本文關(guān)鍵詞： miR-125a-5p miR-145 過(guò)表達(dá) SKOV3 p53　出處：《重慶醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 microRNA（miRNA）是近年來(lái)在動(dòng)植物種體內(nèi)發(fā)現(xiàn)的一種內(nèi)源性非編碼RNA，可以通過(guò)與mRNA3’-UTR結(jié)合，發(fā)揮轉(zhuǎn)錄后調(diào)控作用，起到癌基因或抑癌基因的作用。研究表明，miRNA在大多數(shù)腫瘤中異常表達(dá)。在卵巢癌組織中，miR-125a表達(dá)水平升高，miR-145的表達(dá)水平降低，提示miRNA可能與卵巢癌的發(fā)生有關(guān)，但通過(guò)何種途徑參與卵巢癌的發(fā)生未見報(bào)道。p53作為腫瘤中常見的抑癌基因，其編碼p53蛋白是細(xì)胞周期中的負(fù)調(diào)節(jié)因子，故推測(cè)miR-125a-5p可能通過(guò)下調(diào)p53基因的表達(dá)來(lái)提高細(xì)胞的增殖與侵襲能力，miR-145可能通過(guò)上調(diào)p53基因的表達(dá)來(lái)降低細(xì)胞的增殖與侵襲能力。本研究通過(guò)建立過(guò)表達(dá)miR-125a-5p和miR-145的卵巢癌SKOV3細(xì)胞，研究miR-125a-5p、miR-145對(duì)細(xì)胞增殖、轉(zhuǎn)移、侵襲及凋亡能力的影響，為進(jìn)一步探討miR-125a-5p和miR-145與卵巢癌相關(guān)作用的靶基因提供依據(jù)。 方法 通過(guò)慢病毒轉(zhuǎn)染技術(shù)將含miR-125a-5p和miR-145的質(zhì)粒分別轉(zhuǎn)入兩組SKOV3細(xì)胞中，經(jīng)嘌呤霉素篩選建立穩(wěn)定過(guò)表達(dá)miR-125a-5p和miR-145的SKOV3細(xì)胞株。通過(guò)實(shí)時(shí)熒光定量PCR驗(yàn)證兩種基因在細(xì)胞中的表達(dá)情況，MTT實(shí)驗(yàn)、流式細(xì)胞技術(shù)、transwell實(shí)驗(yàn)、劃痕實(shí)驗(yàn)檢測(cè)上調(diào)miR-125a-5p和miR-145后的SKOV3細(xì)胞在增殖、凋亡、侵襲及轉(zhuǎn)移能力方面的改變，并用western-blot檢測(cè)過(guò)表達(dá)miR-125a-5p和miR-145后SKOV3細(xì)胞中p53蛋白表達(dá)量的變化。 結(jié)果 1.MTT實(shí)驗(yàn)表明，過(guò)表達(dá)miR-125a-5p的SKOV3細(xì)胞增殖能力較對(duì)照組有顯著提高，過(guò)表達(dá)miR-145的SKOV3增殖能力顯著降低（p＜0.05）。 2.流式細(xì)胞檢測(cè)顯示過(guò)表達(dá)miR-125a-5p的SKOV3細(xì)胞處于S期的細(xì)胞比例增大，G0/G1期比例減少，細(xì)胞凋亡率降低；過(guò)表達(dá)miR-145的SKOV3細(xì)胞處于S期的細(xì)胞比例減小，G0/G1期比例減小，，細(xì)胞凋亡率增高（p＜0.05）。 3.劃痕實(shí)驗(yàn)表明過(guò)表達(dá)miR-125a-5p的SKOV3細(xì)胞24h和48h遷移程度分別高出對(duì)照組2.37倍和4.28倍，過(guò)表達(dá)miR-125a-5p的實(shí)驗(yàn)組細(xì)胞遷移能力明顯提高（P＜0.05）。 4.transwell實(shí)驗(yàn)結(jié)果顯示，過(guò)表達(dá)miR-125a-5p的SKOV3細(xì)胞侵襲能力明顯提高；過(guò)表達(dá)miR-145的SKOV3細(xì)胞侵襲能力明顯降低（P＜0.05）。 5.western-blot實(shí)驗(yàn)結(jié)果表明，過(guò)表達(dá)miR-125a-5p后的SKOV3細(xì)胞p53蛋白表達(dá)量降低；過(guò)表達(dá)miR-145的SKOV3細(xì)胞p53蛋白表達(dá)量升高（P＜0.05）。 結(jié)論 miR-125a-5p過(guò)表達(dá)可促進(jìn)SKOV3細(xì)胞增殖、侵襲和遷移能力，而抑制細(xì)胞凋亡水平，其可能通過(guò)降低卵巢癌SKOV3細(xì)胞中抑癌基因p53的表達(dá)來(lái)增加細(xì)胞的腫瘤惡性。miR-145過(guò)表達(dá)可抑制SKOV3細(xì)胞增殖、侵襲和遷移能力，促進(jìn)細(xì)胞凋亡水平，其可能通過(guò)增加卵巢癌SKOV3細(xì)胞中抑癌基因p53的表達(dá)來(lái)降低細(xì)胞的腫瘤惡性。
[Abstract]:Purpose. MicroRNAs miRNAs are a kind of endogenous non-coding RNAs found in animal and plant species in recent years, which can play a role in post-transcriptional regulation by binding to mRNA3'-UTR. The expression level of miR-125a in ovarian cancer tissues increased and the expression level of miR-145 decreased, suggesting that miRNA may be related to the occurrence of ovarian cancer. However, there is no report on how to participate in the development of ovarian cancer. P53 is a common tumor suppressor gene, which encodes p53 protein as a negative regulatory factor in cell cycle. It is speculated that miR-125a-5p may increase the ability of proliferation and invasion by down-regulating the expression of p53 gene. MiR-145 may decrease the proliferation and invasion of ovarian cancer cells by up-regulating the expression of