miR-125a-5p与miR-145对卵巢癌SKOV3细胞增殖和侵袭的影响及对p53表达的调控

发布时间：2018-02-24 01:06

本文关键词： miR-125a-5p miR-145 过表达 SKOV3 p53　出处：《重庆医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的 microRNA（miRNA）是近年来在动植物种体内发现的一种内源性非编码RNA，可以通过与mRNA3’-UTR结合，发挥转录后调控作用，起到癌基因或抑癌基因的作用。研究表明，miRNA在大多数肿瘤中异常表达。在卵巢癌组织中，miR-125a表达水平升高，miR-145的表达水平降低，提示miRNA可能与卵巢癌的发生有关，但通过何种途径参与卵巢癌的发生未见报道。p53作为肿瘤中常见的抑癌基因，其编码p53蛋白是细胞周期中的负调节因子，故推测miR-125a-5p可能通过下调p53基因的表达来提高细胞的增殖与侵袭能力，miR-145可能通过上调p53基因的表达来降低细胞的增殖与侵袭能力。本研究通过建立过表达miR-125a-5p和miR-145的卵巢癌SKOV3细胞，研究miR-125a-5p、miR-145对细胞增殖、转移、侵袭及凋亡能力的影响，为进一步探讨miR-125a-5p和miR-145与卵巢癌相关作用的靶基因提供依据。方法通过慢病毒转染技术将含miR-125a-5p和miR-145的质粒分别转入两组SKOV3细胞中，经嘌呤霉素筛选建立稳定过表达miR-125a-5p和miR-145的SKOV3细胞株。通过实时荧光定量PCR验证两种基因在细胞中的表达情况，MTT实验、流式细胞技术、transwell实验、划痕实验检测上调miR-125a-5p和miR-145后的SKOV3细胞在增殖、凋亡、侵袭及转移能力方面的改变，并用western-blot检测过表达miR-125a-5p和miR-145后SKOV3细胞中p53蛋白表达量的变化。结果 1.MTT实验表明，过表达miR-125a-5p的SKOV3细胞增殖能力较对照组有显著提高，过表达miR-145的SKOV3增殖能力显著降低（p＜0.05）。 2.流式细胞检测显示过表达miR-125a-5p的SKOV3细胞处于S期的细胞比例增大，G0/G1期比例减少，细胞凋亡率降低；过表达miR-145的SKOV3细胞处于S期的细胞比例减小，G0/G1期比例减小，，细胞凋亡率增高（p＜0.05）。 3.划痕实验表明过表达miR-125a-5p的SKOV3细胞24h和48h迁移程度分别高出对照组2.37倍和4.28倍，过表达miR-125a-5p的实验组细胞迁移能力明显提高（P＜0.05）。 4.transwell实验结果显示，过表达miR-125a-5p的SKOV3细胞侵袭能力明显提高；过表达miR-145的SKOV3细胞侵袭能力明显降低（P＜0.05）。 5.western-blot实验结果表明，过表达miR-125a-5p后的SKOV3细胞p53蛋白表达量降低；过表达miR-145的SKOV3细胞p53蛋白表达量升高（P＜0.05）。结论 miR-125a-5p过表达可促进SKOV3细胞增殖、侵袭和迁移能力，而抑制细胞凋亡水平，其可能通过降低卵巢癌SKOV3细胞中抑癌基因p53的表达来增加细胞的肿瘤恶性。miR-145过表达可抑制SKOV3细胞增殖、侵袭和迁移能力，促进细胞凋亡水平，其可能通过增加卵巢癌SKOV3细胞中抑癌基因p53的表达来降低细胞的肿瘤恶性。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

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