| Part One Effects of G1896 A and C2288 A mutations in HBV preC/C on biological function of HCC Objective: To explore the effect of HBV preC/C G1896 A and C2288 A mutations on HCC biological function.Methods:1.The wild type,G1896 A and C2288 A mutant HBV open reading frame C were inserted into the expression vector p CDH using restriction endonucliase,and the lentivirus was packaged in 293 T cells.2.Hep3 B,HepG2,Huh-7 and PLC/PRF/5 cells were infected with lentivirus overexpressing wild type,G1896 A or C2288 A mutant HBV preC/C gene respectively,and stable cell lines were selected by expression of green fluorescent proteins and puromycin resistant marker.HBcAg and HBeAg expression were verified by immunoblotting,immunofluorescence and ELISA.3.The proliferative ability of HCC cells in control,wild type,G1896 A and C2288 A mutant groups was determined by CCK8 assay and colony formation experiment.EdU incorporation assay was used to detect the DNA synthesis rate of HCC cells in each group.4.The invasion ability of HCC cells in control,wild type,G1896 A and C2288 A mutant groups was compared by transwell assay.5.Scratch wound healing experiment was used to compare the migration ability of HCC cells in control,wild type,G1896 A and C2288 A mutant groups.6.The apoptosis rate of HCC cells in control,wild type,G1896 A and C2288 A mutant groups was determined by flow cytometry.7.The tumor growth rate in vivo of HepG2 cells in control,wild type,G1896 A and C2288 A mutant groups was analyzed using mouse xenograft tumor model.8.The inhibitory ratio of sorafenib against HCC cells in control,wild type,G1896 A and C2288 A mutant groups was analyzed by cell viability detection.Results:1.Wild type,G1896 A and C2288 A mutant HBV preC/C gene expression vectors were constructed and verified by restriction endonuclease and gene sequencing,and recombinant lentiviruses were successfully constructed.2.The stable cell lines of HCC cells of control,wild-type,G1896 A and C2288 A mutant groups were successfully constructed by lentivirus infection.HCC cells in wild type,G1896 A and C2288 A mutant groups expressed HBcAg,while HBeAg was expressed in wild type and C2288 A mutant group,but not in G1896 A mutant group.3.CCK8 and colony formation experiment showed that HCC cells in wild type group showed higher viability and more colonies formation than those in control group.HCC cells in G1896 A and C2288 A mutant groups showed higher viability and more colonies formation than those in wild type group.The proportion of EdU positive cells in wild type group was higher than that in control group,and the proportion of EdU positive cells in G1896 A and C2288 A mutant groups was higher than that in wild type group.4.Transwell experiment showed that more HCC cells moved through the matrix in wild type group than control group,and more HCC cells moved through the matrix in G1896 A and C2288 A mutant groups than wild type group.5.Scratch wound healing experiment showed that migration rate of wild type HCC cells was faster than that of control group,and migration rate of G1896A and C2288 A mutant HCC cells was faster than that of wild type group.6.The apoptosis rate of wild type HCC cells was lower than that of control group,and the apoptosis rate of G1896 A and C2288 A mutant HCC cells was lower than that of wild type HCC cells.7.Wild type HepG2 cells grew into larger and heavier tumors in vivo than cells in control group.HepG2 cells in G1896 A and C2288 A mutant groups grew into larger and heavier tumors in vivo than those in wild type group.HBcAg expression pattern is consistent with in vitro experiment.The level of KI67 increased gradually in control,wild type and mutant groups of HepG2 cells.8.The inhibition rate of sorafenib on HepG2,Huh-7 and PLC/PRF/5 in control,wild-type and mutant groups showed a decreasing trend.Conclusions:G1896A and C2288 A mutation in HBV preC/C gene promote HCC cell proliferation,invasion and migration,inhibit cell apoptosis in vitro,promote tumor formation in vivo,and enhance the resistance of HCC cells to sorafenib.Part Two G1896 A and C2288 A mutations in HBV preC/C gene promote HBV replication Objective: To explore the effect of G1896 A and C2288 A