本文關鍵詞：miRNA-146a對乙型肝炎病毒生活周期的影響及機制研究　出處：《重慶醫(yī)科大學》2017年碩士論文　論文類型：學位論文

  更多相關文章： 乙型肝炎病毒 微小RNA-146a 硫酸類肝素3氧磺基轉移酶 microarray芯片 雙熒光素酶報告基因

【摘要】：目的:研究微小RNA-146a(microRNA-146a,miR-146a)對乙型肝炎病毒(hepatitis B virus,HBV)生活周期的影響及其可能機制。方法:通過miRNA芯片比較HepG2.2.15細胞與HepG2細胞miRNA表達譜差異,選擇miR-146a為研究對象,RT-PCR驗證芯片結果。在HepG2.2.15細胞中分別轉染miR-146a mimic和inhibitor,RT-PCR檢測HBV復制水平,ELISA和Western blot檢測蛋白水平。雙熒光素酶報告系統(tǒng)進一步驗證miR-146a與潛在靶點HS3ST3B1相互作用。HepG2.2.15細胞中轉染miR-146a mimic,RT-PCR和western blot分別檢測HS3ST3B1 mRNA和蛋白水平。結果:(1)miRNA芯片發(fā)現(xiàn)74條miRNAs在HepG2.2.15中表達發(fā)生變化,其中29條上調(diào),45條下調(diào),RT-PCR證實,miR-146a在HepG2.2.15水平明顯高于HepG2細胞(P0.05)。(2)HepG2.2.15細胞轉染miR-146a mimic后,HBV復制和蛋白水平較對照組明顯升高(P0.05);轉染miR-146a inhibitor后,HBV復制和蛋白表達水平較對照組明顯降低(P0.05)。(3)生物信息學預測發(fā)現(xiàn)HBV抑制因子HS3ST3B1是miR-146a的潛在靶點,雙熒光素酶報告系統(tǒng)顯示,HS3ST3B1野生型載體的報告熒光較對照組明顯下調(diào)(P0.05),突變預測靶位點后,HS3ST3B1突變型載體的報告熒光與對照組無明顯差異(P0.05)。HepG2.2.15細胞中轉染miR-146a mimic后,HS3ST3B1 m RNA水平較對照組無明顯變化(P0.05),HS3ST3B1蛋白水平較對照組明顯下調(diào)。結論:本研究證實miR-146a能影響HBV生活周期,研究發(fā)現(xiàn)miR-146a可能通過作用于HBV抑制因子HS3ST3B1 3′UTR抑制其翻譯從而影響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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R373.21

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本文編號：1413102