本文選題：miR-935 + 靶基因��； 參考：《山東大學(xué)》2017年博士論文

【摘要】：研究目的檢測miR-935在胰腺癌組織及細(xì)胞中的表達(dá)情況,并研究其對胰腺癌細(xì)胞增殖、遷移、凋亡的影響。預(yù)測miR-935作用的直接靶基因,并探討其影響胰腺癌細(xì)胞惡性生物學(xué)行為的作用機制。研究方法(1)收集新鮮胰腺癌組織及癌旁正常組織標(biāo)本,選取正常胰腺導(dǎo)管HPDE6-C7細(xì)胞及胰腺癌PANC-1細(xì)胞進(jìn)行培養(yǎng);(2)以實時定量PCR(Real-time PCR)方法測定胰腺癌組織及胰腺癌PANC-1細(xì)胞中miR-935表達(dá)情況;(3)利用生物學(xué)信息學(xué)數(shù)據(jù)庫預(yù)測miR-935直接靶基因,并應(yīng)用熒光素酶基因報告實驗驗證靶基因的合理性;(4)分別應(yīng)用miR-935類似物,mir-935抑制物和miR-935拮抗物對胰腺癌PANC-1細(xì)胞進(jìn)行轉(zhuǎn)染,MTT法及克隆形成法檢測轉(zhuǎn)染細(xì)胞增殖情況、Transwell法檢測轉(zhuǎn)染細(xì)胞遷移能力、流式細(xì)胞儀檢測轉(zhuǎn)染細(xì)胞凋亡情況、通過qRT-PCR及Western blot檢測P21、P27、EMT相關(guān)分子及凋亡相關(guān)分子在轉(zhuǎn)染細(xì)胞中的表達(dá)情況、沉默靶基因表達(dá)以探討miR-935與靶基因之間的調(diào)控方式。結(jié)果(1)與正常胰腺組織相比,miR-935在胰腺癌組織中表達(dá)顯著上調(diào),而INPP4A的表達(dá)顯著下調(diào);(2)與正常胰腺導(dǎo)管HPDE6-C7細(xì)胞相比,miR-935在胰腺癌PANC-1細(xì)胞中表達(dá)顯著上調(diào),而INPP4A的表達(dá)顯著下調(diào);(3)胰腺癌PANC-1細(xì)胞轉(zhuǎn)染miR-935抑制物后,miR-935表達(dá)被明顯抑制,表達(dá)下調(diào)的miR-935顯著抑制細(xì)胞的增殖、遷移,并促進(jìn)誘導(dǎo)細(xì)胞的凋亡;(4)miR-935表達(dá)抑制導(dǎo)致P27表達(dá)水平顯著上調(diào);導(dǎo)致EMT相關(guān)分子表達(dá)水平顯著變化:E-cadherin的表達(dá)水平明顯上調(diào),N-cadherin、Snail和Vimentin的表達(dá)水平均明顯下調(diào);導(dǎo)致凋亡相關(guān)分析表達(dá)水平顯著變化:Bax的表達(dá)水平明顯上調(diào),Bcl-2、pro-caspase-3及active-caspase-3的表達(dá)水平明顯下調(diào);(5)胰腺癌PANC-1細(xì)胞轉(zhuǎn)染miR-935類似物后,miR-935表達(dá)被明顯上調(diào),表達(dá)上調(diào)的miR-935顯著促進(jìn)細(xì)胞的增殖、遷移,并抑制誘導(dǎo)細(xì)胞的凋亡;(6)miR-935表達(dá)上調(diào)導(dǎo)致P27表達(dá)水平顯著下調(diào);導(dǎo)致EMT相關(guān)分子表達(dá)水平顯著變化:E-cadherin的表達(dá)水平明顯下調(diào),N-cadherin、Snail和Vimentin的表達(dá)水平均明顯上調(diào);導(dǎo)致凋亡相關(guān)分析表達(dá)水平顯著變化:Bax的表達(dá)水平明顯下調(diào),Bcl-2、pro-caspase-3及active-caspase-3的表達(dá)水平明顯上調(diào);(7)INPP4A是miR-935直接作用靶基因。沉默INPP4A表達(dá)顯著抵消了miR-935表達(dá)抑制對胰腺癌PANC-1細(xì)胞遷移及凋亡的抑制作用;也顯著抵消了miR-935表達(dá)抑制對EMT及凋亡相關(guān)分子表達(dá)水平的影響。結(jié)論miR-935在胰腺癌組織及胰腺癌PANC-1中均有表達(dá),上調(diào)miR-935表達(dá)水平可以促進(jìn)胰腺癌細(xì)胞增殖及遷移能力,并抑制細(xì)胞凋亡。miR-935可能通過直接作用于靶基因INPP4A調(diào)控胰腺癌細(xì)胞惡性生物學(xué)行為。miR-935及INPP4A可能能成為胰腺癌基因治療的潛在靶點。
[Abstract]:Objective to investigate the expression of miR-935 in pancreatic carcinoma and its effect on proliferation, migration and apoptosis of pancreatic cancer cells. To predict the direct target gene of miR-935 and to explore the mechanism of its influence on malignant biological behavior of pancreatic cancer cells. Methods 1) fresh pancreatic cancer tissues and adjacent normal tissues were collected. Normal pancreatic duct HPDE6-C7 cells and pancreatic cancer PANC-1 cells were selected for culture. The expression of miR-935 in pancreatic carcinoma tissues and pancreatic cancer PANC-1 cells was detected by real-time quantitative PCR(Real-time PCR. The direct target gene of miR-935 was predicted by bioinformatics database. Using luciferase gene report experiment to verify the rationality of target gene. (4) using miR-935 analogues (miR-935 analogs) and miR-935 antagonists to detect the proliferation of pancreatic cancer PANC-1 cells by MTT assay and clone formation method respectively. The ability of transfection cells to migrate was detected by Elisa. Apoptosis of transfected cells was detected by flow cytometry. The expression of P21 P27EMT related molecules and apoptosis-related