HBV蛋白通过转录因子YY1调节miRNAs形成相关因子DGCR8的表达

发布时间：2018-07-01 19:57

  本文选题：HBV + DGCR8　； 参考：《重庆医科大学》2012年硕士论文

【摘要】：目的 研究HBV蛋白对miRNAs形成相关因子DGCR8表达的调控作用，并对其作用机制进行初步探讨，为研究HBV引起的miRNAs异常表达机制提供一种新的思路。 方法 1.用RT-PCR和Real-Time PCR的方法检测DGCR8在HepG2细胞及HepG2.2.15细胞中的差异表达，并将HBV表达质粒(pCH9/3091)瞬时转染HepG2细胞，进一步确证HBV对DGCR8表达的影响，最后用Western blot验证上述结果。 2.双荧光素酶报告系统分析HBV是否对DGCR8启动子有调节作用。首先构建DGCR8启动子质粒（pGL3-DGCR8-P），将其与pCH9/3091共转染HepG2细胞，以此检测HBV对DGCR8启动子活性的影响。 3.构建HBV的四种蛋白的表达质粒HBc、HBp、HBs及HBx，分别与pGL3-DGCR8-P共转染HepG2细胞，分析HBV蛋白对DGCR8启动子活性的影响情况。在HepG2.2.15细胞中干扰掉相应蛋白，用双荧光素酶报告系统及Western blot验证上述蛋白对DGCR8表达的影响。 4.寻找DGCR8启动子上可能结合的转录因子，通过RT-PCR及Western blot的方法检测HBV对相关转录因子的mRNA和蛋白表达的影响。通过双荧光素酶报告系统检测相关转录因子对DGCR8启动子活性的影响，Western blot检测转录因子对DGCR8蛋白表达的影响，并用shRNA在HepG2.2.15细胞中干扰相应转录因子的表达，，通过双荧光素酶报告系统和Western blot验证上述结果。 结果 1. RT-PCR和Real-Time PCR结果显示， DGCR8mRNA在HepG2.2.15细胞中的表达较HepG2细胞降低，将HBV表达质粒以一定的浓度梯度转染入HepG2细胞后可见DGCR8的表达随HBV转染量的增高而降低，Western blot结果证实HBV可以下调DGCR8蛋白表达水平。 2.构建了DGCR8启动子表达质粒，并证明了其活性。将DGCR8启动子质粒与pCH9/3091共转HepG2细胞，发现HBV能降低DGCR8启动子的活性，并且HBV对启动子的抑制作用呈剂量依赖性，最后我们通过双荧光素酶报告系统进一步在稳定表达HBV的HepG2.2.15细胞中验证了HBV对DGCR8启动子活性的抑制作用。 3.将HBV四种蛋白的表达质粒与DGCR8启动子质粒共染转HepG2细胞，发现HBs和HBx对DGCR8启动子活性影响最为明显。在HepG2.2.15细胞中分别抑制HBs和HBx的表达后，双荧光素酶报告系统检测显示DGCR8启动子活性有所恢复，Western blot检测显示DGCR8蛋白表达水平有所升高。 4.通过生物信息学的方法搜寻到DGCR8启动子区域有转录因子YY1的结合位点。首先我们通过RT-PCR和Western blot的方法证实了HBV可以促进YY1的表达。然后将YY1的表达质粒与DGCR8启动子共转染HepG2细胞后发现YY1能下调DGCR8启动子活性，Western blot实验也验证了这一结果。随后在HepG2.2.15细胞中抑制YY1的表达，DGCR8启动子活性有所升高，DGCR8蛋白表达水平也升高。 结论 HBV能通过减弱DGCR8启动子活性降低其转录水平和蛋白水平的表达，其中HBs和HBx蛋白起主要作用，这一过程可能是通过转录因子YY1介导的。本研究探讨了HBV对DGCR8表达的调节机制，为研究HBV导致的miRNA的异常表达提供了新的思路，进一步阐明了HBV的生物学作用。
[Abstract]:Purpose

To study the effect of HBV protein on the expression of DGCR8 , a new idea for the study of the mechanism of HBV - induced abnormal expression of the expression of DGCR8 .

method

1 . The differential expression of DGCR8 in HepG2 cells and HepG2.2 . 15 cells was detected by RT - PCR and Real - Time PCR , and HBV expression plasmid ( pCH9 / 3091 ) was transiently transfected into HepG2 cells . The effect of HBV on DGCR8 expression was further confirmed . Finally , Western blot was used to validate the results .

2 . The effect of HBV on the activity of DGCR8 promoter was detected by constructing DGCR8 promoter plasmid ( pGL3 - DGCR8 - P ) , and co - transfected HepG2 cells with pCH9 / 3091 .

3 . The expression plasmids of the four proteins of HBV were constructed by cotransfection of HepG2 cells with pGL3 - DGCR8 - P , respectively . The effect of HBV protein on the activity of DGCR8 promoter was analyzed . The effect of the above proteins on DGCR8 expression was verified by dual luciferase reporter system and Western blot in HepG2.2 . 15 cells .

4 . The transcription factors which may bind to DGCR8 promoter were found . The effects of HBV on the mRNA and protein expression of the related transcription factors were detected by RT - PCR and Western blot . The effect of transcription factors on the expression of DGCR8 promoter was detected by double luciferase reporter system . Western blot was used to detect the effect of transcription factors on the expression of DGCR8 promoter , and the results were verified by double luciferase reporter system and Western blot .

Results

1 . The results of RT - PCR and Real - Time PCR showed that the expression of DGCR8 mRNA in HepG2.2 . 15 cells was lower than that in HepG2 cells , and the expression of DGCR8 decreased with the increase of HBV transfection .

2 . The expression plasmid of DGCR8 promoter was constructed and its activity was demonstrated . The DGCR8 promoter plasmid was co - transfected with pCH9 / 3091 . It was found that HBV could decrease the activity of DGCR8 promoter , and the inhibitory effect of HBV on the promoter was dose dependent . Finally , we demonstrated the inhibitory effect of HBV on the promoter activity of DGCR8 in HepG2.2 . 15 cells stably expressing HBV .

3 . The expression plasmid of HBV four proteins was co - stained with DGCR8 promoter plasmid to HepG2 cells , and it was found that HBs and HBx were most affected by DGCR8 promoter activity . After the expression of HBsAg and HBx were respectively inhibited in HepG2.2 . 15 cells , the double luciferase reporter system detected that DGCR8 promoter activity was restored , and Western blot analysis showed that the expression level of DGCR8 was increased .

4 . The transcription factor YY1 binding site was found in the DGCR8 promoter region by bioinformatics . First , we confirmed that HBV could promote the expression of YY1 by RT - PCR and Western blot . After co - transfection of the expression plasmid of YY1 with DGCR8 promoter , YY1 could downregulate DGCR8 promoter activity . Western blot assay also demonstrated this result .

Conclusion

HBV can reduce its transcriptional level and protein level by reducing the activity of DGCR8 promoter , in which HBs and HBx proteins play a major role , which may be mediated by transcription factor YY1 . This study discusses the regulation mechanism of HBV on DGCR8 expression , and provides a new idea for studying the abnormal expression of HBV - induced miRNA , and further clarifies the biological function of HBV .
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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