本文選題：HBV + DGCR8��； 參考：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的 研究HBV蛋白對miRNAs形成相關(guān)因子DGCR8表達(dá)的調(diào)控作用，并對其作用機(jī)制進(jìn)行初步探討，為研究HBV引起的miRNAs異常表達(dá)機(jī)制提供一種新的思路。 方法 1.用RT-PCR和Real-Time PCR的方法檢測DGCR8在HepG2細(xì)胞及HepG2.2.15細(xì)胞中的差異表達(dá)，并將HBV表達(dá)質(zhì)粒(pCH9/3091)瞬時(shí)轉(zhuǎn)染HepG2細(xì)胞，進(jìn)一步確證HBV對DGCR8表達(dá)的影響，最后用Western blot驗(yàn)證上述結(jié)果。 2.雙熒光素酶報(bào)告系統(tǒng)分析HBV是否對DGCR8啟動(dòng)子有調(diào)節(jié)作用。首先構(gòu)建DGCR8啟動(dòng)子質(zhì)粒（pGL3-DGCR8-P），將其與pCH9/3091共轉(zhuǎn)染HepG2細(xì)胞，以此檢測HBV對DGCR8啟動(dòng)子活性的影響。 3.構(gòu)建HBV的四種蛋白的表達(dá)質(zhì)粒HBc、HBp、HBs及HBx，分別與pGL3-DGCR8-P共轉(zhuǎn)染HepG2細(xì)胞，分析HBV蛋白對DGCR8啟動(dòng)子活性的影響情況。在HepG2.2.15細(xì)胞中干擾掉相應(yīng)蛋白，用雙熒光素酶報(bào)告系統(tǒng)及Western blot驗(yàn)證上述蛋白對DGCR8表達(dá)的影響。 4.尋找DGCR8啟動(dòng)子上可能結(jié)合的轉(zhuǎn)錄因子，通過RT-PCR及Western blot的方法檢測HBV對相關(guān)轉(zhuǎn)錄因子的mRNA和蛋白表達(dá)的影響。通過雙熒光素酶報(bào)告系統(tǒng)檢測相關(guān)轉(zhuǎn)錄因子對DGCR8啟動(dòng)子活性的影響，Western blot檢測轉(zhuǎn)錄因子對DGCR8蛋白表達(dá)的影響，并用shRNA在HepG2.2.15細(xì)胞中干擾相應(yīng)轉(zhuǎn)錄因子的表達(dá)，，通過雙熒光素酶報(bào)告系統(tǒng)和Western blot驗(yàn)證上述結(jié)果。 結(jié)果 1. RT-PCR和Real-Time PCR結(jié)果顯示， DGCR8mRNA在HepG2.2.15細(xì)胞中的表達(dá)較HepG2細(xì)胞降低，將HBV表達(dá)質(zhì)粒以一定的濃度梯度轉(zhuǎn)染入HepG2細(xì)胞后可見DGCR8的表達(dá)隨HBV轉(zhuǎn)染量的增高而降低，Western blot結(jié)果證實(shí)HBV可以下調(diào)DGCR8蛋白表達(dá)水平。 2.構(gòu)建了DGCR8啟動(dòng)子表達(dá)質(zhì)粒，并證明了其活性。將DGCR8啟動(dòng)子質(zhì)粒與pCH9/3091共轉(zhuǎn)HepG2細(xì)胞，發(fā)現(xiàn)HBV能降低DGCR8啟動(dòng)子的活性，并且HBV對啟動(dòng)子的抑制作用呈劑量依賴性，最后我們通過雙熒光素酶報(bào)告系統(tǒng)進(jìn)一步在穩(wěn)定表達(dá)HBV的HepG2.2.15細(xì)胞中驗(yàn)證了HBV對DGCR8啟動(dòng)子活性的抑制作用。 3.將HBV四種蛋白的表達(dá)質(zhì)粒與DGCR8啟動(dòng)子質(zhì)粒共染轉(zhuǎn)HepG2細(xì)胞，發(fā)現(xiàn)HBs和HBx對DGCR8啟動(dòng)子活性影響最為明顯。在HepG2.2.15細(xì)胞中分別抑制HBs和HBx的表達(dá)后，雙熒光素酶報(bào)告系統(tǒng)檢測顯示DGCR8啟動(dòng)子活性有所恢復(fù)，Western blot檢測顯示DGCR8蛋白表達(dá)水平有所升高。 4.通過生物信息學(xué)的方法搜尋到DGCR8啟動(dòng)子區(qū)域有轉(zhuǎn)錄因子YY1的結(jié)合位點(diǎn)。首先我們通過RT-PCR和Western blot的方法證實(shí)了HBV可以促進(jìn)YY1的表達(dá)。然后將YY1的表達(dá)質(zhì)粒與DGCR8啟動(dòng)子共轉(zhuǎn)染HepG2細(xì)胞后發(fā)現(xiàn)YY1能下調(diào)DGCR8啟動(dòng)子活性，Western blot實(shí)驗(yàn)也驗(yàn)證了這一結(jié)果。隨后在HepG2.2.15細(xì)胞中抑制YY1的表達(dá)，DGCR8啟動(dòng)子活性有所升高，DGCR8蛋白表達(dá)水平也升高。 結(jié)論 HBV能通過減弱DGCR8啟動(dòng)子活性降低其轉(zhuǎn)錄水平和蛋白水平的表達(dá)，其中HBs和HBx蛋白起主要作用，這一過程可能是通過轉(zhuǎn)錄因子YY1介導(dǎo)的。本研究探討了HBV對DGCR8表達(dá)的調(diào)節(jié)機(jī)制，為研究HBV導(dǎo)致的miRNA的異常表達(dá)提供了新的思路，進(jìn)一步闡明了HBV的生物學(xué)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 .
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

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本文編號：2088984