs766t-L1和Hs766t-L2),mi R-29c表達(dá)與胰腺癌細(xì)胞轉(zhuǎn)移潛能呈負(fù)相關(guān),而MMP2蛋白表達(dá)與胰腺癌細(xì)胞的轉(zhuǎn)移潛能呈正相關(guān);利用高轉(zhuǎn)移潛能胰腺癌細(xì)胞Hs766t-L2構(gòu)建裸鼠原位胰腺癌動(dòng)物模型,過(guò)表達(dá)miR-29c明顯抑制裸鼠原位胰腺癌自發(fā)性肝轉(zhuǎn)移。5.在胰腺癌臨床樣本中,miR-29c的表達(dá)明顯低于其對(duì)應(yīng)的癌旁組織,且與MMP2蛋白的表達(dá)呈負(fù)相關(guān);miR-29c的表達(dá)與胰腺癌的分化程度、淋巴結(jié)轉(zhuǎn)移和TNM期相關(guān);同時(shí)miR-29c的表達(dá)是影響胰腺癌預(yù)后的獨(dú)立因素。結(jié)論:1.在細(xì)胞水平和活體水平證實(shí)mi R-29c通過(guò)靶向調(diào)控MMP2表達(dá)抑制胰腺癌侵襲轉(zhuǎn)移。2.mi R-29c調(diào)控MMP2是調(diào)控胰腺癌轉(zhuǎn)移的一個(gè)重要機(jī)制,但存在其它機(jī)制調(diào)控胰腺癌侵襲轉(zhuǎn)移;在胰腺癌肝轉(zhuǎn)移過(guò)程中,miR-29c是調(diào)控MMP2的一個(gè)重要因素,但并不是唯一的。3.mi R-29c靶向調(diào)控MMP2表達(dá)為望為胰腺癌肝轉(zhuǎn)移的診斷和治療提供理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.9

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本文編號(hào)：1986136