MiR-193b在胃癌发生发展中的功能与机制研究

发布时间：2018-06-27 07:39

本文选题：微小RNA + miR-193b　；参考：《河南大学》2016年硕士论文

【摘要】：微小RNA(microRNA,miRNA)是一类在动植物中均有表达的长度约21-25nt的非编码RNA,通常与靶基因的mRNAs的3’端非编码区(3’-untranslated region,3’-UTR)互补结合,参与许多编码基因表达的转录后调控;通过形成沉默复合物,而参与RNA沉默。迄今为止,在人类基因组中已发现了数以千计的miRNAs,大约50%的蛋白质编码基因受其调控,形成了一个转录后水平的调控网络。MiRNAs在很多重要的生物学过程中起着调节作用,如细胞增殖、凋亡、分化及迁移等。MiRNAs的表达和代谢异常,通常会引发多种疾病,比如肿瘤,一些miRNAs的异常表达(过表达,低表达)可能导致肿瘤的发生和发展,即其可作为癌基因或抑癌基因而发挥作用。研究表明在胃癌的发生发展过程中miRNAs发挥了至关重要的调节作用。为了进一步研究miRNAs在胃癌发生发展中的功能与机制,通过对胃癌组织和癌旁正常组织中差异表达的miRNAs进行筛选,发现miR-193b的表达具有显著差异。Cyclin D1(CCND1)属于高度保守的细胞周期家族成员。该家族的蛋白丰度在整个细胞周期中具有周期性变化。该基因出现异常表达或突变都会影响细胞周期的进程,这可能是它影响多种肿瘤发生发展的最主要的机制。研究表明miR-193b在肿瘤中可通过靶向CCND1而发挥抑癌基因的作用。目的:研究miR-193b在胃癌发生发展中的功能和机制。材料方法:我们用Real-time PCR的方法对50对胃癌组织(C)和癌旁正常组织(N)中的miR-193b的表达进行检测,并对C/N的值进行统计,取cutoff=1.5。用细胞转染的方法在两种胃癌细胞系HGC-27和MGC-803中转入miR-193b过表达载体,并用Real-time PCR验证其表达上调情况:在此基础之上,我们用细胞增殖实验检测miR-193b对胃癌细胞增殖能力的影响;划痕实验及侵袭实验用于检测miR-193b对胃癌细胞迁移能力的影响。并用Western blot实验检测过表达miR-193b对细胞中CCND1蛋白表达的影响。在miR-193b的机制研究中,我们用RNAi的方法对HGC-27和MGC-803细胞中的CCND1蛋白表达进行干预,并用Western blot验证siCCND1对CCND1的敲低效率,并用细胞增殖实验检测CCND1对HGC-27和MGC-803细胞增殖能力的影响。结果:miR-193b在胃癌组织中的表达比癌旁正常组织中的表达低。而且miR-193b低表达与pTNM分期(stageI/II vs III/IV)相关。我们的进一步研究表明miR-193b可以抑制HGC-27和MGC-803细胞的增殖,迁移和侵袭,对细胞周期也有一定的影响。而且miR-193b可直接作用于CCND1,即miR-193b可抑制CCND1的表达,并且在HGC-27和MGC-803细胞用siCCND1将CCND1表达降低后,两种胃癌细胞系的细胞增殖能力的下降。以上结果提示miR-193b在胃癌中的抗癌作用可能是通过靶向CCND1实现的。结论:miR-193b通过靶向作用于cyclin D1而抑制胃癌细胞的增殖、侵袭、转移能力。
[Abstract]:MicroRNAs (microRNAs) are a class of non-coding RNAs with a length of about 21-25nt expressed in animals and plants, which are usually complementary to the 3'-untranslated region 3'-UTR of the target gene, and participate in the post-transcriptional regulation of the expression of many coding genes. Participate in RNA silencing by forming silencing complex. So far, thousands of miRNAss have been found in the human genome, and about 50% of the protein-coding genes are regulated by them, forming a post-transcriptional regulatory network. MiRNAs play a regulatory role in many important biological processes. The abnormal expression and metabolism of MiRNAs, such as cell proliferation, apoptosis, differentiation and migration, usually lead to many diseases, such as tumor. Abnormal expression (overexpression, low expression) of some miRNAs may lead to the occurrence and development of tumor. That is, it can act as oncogene or tumor suppressor gene. It has been shown that miRNAs play an important role in the development of gastric cancer. In order to further study the function and mechanism of miRNAs in carcinogenesis and development of gastric cancer, the differentially expressed miRNAs were screened in gastric cancer tissues and adjacent normal tissues. It was found that the expression of miR-193b was significantly different. Cyclin D1 (CCND1) belonged to a highly conserved cell cycle family. The protein abundance of the family changes periodically throughout the cell cycle. Abnormal expression or mutation of the gene may affect the progress of cell cycle, which may be the most important mechanism that affects the occurrence and development of many kinds of tumors. It has been shown that miR-193b can act as a tumor suppressor gene by targeting CCND1 in tumors. Objective: to study the function and mechanism of miR-193b in carcinogenesis and development of gastric cancer. Materials: Real-time PCR was used to detect the expression of miR-19b in 50 pairs of gastric cancer tissues (C) and adjacent normal tissues (N). Two gastric cancer cell lines HGC-27 and MGC-803 were transfected into miR-193b overexpression vector by cell transfection, and the upregulation of miR-193b expression was verified by Real-time PCR. On this basis, the effect of miR-193b on the proliferation of gastric cancer cells was detected by cell proliferation assay. The effect of miR-193b on the migration ability of gastric cancer cells was detected by scratch test and invasion assay. The effect of overexpression of miR-19b on the expression of CCND1 protein was detected by Western blot assay. In the study of the mechanism of miR-193b, we used RNAi to interfere with the expression of CCND1 protein in HGC-27 and MGC-803 cells. Western blot was used to verify the knock down efficiency of siCCND1 on CCND1, and the effect of CCND1 on the proliferation of HGC-27 and MGC-803 cells was detected by cell proliferation assay. Results the expression of miR-19b in gastric cancer was lower than that in adjacent normal tissues. And the low expression of miR-193b was associated with pTNM staging (stageI / II vs III / IV). Our further study showed that miR-193b could inhibit the proliferation, migration and invasion of HGC-27 and MGC-803 cells, and had a certain effect on cell cycle. MiR-193b could directly inhibit the expression of CCND1 in CCND1 cells, and the proliferation ability of the two gastric cancer cell lines was decreased after the expression of CCND1 was reduced by siCCND1 in HGC-27 and MGC-803 cells. These results suggest that the anticancer effect of miR-193b in gastric cancer may be mediated by targeting CCND1. Conclusion the ability of proliferation, invasion and metastasis of gastric cancer cells was inhibited by targeting cyclin D1.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2

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