miRNA-146a对乙型肝炎病毒生活周期的影响及机制研究

发布时间：2018-01-12 06:27

本文关键词：miRNA-146a对乙型肝炎病毒生活周期的影响及机制研究　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:研究微小RNA-146a(microRNA-146a,miR-146a)对乙型肝炎病毒(hepatitis B virus,HBV)生活周期的影响及其可能机制。方法:通过miRNA芯片比较HepG2.2.15细胞与HepG2细胞miRNA表达谱差异,选择miR-146a为研究对象,RT-PCR验证芯片结果。在HepG2.2.15细胞中分别转染miR-146a mimic和inhibitor,RT-PCR检测HBV复制水平,ELISA和Western blot检测蛋白水平。双荧光素酶报告系统进一步验证miR-146a与潜在靶点HS3ST3B1相互作用。HepG2.2.15细胞中转染miR-146a mimic,RT-PCR和western blot分别检测HS3ST3B1 mRNA和蛋白水平。结果:(1)miRNA芯片发现74条miRNAs在HepG2.2.15中表达发生变化,其中29条上调,45条下调,RT-PCR证实,miR-146a在HepG2.2.15水平明显高于HepG2细胞(P0.05)。(2)HepG2.2.15细胞转染miR-146a mimic后,HBV复制和蛋白水平较对照组明显升高(P0.05);转染miR-146a inhibitor后,HBV复制和蛋白表达水平较对照组明显降低(P0.05)。(3)生物信息学预测发现HBV抑制因子HS3ST3B1是miR-146a的潜在靶点,双荧光素酶报告系统显示,HS3ST3B1野生型载体的报告荧光较对照组明显下调(P0.05),突变预测靶位点后,HS3ST3B1突变型载体的报告荧光与对照组无明显差异(P0.05)。HepG2.2.15细胞中转染miR-146a mimic后,HS3ST3B1 m RNA水平较对照组无明显变化(P0.05),HS3ST3B1蛋白水平较对照组明显下调。结论:本研究证实miR-146a能影响HBV生活周期,研究发现miR-146a可能通过作用于HBV抑制因子HS3ST3B1 3′UTR抑制其翻译从而影响HBV生活周期。
[Abstract]:Objective: to study the effects of microRNA-146a simiR-146a on hepatitis B virus. Methods: the difference of miRNA expression profiles between HepG2.2.15 cells and HepG2 cells was compared by miRNA microarray. Select miR-146a as the research object. MiR-146a mimic and inhibitor were transfected into HepG2.2.15 cells by RT-PCR. HBV replication level was detected by RT-PCR. ELISA and Western. Detection of protein level by blot. Double luciferase reporting system further verifies the interaction between miR-146a and potential target HS3ST3B1. HepG2.2.15 cells transfected with miR-. 146a. Mimic. RT-PCR and western blot were used to detect HS3ST3B1 mRNA and protein levels respectively. The expression of 74 miRNAs in HepG2.2.15 was detected by miRNA chip. Among them, 29 upregulated 45 down-regulated and confirmed by RT-PCR. The level of miR-146a in HepG2.2.15 was significantly higher than that in HepG2 cells (P0.05. 2). HepG2.2.15 cells were transfected with miR-146a mimic. The levels of HBV replication and protein were significantly higher than those of the control group (P 0.05). After transfection of miR-146a inhibitor. The levels of HBV replication and protein expression were significantly lower than those of the control group (P 0.05). Bioinformatics predicted that HBV inhibitor HS3ST3B1 was a potential target of miR-146a. The double luciferase report system showed that the reported fluorescence of HS3ST3B1 wild-type vector was significantly lower than that of the control group, and the mutation predicted the target site. There was no significant difference between the HS3ST3B1 mutant vector and the control group after transfection of miR-146a mimic in P0.05G 2.2.15 cells. The level of HS3ST3B1 m RNA was not significantly changed compared with the control group (P 0.05). HS3ST3B1 protein level was significantly decreased compared with the control group. Conclusion: this study confirmed that miR-146a can affect the life cycle of HBV. It has been found that miR-146a may affect the life cycle of HBV by inhibiting the translation of HBV inhibitor HS3ST3B1 3 UTR.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373.21

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