molecules in transfected cells was detected by qRT-PCR and Western blot. The expression of target genes was silenced to explore the regulation between miR-935 and target genes. Results 1) compared with normal pancreatic tissues, the expression of mmiR-935 was significantly up-regulated in pancreatic carcinoma tissues, while the expression of INPP4A was significantly down-regulated in pancreatic carcinoma tissues. Compared with normal pancreatic ductal HPDE6-C7 cells, the expression of mmiR-935 was significantly up-regulated in pancreatic cancer PANC-1 cells. However, the expression of INPP4A was significantly down-regulated in PANC-1 cells of pancreatic cancer. After transfection of miR-935 inhibitor, the expression of miR-935 was significantly inhibited, and the down-regulated miR-935 significantly inhibited the proliferation and migration of the cells. The expression level of P27 and the expression of EMT related molecules were significantly up-regulated and down-regulated by up-regulation of N-cadherin Snail and down-regulation of Vimentin expression, and the expression of P27 was significantly up-regulated by promoting the expression inhibition of apoptosis-inducing cell line 4miR-935, leading to a significant change in the expression level of EMT related molecules, and a marked down-regulation of the expression levels of N-cadherin and Snail. The apoptosis-related analysis showed that the expression level of Bcl-2P pro-caspase-3 and active-caspase-3 were up-regulated significantly (P < 0.05). The expression of miR-935 was upregulated after transfection of miR-935 analogues in PANC-1 cells of pancreatic carcinoma, and the up-regulated miR-935 significantly promoted cell proliferation. Migration and inhibition of apoptosis-induced cell apoptosis resulted in a significant down-regulation of P27 expression, a significant change in the expression level of EMT related molecules, and a marked down-regulation of the expression level of N-cadherin Snail and Vimentin. As a result, the expression level of Bcl-2 pro-caspase-3 and active-caspase-3 were significantly down-regulated in the expression level of Bcl-2P -caspase-3 and active-caspase-3, and the expression level of Bcl-2P -caspase-3 and active-caspase-3 were up-regulated significantly in apoptosis-related analysis, which was the target gene of direct action of miR-935. The silencing of INPP4A significantly counteracted the inhibition of miR-935 expression on the migration and apoptosis of pancreatic cancer PANC-1 cells and the effect of miR-935 inhibition on the expression of EMT and apoptosis-related molecules. Conclusion miR-935 is expressed in pancreatic cancer tissues and PANC-1. Upregulation of miR-935 expression can promote the proliferation and migration of pancreatic cancer cells. Inhibition of apoptosis. MiR-935 may be a potential target for gene therapy of pancreatic cancer by directly acting on target gene INPP4A to regulate malignant biological behavior of pancreatic cancer cells. MiR-935 and INPP4A may be potential targets for gene therapy of pancreatic cancer.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.9

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本文編號：1825911