| ObjectiveHepatocellular carcinoma(HCC)is one of the most common tumors in China,which is a serious threat for human health and life.The occurrence and development of HCC is a complex process that involving multi-factors and multi-steps.Among the risk factors of HCC,chronic infection of hepatitis B virus(HBV)is the main risk factor.It is an important challenge for human beings to explore the role of HBV in the occurrence of HCC.In the first part of this study,we analyzed the distribution and characteristics of HBV S gene mutation in cancer tissues and corresponding adjacent tissues of patients with HBV-related HCC.At the same time,this study will explore the effect of s W172* on autophagy-related genes and apoptosis-related genes in Hep G2 cells.And we detected the apoptosis of Hep G2 cells by flow cytometry.Methods1.Totals of 43 patients with HBV-related HCC who underwent HCC surgery in the Guangxi Medical University Cancer Hospital,were selected as subjects from 2015 to 2016,and the HCC tissues and corresponding adjacent tissues were collected.The HBV S gene fragments were amplified by nest PCR and sequenced.The sequence of S gene was analyzed by Chromas and DNAMAN software.2.HBV wild-type S gene and sW172* gene were used as the target fragments,and pc DNA3.1(+)was used as vector to construct eukaryotic plasmid and then transfected them into Hep G2 cells.The transfected pc DNA3.1(+)-SHBs and pc DNA3.1(+)-s W172* were used as the experimental group,and the transfected empty plasmid pc DNA3.1(+)were used as the control group.3.To determine whether the transfection was successful or not,the method of reverse transcription polymerase reaction(RT-PCR)was used to detect the expression of HBV S gene in each group after transfection.4.The relative expressions of autophagy-related genes(Beclin1,LC3)and apoptosis-related genes(Caspase-3,Bcl-2)m RNA were detected by RT-PCR method.5.The flow cytometry was used to detect the apoptosis ability of cells in the experimental group and the control group.Results1.There was no significant difference in the distribution of hot spot mutations of HBV S gene between cancer tissues and adjacent tissues in patients with hepatocellular carcinoma(P>0.05).2.SHBs stop mutation exists only in cancer tissues,and the mutation sites are s C69*,s W74*,s L94*,s W172* and s W182*,respectively.3.In this study,the expression of S gene m RNA was verified by RT-PCR,which means that the expression of s W172* gene and wild type S gene in Hep G2 cells were successfully constructed.4.The relative expressions of Beclin1 m RNA in pc DNA3.1(+)-SHBs and pc DNA3.1(+)-s W172* was significantly higher than the empty plasmid group[pc DNA3.1(+)],but there was no significant difference between pc DNA3.1(+)-SHBs group and pc DNA3.1(+)-s W172* group.There was no significant difference in the relative expressions of LC3 m RNA among pc DNA3.1(+)-SHBs group,pc DNA3.1(+)-s W172* group and pc DNA3.1(+)group.And there was no significant difference in Caspase-3 m RNA and Bcl-2 m RNA level among pc DNA3.1(+)-SHBs group,pc DNA3.1(+)-s W172* group and pc DNA3.1(+)group.5.The apoptosis rates of pcDNA3.1(+),pc DNA3.1(+)-SHBs and pc DNA3.1(+)-s W172* groups were 4.51 ±1.15,5.84 ±3.46 and 3.92 ±2.47 respectively,and there was no significant difference among the three groups.Conclusions1.The distribution of hot spot mutations of HBV S gene in HBV-related HCC cancer tissues and their adjacent tissues in Guangxi was not specific.2.SHBs stop mutation exists only in HCC cancer tissues,which may affect the occurrence and development of HCC.3.SHBs and sW172* mutations can increase the transcriptional level of Beclin1 m RNA,SHBs and s W172* mutations may be related to the autophagy of Hep G2 cells.4.SHBs and sW172* are not the main factors that regulating the apoptosis of HepG2 cells. |
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