| BackgroundHepatocellular carcinoma(HCC)caused by chronic infection with hepatitis B virus(HBV)accounts for 87.5% of all hepatocellular carcinoma and is one of the major causes of tumor death in China.Liver cancer caused by hepatitis b virus infection(HBV-HCC)obvious gender differences in the incidence and mortality,the ratio of 6.7:1,the application of environmental exposure difference fails to explain the phenomenon,studies have shown that sex hormones and their receptors is the important reason for liver cancer gender differences,androgen receptors(androgen receptor,AR)play a role of promoting cancer,estrogen receptor(estrogen receptor,ER)play a role of tumor suppressor.AR interacts with HBV in chronic carcinogenesis of hepatitis B virus,and AR interacts with immune inflammatory molecules to maintain chronic inflammation and destabilize the balance of apolipoprotein B m RNA editing enzyme polypeptide 3 family(APOBECE3)and DNA glycosylase(UNG).It promotes somatic variation and HBV integration,and interacts with androgen to alter key signaling pathways and survival advantages.This study mainly explores the relationship between somatic mutations of AR and the two key types of mutations associated with HBV integration.Somatic variation is the basis of HCC evolution.The APOBEC3 family is an important molecule that induces somatic and viral mutations and can cause damage to human and viral genomes through deamination.Currently,studies on the functional relationship between APOBEC3 family genes and AR are still lacking.HBV integration is a unique mutation type of HBV-HCC and a way for HBV to directly damage human genome.Telomerase reverse transcriptase(TERT)has been confirmed as a common HBV integration gene.The function of HBV integration insertion into TERT fragments and the effect of AR on it are still to be studied.ObjectiveHBV-HCC has obvious gender differences,suggesting that AR plays a significant role in the evolution of HCC.The study started from two aspects: the imbalance of APOBEC-UNG and the HBV integrated mutation to explain the role of AR in the evolution of HCC through interaction with somatic mutation factors.Methods1.To evaluate the effect of AR/ER and gender difference on prognosis by using public database information,to construct AR/ER overexpression hepatoma and normal hepatoma cell lines,and to observe the cell proliferation under the stimulation of androgen(DHT),estrogen(E2)and androgen receptor inhibitor(FLU).2.Analyze the relationship between AR and APOBEC family genes,and study the role of AR in the balance of APOBEC family genes and UNG in over expressed AR/ER cell lines.3.The results of bioinformatics analysis were verified by transcriptome sequencing,and the key HBV integrated mutations related to AR function were explored by capture sequencing.The synergistic effect of AR on HBV high frequency integrated mutations was studied in AR overexpression cell lines.4.To construct PDX integration mice,castration mice,AR overexpression mice and AID-UNG imbalance mice,and explore the role of hormones and receptors in tumor formation.Results1.Effect of AR on prognosis and proliferation of HCC cellsIn GSE14520 and TCGA-LIHC cohorts,gender differences and the influence of AR/ER on the prognosis of HBV-HCC were analyzed.It was found that gender differences were significantly correlated with the prognosis differences in GSE14520 cohorts,suggesting that the prognosis of male HCC was poor(P=0.0467);gender differences in TCGA-LIHC cohorts were not statistically significant with the prognosis of HCC.Low expression of AR in cancer tissues of the TCGA-LIHC cohort and non-HBV-infected populations indicates a poor prognosis of HCC(P=0.0045,P=0.0058).Among patients with HBV infection in the TCGA-LIHC cohort and female tumor patients in the GSE14520 cohort,there is marginal statistical significance between high expression and poor HCC prognosis(P=0.0863,P=0.065).In the TCGA-LIHC cohort and its tumor and para-cancer samples,the low expression of ER indicates a poor prognosis of HCC(P=0.0202,P=0.0358,P=0.0108);the low expression of ER and the poor prognosis of HCC in the GSE14520 cohort have marginal statistics Significance(P=0.0572).The high expression of ER in its tumor samples indicates a poor prognosis of HCC(P=0.0234).The prognosis of ER and HCC in its adjacent samples is not statistically significant(P=0.4327).The AR/ER cell line(Hep G2/L02)was successfully constructed.The cell proliferation experiment was carried out under the effect of hormone.The results showed that: the addition of androgen alone had no significant effect on the proliferation of Hep G2 cells,the addition of estrogen alone had a certain effect on the proliferation of L02 cells(P<0.001),and the increase of androgen receptor level and the application of androgen stimulation had significant effect on cell proliferation(Hep G2,P=0.019;L02,P=0.009).In untreated cell lines,androgen receptor antagonists could significantly inhibit cell growth(P<0.001),and overexpression of AR could antagonize the effect of androgen receptor inhibitors.The results showed that over expression of AR can promote the growth of HCC,and maintain a certain level of AR base is a necessary condition for the proliferation of HCC.2.AR promotes the