Effect of DHX15 on the replication of HBV and its mechanism
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Effect of DHX15 on the replication of HBV and its mechanism
Journal of Chongqing Medical UniversityVol. 43, Issue 11, Pages: 1422-1426(2018)
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重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆,400016
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Published:2018,
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Yang Liuqing, Wei Xiafei, Gan Chunyang, et al. Effect of DHX15 on the replication of HBV and its mechanism. [J]. Journal of Chongqing Medical University 43(11):1422-1426(2018)
DOI:
Yang Liuqing, Wei Xiafei, Gan Chunyang, et al. Effect of DHX15 on the replication of HBV and its mechanism. [J]. Journal of Chongqing Medical University 43(11):1422-1426(2018)DOI:
Effect of DHX15 on the replication of HBV and its mechanism
Objective:To screen DDX family for genes that affect the transcription activity of HBV core promoter,and to research the relationship between DHX15 and HBV replication in vitro. Methods:The DDX family genes were cloned into the expression plasmid PCH9 respectively. The HBV core promoter was cloned into a luciferase reporter vector to generate pcore-Rluc. HepG2 cells were tran-siently co-transfected with each of the DEx D/H-box expressing plasmids and pCH9-pcore-Rluc. The luciferase activity was measured with luciferase assay system. HepG2 cells were transiently co-transfected with DHX15 and HBV1.3 cells were lysed and subjected to Western blot analysis. The effects of DHX15 overexpression on the HBV DNA replication and the HBV RNA transcription were tested by using Southern blot and Northern blot respectively. HBV core promoter truncated mutants were constructed to dissect the sequence might interacting with DHX15. Results:Thirty DEx D/h-box family genes were successfully cloned. DHX15 significantly influenced the activity of HBV core promoter(P=0.042). Compared with the control,overexpression of DHX15 promoted the replica-tion of HBV DNA and increased the transcription level of HBV RNA in HepG2 cells. Both of the 2 HBV enhancers seem to be partially responsible for the upregulation effect of DHX15 on the activity of HBV core promoter(t=8.292,P=0.001;t=5.215,P=0.006). Conclusion:Overexpression of DHX15 promotes HBV replication in HepG2 cells by affecting the activity of HBV enhancer.(qRT-PCR) and Western blot. Cell counting Kit-8(CCK-8) assays were used to evaluate the sensibility to gefitinib of different group cells. The invasion ability and migration ability of different groups were detected by Transwell assay. Results:Nicotine in-duced epithelial-mesenchymal transition(EMT) of PC-9 cells in a time and concentration dependent manner. Down-regulation of E-cadherin and up-regulation of Vimentin were most significant with a treatment of 10 μmol/L nicotine for 10 h,so further experi-ments were completed under this condition. CCK-8 displayed reduction of gefitinib sensitivity of PC-9 cells in the NIC group com-pared with the control group,especially with a gefitinib concentration of 0.05 μmol/L(F=82.005,P=0.000,P=0.000). In invasion as-says,more penetrating cells were observed in the NIC group than in the control group[(15.70±1.53) vs. (32.70±2.08),F=75.406,P=0.000,P=0.000]. Migration assays presented similar results [(102.70±7.02) vs. (18.70±2.08),F=204.038,P=0.000,P=0.000]. E-cadherin mRNA expression decreased[(0.932±0.100) vs. (0.459±0.024),F=25.924,P=0.000,P=0.000] and Vimentin mRNA expression increased[(1.200±0.200) vs. (1.973±0.129),F=20.998,P=0.000,P=0.000] in the NIC group compared with the control group,and Western blot showed the same results. Cells in the MET+NIC group displayed higher sensitivity to gifitinib,which is statis-tically significant with a gifitinib concentration of 0.05,0.10,0.50,1.00 and 5.00 μmol/L. Fewer cells penetrated the Matrigel in the NIC+MET group[(17.00±2.00) vs. (32.70±2.08),F=75.406,P=0.000,P=0.000] and migration assays presented similar results [(51.70±5.03) vs. (102.70±7.02),F=204.038,P=0.000,P=0.000]. E-cadherin mRNA expression was upregulated[(0.459±0.024) vs. (0.838±0.058),F=25.924,P=0.000,P=0.000],and Vimentin mRNA expression was downregulated [(1.973±0.129) vs. (1.467±0.115),F=20.998,P=0.000,P=0.003] in the NIC+MET group compared with the NIC group. Western blot showed the same results. Conclusion:Nicotine induced EGFR-TKI resistance in PC-9 cells by EMT in non-small cell lung cancer,which could be reversed by metformin.
关键词
乙型肝炎病毒DDX家族DHX15启动子增强子
Keywords
hepatitis B virusDEx D/H-boxDHX15HBV core promoterenhancer
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Quantification of intrahepatic cccDNA,DNA,serum HBV DNA and its relationship with HBeAg in hepatitis B virus-related hepatocellular carcinoma
S gene variations of hepatitis B virus in children with vaccination failure
Association between intrahepatic levels and mutation of hepatitis B virus covalently closed circular DNA in hepatitis B virus-related hepatocellular carcinoma
Research progress of breast cancer chemotherapy related HBV reactivation
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