p53 gene. In this study, we established ovarian cancer SKOV3 cells which overexpressed miR-125a-5p and miR-145. To study the effects of miR-125a-5pmmiR-145 on cell proliferation, metastasis, invasion and apoptosis, and to provide a basis for further study on the target genes of miR-125a-5p and miR-145 related to ovarian cancer. Method. The plasmids containing miR-125a-5p and miR-145 were transfected into two groups of SKOV3 cells by lentivirus transfection. A stable SKOV3 cell line with overexpression of miR-125a-5p and miR-145 was established by purine mycin screening. The expression of the two genes in the cells was verified by real-time fluorescence quantitative PCR. The changes of proliferation, apoptosis, invasion and metastasis of SKOV3 cells after up-regulation of miR-125a-5p and miR-145 were detected by scratch assay, and the expression of p53 protein in SKOV3 cells after miR-125a-5p and miR-145 overexpression was detected by western-blot. Results. 1. The proliferative ability of SKOV3 cells with overexpression of miR-125a-5p was significantly higher than that of control group, and the proliferative ability of SKOV3 with overexpression of miR-145 was significantly lower than that of control group (P < 0.05). 2. Flow cytometry showed that the proportion of SKOV3 cells in S phase was increased, the proportion of G _ 0 / G _ 1 phase was decreased and the apoptosis rate of SKOV3 cells was decreased, while that of SKOV3 cells over-expressing miR-145 in S phase was decreased, and the apoptosis rate was increased (p < 0.05). 3. Scratch test showed that the migration degree of SKOV3 cells with overexpression of miR-125a-5p was 2.37 times and 4.28 times higher than that of control group at 24 h and 48 h, respectively. The migration ability of SKOV3 cells over-expressed with miR-125a-5p increased significantly (P < 0.05). 4. The results of transwell experiment showed that the invasive ability of SKOV3 cells with overexpression of miR-125a-5p was significantly increased, and that of SKOV3 cells with overexpression of miR-145 was significantly lower than that of SKOV3 cells (P < 0.05). 5. The results of western-blot analysis showed that the expression of p53 protein decreased in SKOV3 cells after overexpression of miR-125a-5p, but increased in SKOV3 cells with overexpression of miR-145 (P < 0.05). Conclusion. Overexpression of miR-125a-5p can promote the proliferation, invasion and migration of SKOV3 cells, but inhibit the level of apoptosis. It may inhibit the proliferation of SKOV3 cells by decreasing the expression of tumor suppressor gene p53 in SKOV3 cells. The ability of invasion and migration promotes the level of cell apoptosis, which may reduce the tumor malignancy by increasing the expression of tumor suppressor gene p53 in ovarian cancer SKOV3 cells.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.31

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 胡士軍;楊增明;;miRNAs的表達(dá)調(diào)控機(jī)制[J];生物化學(xué)與生物物理進(jìn)展;2008年05期

本文編號(hào)：1528291