mutations in HBV preC/C gene on HBV replication.Methods:1.The G1896 and C2288 in wild type pCS-HBV1.3 plasmid were mutated respectively by homologous recombination.2.To construct in vitro HBV replication model,wild type,G1896 A and C2288 A mutant pCS-HBV1.3 plasmid were transfected into HepG2 and Huh-7 cells,respectively.After 24 h,48 h and 72 h of transfection,supernatant of each group was collected for detection of HBeAg,HBsAg and HBV DNA,and cells in each group were collected for pg RNA detection.3.The concentration of HBeAg and HBsAg in supernatant of wild type,G1896 A and C2288 A mutant HCC cells were determined by ELISA.4.The HBV DNA in supernatant of with wild type,G1896 A and C2288 A mutant HepG2 and Huh-7 cells were determined by qPCR.5.The relative expression of HBV pg RNA in HepG2 and Huh-7 cells with wild type,G1896 A and C2288 A mutant pCS-HBV1.3 plasmid transfected were determined by RT-qPCR.Results:1.pCS-HBV1.3 plasmid with G1896 A and C2288 A point mutation were constructed successfully and verified by gene sequencing.2.Wild type,G1896 A and C2288 A mutant HBV cell replication model was established successfully.3.The G1896 A mutation of preC/C gene prevented HBeAg expression,while the C2288 A mutation promoted HBeAg expression.Both G1896 A and C2288 A mutations in preC/C gene could promote HBsAg expression.The expression of HBeAg and HBsAg in the supernatant increased in a time dependent manner after transfection.4.Both G1896 A and C2288 A mutations in preC/C gene could promote the synthesis of HBV DNA,and concentration of HBV DNA in cell supernatant increased in a time dependent manner after transfection.5.Both G1896 A and C2288 A mutations in preC/C gene could promote HBV pg RNA transcription,and expression of HBV pg RNA in HCC cells increased in a time dependent manner after transfection.Conclusions:G1896A and C2288 A mutations in preC/C gene promote HBV replication.Part Three G1896 A and C2288 A mutations promote growth of hepatocellular carcinoma cells by activating ERK/MAPK pathway Objective: To explore the relationship between HBV preC/C gene G1896 A and C2288 A mutation and activation of ERK/MAPK signaling pathway in HCC progression.Methods:1.PCR array was used to find differentially expressed genes in MAPK signaling pathway between G1896 A,C2288A mutant group and wild type group of HCC cells.c-myc,BAX and BCL2 m RNA in wild type,G1896 A and C2288 A mutant group of HCC cells were verified by q RT-PCR.2.The protein levels of p-ERK1/2,ERK1/2,p-MEK1/2,MEK1/2,c-myc,BAX and BCL2 in control,wild type,G1896 A and C2288 A mutant groups of Hep3B,HepG2,Huh-7 and PLC/PRF/5 cells were detected by immunoblotting.3.Immunoblotting was used to detect the effect of ERK signaling pathway inhibitor PD 98059 treatment on protein expression of p-ERK1/2,ERK1/2,pMEK1/2,MEK1/2,c-myc,BAX and BCL2 in control,wild type,G1896 A and C2288 A mutant groups of HepG2 and PLC/PRF/5 cells.4.CCK8 assay,colony formation experiment and EdU incorporation assay were used to examine the effect of PD 98059 on cell proliferation of control, wild type,G1896 A and C2288 A mutant groups of HepG2 and PLC/PRF/5 cells.5.Flow cytometry was used to determine the effect of PD 98059 treatment on cell apoptosis of control,wild type,G1896 A and C2288 A mutant groups of HepG2 and PLC/PRF/5 cells.Results:1.Compared with wild type group of HCC cells,the expression level of apoptosis suppressor genes c-myc and BCL2 were increased,while the expression level of apoptosis promoting gene BAX were decreased in G1896 A and C2288 A mutant groups of HCC cells.2.The protein level of p-MEK1/2,p-ERK1/2,c-myc and BCL2 were increased while BAX level was decreased in wild type group of HCC cells compared with control group.The protein level of p-MEK1/2,p-ERK1/2,cmyc and BCL2 were increased while BAX level was decreased in G1896 A and C2288A mutant groups of HCC cells compared with those in wild type group.3.PD 98059 could significantly inhibit the phosphorylation of MEK1/2and ERK1/2 and protein level of c-myc and BCL2 in control,wild type,G1896 A and C2288 A mutant groups of HepG2 and PLC/PRF/5 cells,and BAX was significantly upregulated in PD 98059 treated HCC cells.4.PD 98059 significantly