Expression And Correlation Of Hepatitis B Virus X Mutation And Exdoplasmic Reticulum Stress Molecules GRP78、CHOP In Hepatocellular Carcinoma

AimsTo investigate the characteristics of X region mutation in hepatitis B virus（HBV）.To analysis the correlation between HBV X region mutation and endoplasmic reticulum stress（ERS）molecules GRP78 and CHOP.To explore the relationship between HBV X region mutation,GRP78,CHOP in HBV associated hepatocellular carcinoma（HCC）.Methods1.57 pairs of cancerous and paracancerous tissue of HBV-ralated HCC patients receiving surgery between January 2014 to December 2016 from the Department of Hepatobiliary surgery,affiliated tumor Hospital of Guangxi Medical University.The DNA of HCC cancerous tissues and paracancerous tissues were extracted.The fragments of HBV X region were amplified by nested PCR and sequenced bidirectionally by Sanger.Taking HBV X gene（NC₀03977.1）,which is widely used in NCBI database,as a reference sequence.The HBV X gene was analyzed by using the software Mutation Surveyor 5.1.1.The mutation frequency of HBV X region in HCC cancerous and paracancerous tissues was analyzed by Chi-square test.2.Immunohistochemical method was used to detect the expression of GRP78 and CHOP in cancerous and paracancerous tissues.The difference of high expression rate of GRP78 and CHOP between cancerous and paracancerous tissues was analyzed by Chi-square test.The Mann-Whitney rank sum test was used to analyze the correlation between the expression level of GRP78 and CHOP and the hot spot mutation and joint mutation of HBV X region.3.The correlation between ERS molecules GRP78,CHOP and clinicopathological features was analyzed.The survival of GRP78 high expression group and low expression group,CHOP high expression group and low expression group,hot spot mutation wild type group and mutant group of HBV X region were analyzed by Kaplan-Meier method.Results1.There were significant differences in the distribution of hot spot mutations G1461C,C1470T,T1544A,A1574T,C1653T,G1742A,A1762T and A1762T/G1764A between cancerous and paracancerous tissues.G1461C caused amino acid 30 in HBx to mutate from valine to leucine.C1470T caused amino acid 33 in HBx to mutate from proline to serine.T1544A caused Synonymous mutation of amino acid 57 in HBx.A1574T caused Synonymous mutation of amino acid 67 in HBx.G1742A caused Synonymous mutation of amino acid 123 in HBx.C1653T caused amino acid 94 in HBx to mutate from histidine to tyrosine.A1762T caused amino acid 130 in HBx to mutate from lysine to methionine:A1762T/G1764A caused amino acid 130 in HBx to mutate from lysine to methionine/131 amino acid in HBx to mutate from valine to isoleucine.2.IHC showed that GRP78 and CHOP proteins were mainly expressed in the cytoplasm of cells.The constituent ratio of cancerous tissues with GRP78 high expression was 78.95%（45/57）.And the constituent ratio of paracancerous tissues with GRP78 high expression was 80.70%（46/57）.Respectively,there was no significant difference between the two groups（P>0.05）.The constituent ratio of cancerous tissues with CHOP high expression was 28/57（49.12%）.And the constituent ratio of paracancerous tissues with CHOP high expression was 78.95%（45/57）.Respectively,there was significant difference between the two groups（P<0.05）.3.Among the 8 hot spot mutations in HBV X region,the expression of GRP78 was lower in G1461C mutation group and A1574T mutation group（P=0.001,P=0.012）;the expression of CHOP was lower in A1574T mutation group（P=0.027）.In paracancerous tissues,the expression of CHOP was only lower in C1470T mutation group（P=0.039）.4.In HCC,the expression of GRP78 in G1461C/C1470T and G1461C/A1574T joint mutation group was lower than that in non-mutation group（P=0.007,P=0.001）.The expression of CHOP in G1461C/A1574T joint mutation group was lower than that in non-mutation group（P=0.032）.5.The expression of GRP78 was related to liver cirrhosis,recurrence,total bilirubin and urea nitrogen.The expression of CHOP was related to alanine transferase.(DGRP78 was significantly correlated with liver cirrhosis（P=0.023）.Among patients with liver cirrhosis,the patients with GRP78 high expression in cancerous tissues is higher than thouse without liver cirrhosis.②GRP78 was significantly correlated with recurrence（P=0.044）.Among the patients with recurrence,the patients with GRP78 high expression in cancerous tissues is higher than those without recurrence.③GRP78 was significantly correlated with total bilirubin（P=0.041）.The total bilirubin of patients with high GRP78 expression is higher than those with low GRP78 expression..GRP78 was significantly correlated with urea nitrogen（P=0.035）.Urea nitrogen in patients with high GRP78 expression is lower than those with low GRP78 expression⑤CHOP was significantly correlated with Alanine aminotransferase（P=0.42）.Alanine aminotransferase in patients with high CHOP expression lower than those with low CHOP expression.Conclusions1.The mutations of G1461C,C1470T,T1544A,A1574T,C1653T,G1742A,A1762T and A1762T/G1764A in HBV X gene may be related to HCC.2.GRP78 and CHOP are widely distributed in the cytoplasm of hepatoma cells and adjacent hepatoma paracancerous cells.There was no significant difference in the expression of GRP78 between cancerous tissues and paracancerous tissues.The expression of CHOP in cancerous tissues was lower than that in paracancerous tissues.It is suggested that the ability of inducing apoptosis in cancerous tissues is decreased.3.The expression of GRP78 was lower in G1461C,A1574T,G1461C/C1470T and G1461C/A1574T mutation group.The expression of CHOP was lower in A1574T and G1461C/A1574T mutation group.It is suggested that A1574T and G1461C/A1574T may promote the occurrence of HCC by inhibiting the ERS-apoptosis pathway.4.The expression of GRP78 was related to liver cirrhosis,recurrence,total bilirubin and urea nitrogen.The expression of CHOP was related to glutamic pyruvic transaminase.It is suggested that the up-regulation of GRP78 can increase the production of total bilirubin,weaken the catabolism of protein and decrease the production of urea nitrogen in ERS,.CHOP was up-regulated,apoptosis was induced,hepatocyte destruction was reduced and glutamic pyruvic transaminase was decreased.