miR-29a-3p对胃癌腹膜转移的影响及其机制研究

发布时间：2017-12-31 07:31

本文关键词：miR-29a-3p对胃癌腹膜转移的影响及其机制研究　出处：《宁夏医科大学》2016年硕士论文　论文类型：学位论文

更多相关文章： miR-29a-3p 胃癌 增殖转移 HAS3

【摘要】：背景:胃癌是最常见的恶性肿瘤之一,其发病率在世界范围恶性肿瘤排第四位,死亡率居第二位,在我国,每年新发病例约为30万,严重威胁着国人健康,而转移是胃癌患者最常见的致死原因,其发生的分子机制一直是肿瘤研究领域的热点。微小RNA(micro RNAs,mi RNAs)是一大类内源性的、在进化过程中高度保守的单链非编码小RNA,参与了人体多种生命活动的调控,如细胞的增殖、分化、凋亡等。mi RNA的表达失调可导致多种疾病的发生,并且与多种恶性肿瘤的发生、发展有着极为密切的关系。近年来许多研究表明,在胃癌中也存在mi RNA表达失调的现象,说明mi RNA也参与了胃癌的发生。本实验将在前期的基础上,对mi R-29a-3p在胃癌细胞系的表达水平、生物学功能及可能的作用机制进行初步的研究和探讨。目的:探索hsa-mi R-29a-3p(mi R-29a)对胃癌腹膜转移的影响并初步研究其作用机制。方法:1.利用q RT-PCR验证mi R-29a-3p在三对胃癌高低转移细胞系GC9811-P/GC9811,MKN28-M/MKN-28NM,SGC7901-M/SGC7901-NM中的表达情况。2.构建过表达及抑制mi R-29a-3p的慢病毒表达载体,将过表达mi R-29a-3p的慢病毒及其无关阴性对照转染入胃癌腹膜高转移细胞系GC9811-P中,相反,将抑制mi R-29a-3p表达的慢病毒及其无关阴性对照转入亲本细胞系GC9811中。3.用MTT比色实验、平板克隆形成实验、Transwell、划痕实验及流式细胞学技术分别观察mi R-29a-3p对胃癌细胞的增殖、转移及凋亡能力的影响。4.采用生物信息学的方法预测mi R-29a-3p的靶基因,查阅文献选出与转移相关的基因并采用q RT-PCR及western blot验证与mi R-29a-3p的关系。5.转染si RNA及空载体沉默基因表达,进一步行功能试验验证其功能及是否为mi R-29a-3p的靶基因。结果:1.mi R-29a-3p在胃癌高转移细胞系(GC9811-P、MKN28-M、SGC7901-M)中高表达。2.成功构建高低表达mi R-29a-3p的细胞系,q RT-PCR验证转染成功。3.功能试验结果表明,过表达mi R-29a-3p可以抑制细胞的增殖及转移能力而促进细胞的凋亡能力;相反,抑制mi R-29a-3p的表达可以促进细胞的增殖及转移能力而抑制细胞的凋亡能力。4.生物信息学预测提示HAS3可能为mi R-29a-3p的潜在靶基因之一,q RT-PCR结果显示,在上调mi R-29a-3p的细胞系中,HAS3表达有降低的趋势但无统计学意义;在下调mi R-29a-3p的细胞系中,HAS3表达有升高的趋势但无统计学意义。Western blot结果显示,上调mi R-29a-3p的表达,HAS3表达下调,下调mi R-29a-3p的表达,HAS3表达上调。5.沉默HAS3的表达,胃癌细胞增殖及迁移能力减弱,说明HAS3促进胃癌细胞的增殖与转移。在高表达mi R-29a-3p的细胞系中沉默HAS3的表达后,细胞的增殖及转移能力减弱,与过表达mi R-29a-3p有类似的效果,说明mi R-29a-3p可负性调控HAS3,进一步证实HAS3可为mi R-29a-3p的潜在靶基因之一。结论:mi R-29a-3p抑制胃癌细胞的增殖与转移而促进凋亡,并可能通过其下游靶分子之一HAS3发挥其作用。
[Abstract]:Background: gastric cancer is one of the most common malignant tumors, the incidence of cancer ranked 4th in the world, the death rate ranked second. In China, the annual new cases are about 300,000, which seriously threaten the health of people. Metastasis is the most common cause of death in patients with gastric cancer, and its molecular mechanism has been a hot spot in the field of cancer research. Small RNA(micro RNAs. Mi RNAss are a large class of endogenous, highly conserved, single-stranded, non-coding RNAs that participate in the regulation of a variety of human life activities, such as cell proliferation and differentiation. The expression imbalance of .mi RNA, such as apoptosis, can lead to the occurrence of many diseases, and is closely related to the occurrence and development of many malignant tumors. There is also an imbalance in the expression of mi RNA in gastric cancer, indicating that mi RNA is also involved in the development of gastric cancer. The expression level of mi R-29a-3p in gastric cancer cell line. Biological function and possible mechanism of action were preliminarily studied and discussed. Objective: to explore hsa-mi R-29a-3pmmi R-29a. Effect on peritoneal metastasis of gastric cancer and preliminary study of its mechanism. Methods: 1. Using Q RT-PCR to verify mi. R-29a-3p was detected in three pairs of gastric cancer cell