downregulated HCC cell viability,number of colonies formed and proportion of EdU positive cells in G1896 A and C2288 A mutant groups of HepG2 and PLC/PRF/5 cells,while PD 98059 did not affect the proliferation of HCC cells in control and wild type groups.5.PD 98059 treatment significantly upregulated the apoptosis of G1896 A and C2288 A mutant groups of HCC cells,while PD 98059 did not affect the apoptosis rate of control and wild type groups of HepG2 and PLC/PRF/5 cells.Conclusions:G1896A and C2288 A mutation in HBV preC/C gene regulates the expression of oncogene c-myc and apoptosis related genes BAX and BCL2 by activating ERK/MAPK signaling pathway,whereby the proliferation of HCC cells upregulated and the apoptosis of HCC cells downregulated.Part Four G1896 A and C2288 A mutations promote growth of hepatocellular carcinoma cells via inducing endoplasmic reticulum stress Objective: To explore the relationship between HBV preC/C gene G1896 A and C2288 A mutation and induction of endoplasmic reticulum stress in HCC progression.Methods:1.Gene expression profile in wild type,G1896 A and C2288 A mutant group of HepG2 cells were analyzed by transcriptome sequencing.KEGG enrichment analysis of differentially expressed genes was implemented.2.The protein levels of BIP,p-IRE1,XBP1,p-PERK,CHOP and ATF6 in control,wild type,G1896 A and C2288 A mutant groups of Hep3 B,HepG2,Huh-7 and PLC/PRF/5 cells were examined by immunoblotting.ROS production of HCC cells in control,wild type,G1896 A and C2288 A mutant groups was determined.The endoplasmic reticulum localization of HBcAg or HBeAg precursor in control,wild type,G1896 A and C2288 A mutant groups of HepG2 and PLC/PRF/5 cells was compared by immunofluorescence assay.3.Immunoblotting was used to detect the effect of ERS inhibitor TUDCA on expression of BIP,p-IRE1,XBP1,p-PERK,CHOP and ATF6 in control, wild type,G1896 A and C2288 A mutant groups of HepG2 and PLC/PRF/5 cells.4.CCK8 assay,colony formation experiment and EdU incorporation assay were used to examine the effect of TUDCA on cell proliferation of control,wild type,G1896 A and C2288 A mutant groups of HepG2 and PLC/PRF/5 cells.5.Flow cytometry was used to determine the effect of TUDCA treatment on cell apoptosis of control,wild type,G1896 A and C2288 A mutant groups of HepG2 and PLC/PRF/5 cells.Results:1.Transcriptome sequencing suggested that genes related to protein processing in endoplasmic reticulum was upregulated in G1896 A and C2288 A mutant groups of HepG2 cells compared with wild type cells.2.The protein level of p-IRE1,XBP1,p-PERK and CHOP were increased while BIP and ATF6 level did not change in wild type group of HCC cells compared with control group.The protein level of BIP,p-IRE1,XBP1,p-PERK and CHOP in G1896 A and C2288 A mutant groups were increased compared with wild type group,but ATF6 level was not significantly changed.ROS levels in G1896 A and C2288 A mutant groups of HCC cells were significantly higher than those in the wild type group.G1896 A mutation promoted the localization of the N-terminal peptide of HBeAg precursor and BIP in the endoplasmic reticulum.C2288 A mutation induced BIP expression in HCC cells and increased colocalization of mutated HBcAg and HBeAg with BIP was detected.3.TUDCA significantly inhibited the protein level of BIP,p-IRE1,XBP1,p-PERK and CHOP in control,wild type,G1896 A and C2288 A mutant groups of HepG2 and PLC/PRF/5 cells,while ATF6 were not significantly changed.4.TUDCA significantly downregulated HCC cell viability,number of colonies formed and proportion of EdU positive cells in G1896 A and C2288 A mutant groups of HepG2 and PLC/PRF/5 cells,while TUDCA did not affect the proliferation of HCC cells in control and wild type groups.5.TUDCA treatment significantly upregulated the apoptosis of G1896 A and C2288 A mutant groups of HCC cells,while TUDCA did not affect the apoptosis rate of control and wild type groups of HepG2 and PLC/PRF/5 cells.Conclusions:G1896A and C2288 A mutation in HBV preC/C gene promote proliferation and inhibit apoptosis in HCC cells by inducing endoplasmic reticulum stress and subsequent unfolded protein responses. |
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