| Objective:To analyze the characteristics of HBX mutation in cancer and adjacent tissues of patients with HBV-related HCC,and to evaluate the autophagic activity of HCC by detecting the expression of autophagy-related proteins(Beclin-1,LC3B and P62),and to explore whether autophagy is involved in the process of HBX mutation-induced HCC,so as to provide experimental basis for the mechanism of hepatocarcinogenesis of HBX.Method:From July 1,2015 to December 30,2016,underwent hepatobiliary surgery in the Tumor Hospital Affiliated to Guangxi Medical University were collected.Their HCC tissues and corresponding adjacent tissues(more than 2cm from the incision margin)were taken and their clinical data were collected.HBX gene sequence was amplified by nested PCR method,and the purified products were determined by Sanger sequencing method for HBX mutation types in cancer tissues and adjacent tissues.Immunohistochemistry was used to detect the expression of autophagy related proteins(beclin-1,LC3B and P62)in cancer tissues and adjacent tissues.Analyze data using SPSS22.0 software package.Normality test was performed on all quantitative data,and paired t-test was used to analyze the mean differences of proteins between cancer and adjacent tissues.The relationship between autophagy protein expression and clinicopathological factors was tested by independent sample t test,one-way anova was used for comparison between groups,and LSD-t test was used for comparison between groups.Chi-square test was used to compare the rate of counting data with that of composition.Pearson correlation analysis of autophagy protein expression correlation;Sperman correlation analysis the correlation between HBX mutation and autophagy protein expression;P<0.05was considered statistically significant(bilateral).Result:1.Deletion of carboxyl terminal sequence of HBX geneIn cancer tissues,the deletion rate of HBX carboxyl terminal sequence was25.0%(15/60),higher than 6.7%(4/60)in adjacent tissues(?~2=7.566,P=0.006<0.05).The deletion length of HBX carboxyl terminal sequence in cancer tissues ranged from(6-31)bp to(9-25)bp.2.The insertion mutation of HBX geneThere was no significant difference in the distribution of insertion mutation between 6 sequences of hepatocellular carcinoma and 3 sequences of adjacent tissues(P>0.05),but especially in the insertion mutation sequence,AFP>20ng/ml existed.In addition,89%of insertion mutation sequences were located at the carboxyl end of HBX3.Point mutation of HBX geneThe hotspot amino acid point mutations found in this study were not unique to a particular tissue but existed in both cancer and adjacent tissues.Among non-differentially hotspot mutations,the detection rates of S78R and P152T mutations were about 90%,which were the hotspot mutations with higher detection rates among all mutations.The mutation rates of 47,118,130,131,130+131,132,143,30+144 amino acid points in cancer tissues were 53.6%?32.1%?78.6%?78.6%?76.8%?30.4%?28.6%?12.5%,higher than 33.9%?14.3%?58.9%?58.9%?55.4%?10.7%?12.5%?1.8%of the adjacent tissues(P<0.05).There was no significant difference in the detection rate of other hot spot mutations between the tissues and the adjacent tissues(P>0.05).4.Effect of Differential Point Mutation on Protein StructureThe results showed that all the six mutation sites were located at the carboxyl end of HBX and five of them were located in EnhII(K118T)or BCP(K130M,V131I,F132Y/E,C143R/H),A47T was located at the important T_B cell epitope,K118T,K130M,V131I,F132Y/E were located at the important T_H cell epitope(aa111-135),K130M,V131I,F132Y/E were located at the T_C cell epitope(aa126-134).5.Expression of Beclin-1,LC3B and P62 proteins in cancer tissues and adjacent tissuesBeclin-1,LC3B and P62 proteins were mainly localized in the cytoplasm and diffusely distributed in yellow and brown-yellow stained granules.Compared with the adjacent tissues,the expression of Beclin-1 and LC3B protein in cancer tissues decreased,while the expression of P62 protein increased,the difference was statistically significant(P<0.05).6.The relationship between the expression of Beclin-1,LC3B and P62protein and clinicopathological featuresThe expression level of Beclin-1 protein was correlated with the size of tumors(F=3.474,P=0.030);The expression level of LC3B protein was correlated with BCLC staging(A/B)(t=5.960,P<0.0001);The expression level of P62 protein was correlated with Edmondson grade and the presence of cancer thrombus(F=4.274,t=2.459,P<0.05);There was no significant difference with other clinical and pathological features(P>0.05).7.Analysis of the correlation between HBX mutation and Beclin-1,LC3B and P62 proteins in cancer tissues and adjacent tissuesBased on the above results of HBX mutant detection,combined with the previous serological results of our group and other research results of our group,several different HBX gene mutants were screened out.They are carboxyl end deletion,V30L,A47T,V88N/D,K118T,K130M,V131I,F132Y/E,C143R/H,S144Y,V30L+S144Y,K130M+V131I,triple mutation K130M+V131I+F132Y/E,and III.Co-mutation K130M+V131I+C143R/H;Correlation analysis showed that double mutation of F132Y/E,double mutation of V30L+S144Y,triple mutation of K130M+V131I+F132Y/E were positively correlated with LC3B(r_s=0.278,0.279,0.278,P<0.05),while in adjacent tissues,A47T was positively correlated with LC3B(r_s=0.270,P=0.045);The mutation of base acid sites was negatively correlated with the expression of Beclin-1 protein(r_s=-0.325,P=0.014),while the other HBX mutations were not correlated with the expression of Beclin-1,LC3B and P62 protein(P>0.05).8.Analysis of correlation between expression of Beclin-1,LC3B and P62proteins in tissuesThe expression of Beclin-1 was positively correlated with that of LC3B(r=0.416,P=0.001),Beclin-1 was positively correlated with that of P62(r=0.361,P=0.006),and there was no significant correlation between the expression of LC3B and P62(P>0.05).There was no significant correlation among Beclin-1,LC3B and P62 in cancer tissues(P>0.05);The expression of Beclin-1 and LC3B was positively correlated in all tissues(r=0.297,P=0.001);The expression of Beclin-1,LC3B and P62 was not significantly correlated(P>0.05).Conclusion:1.HBX gene mutation has polymorphism.2.Despite the large number of point mutations in HBX sequence,only some piont mutations affect the occurrence of HCC.3.Decreased autophagic activity may be related to the occurrence and development of HCC.4.HBX F132Y/E,V30L+S144Y double mutation,and K130M+V131I+F132Y/E triple mutation may have effects on autophagy prophysis,which may be related to the occurrence and development of HCC. |
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