lines GC9811-P/GC9811. MKN28-M/MKN-28NM. The expression of SGC7901-M/SGC7901-NM. 2. Construct the expression vector of lentivirus which overexpressed and inhibited mi R-29a-3p. The overexpression of mi R-29a-3p lentivirus and its unrelated negative control were transfected into the peritoneal high metastasis cell line GC9811-P of gastric cancer. The lentivirus which inhibited the expression of mi R-29a-3p and its unrelated negative control were transferred into the parental cell line GC9811. 3. MTT colorimetric assay and plate clone formation test were used. The proliferation of gastric cancer cells induced by miR-29a-3p was observed by flow cytometry and scratch test. Effects of Metastasis and apoptosis. Bioinformatics was used to predict the target gene of mi R-29a-3p. Search the literature to select genes related to metastasis and verify the relationship with mi R-29a-3p by Q RT-PCR and western blot .5.transfect si. RNA and empty vector silenced gene expression. Further functional tests were performed to verify its function and whether it was the target gene of mi R-29a-3p. Results: 1. Mi R-29a-3p was detected in gastric cancer cell line GC9811-P. High expression of MKN28-MGC7901-M2.The cell line with high and low expression of miR-29a-3p was successfully constructed. The results of functional test showed that overexpression of mi R-29a-3p could inhibit cell proliferation and metastasis and promote cell apoptosis. On the contrary. Inhibition of the expression of mi R-29a-3p could promote cell proliferation and metastasis and inhibit cell apoptosis. 4. Bioinformatics prediction suggested that HAS3 might be mi. One of the potential target genes of R-29a-3p. The results of Q RT-PCR showed that the expression of HAS3 decreased in the cell line of miR-29a-3p, but had no statistical significance. The down-regulation of the expression of HAS3 in the cell line of miR-29a-3p showed a tendency to increase, but no statistical significance. Western blot results showed. Up regulated the expression of miR-29a-3p and down-regulated the expression of miR-29a-3p and up-regulated the expression of HAS3. Silenced the expression of HAS3. The ability of proliferation and migration of gastric cancer cells was decreased, which indicated that HAS3 promoted proliferation and metastasis of gastric cancer cells. After silencing the expression of HAS3 in the cell lines with high expression of miR-29a-3p. The ability of cell proliferation and metastasis was decreased, which was similar to that of overexpression of mi R-29a-3p, indicating that mi R-29a-3p could negatively regulate HAS3. It is further confirmed that HAS3 may be one of the potential target genes of mi R-29a-3p. Conclusion: MiR-29a-3p inhibits the proliferation and metastasis of gastric cancer cells and promotes apoptosis. And it may play its role through one of its downstream target molecules, HAS3.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2

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