expression of APOBEC3 family and UNGThe expression level of APOBEC3 A and APOBEC3 B,APOBEC3C,APOBEC3 D and UNG were all increased by AR.the expression level of APOBEC3 D,APOBEC3F,APOBEC3 G and APOBEC3 H were increased by AR overexpression in normal hepatocytes.Under the effect of androgen,the expression level of APOBEC3 A was significantly increased by AR overexpression in hepatoma cells The expression of APOBEC3 A and UNG in normal hepatocytes increased significantly.ER inhibited the expression of APOBEC3 B and UNG in hepatoma cells,while APOBEC3 A,APOBEC3C and APOBEC3 D increased,APOBEC3 B,APOBEC3D and UNG increased in normal hepatocytes;ER over expressed hepatoma cells decreased significantly in UNG,and APOBEC3 A increased in normal hepatocytes.3.Prognosis and correlation analysis of APOBEC3 familyIn the TCGA-LIHC cohort,correlation analysis showed that AR was significantly negatively correlated with APOBEC3 D,APOBEC3G,and APOBEC3H(P=0.005,P=0.021,P=0.01).Prognostic analysis showed that: in the TCGA-LIHC cohort cancer tissues,high expression of APOBEC3 B indicates poor prognosis of HCC patients(P=0.0305),low expression levels of APOBEC3 G and APOBEC3 H indicates poor prognosis of HCC patients(P=0.0033,P=0.0037);Among the HBV-infected people in the TCGA-LIHC cohort,high expression of APOBEC3 A and APOBEC3 B in cancer tissues of HBV-infected patients indicates a poor prognosis in HCC patients(P=0.0137,P=0.0017),and low expression of APOBEC3 H indicates a poor prognosis in HCC patients(P=0.0388);The high expression of APOBEC3 B and UNG in the cancer tissues of HBV-infected people indicates a poor prognosis of HCC patients(P=0.0472,P=0.0008);prognostic analysis of tumor samples from the GSE14520 cohort of all HBV-infected patients indicates that the high expression of APOBEC3 A and UNG suggests HCC has a poor prognosis(P=0.0283,P=0.009),and low expression of APOBEC3 C indicates a poor HCC prognosis(P=0.0304).4.Integrated mutation of high frequency HBV in HCC and its synergistic effect with ARThe most frequent integrated mutation region in HCC was identified as the upstream of TERT by HBV capture and sequencing.It was found that there was diversity in TERT integration,among which the frequency of HBV X fragment integration was the highest,which indicated that HBV X fragment was the main fragment integrated into human genome,and part of P,S and C regions were integrated at the same time.At the DNA level,the detection rate of HBV integration mutation in TERT is the highest(16%),and all of them are distributed in the promoter region.The HBV integration in the promoter region of TERT is related to the increased expression of TERT gene in cancer tissues.Functional verification of HBV fragment insertion into TERT integration.The insertion of each HBV region has an effect on the function of the TERT promoter,and the P region(Pro-P1)inserted in the TERT promoter is the most significant.The functional activity of the TERT promoter fragment integrated in the Pro-P1 region is 20-5000 times that of other integrated promoters.Other promoter region insertion fragments,such as the X region(Pro-X)inserted in the promoter region,reverse inserted X-region(Pro-Xr)and promoter inserted S-region(Pro-S)inserts are 3-7 times higher in fluorescence expression than the control;X region(Up-X)inserted upstream of the promoter and The reverse insertion(Up-Xr)fragment is approximately 15-fold and 35-fold higher than the promoter upstream control fragment(Up-co);the expression amount of the Pre S(Prepro-Pre S)fragment inserted in the pre-promoter region is higher than that of the control fragment 50 times lower,Prepro-S and Prepro-X are 2-3 times higher than the control;in the fragment inserted into the enhancer,the functional activity of the P region(Enhancer-P)inserted in the enhancer region is about 600 times that of the control,enhanced The fluorescence intensity of other inserts(Enhancer-Pre S,Enhancer-S1,Enhancer-S2,Enhancer-P2,Enhancer-X,and Enhancer-Xr)in the sub-regions is 10-30 times higher than that of the control fragments.After androgen stimulation,the non-integrated TERT fragment(TERT-co)is affected,and the functional activity is increased by 2 times.The P region inserted into the promoter region of the integrated fragment(Pro-P1)and the reverse insertion of HBX(Pro-Xr)fragment and S-segment(Pro-S)were affected.Pro-P1 fragment functional activity increased by 1.2 times,Pro-Xr fragment functional activity increased by 2 times,and Pro-S fragment functional activity decreased by 9 times.It shows that the combination of AR and androgen has a positive regulation effect on TERT-co,Pro-P1,Pro-Xr fragments,and a reverse regulation function on Pro-S.After knocking out AR in the overexpression cell line,the functional activity of the Pro-P1 insert was reduced by 1.38 times,the Pro-X region insert was increased by 2.11 times,and the Pro-Xr insert was increased by 7 times,indicating that Pro-P1 Subject to the positive regulation of AR,AR has a negative regulation effect on Pro-X zone and Pro-Xr.5.Prognostic analysis of TERT expression and HCC and its relationship with telomere lengthThe low expression of TERT in the TCGA-LIHC cohort indicates a poor prognosis(P=0.0406),and further screening for HBV-infected patients.Similarly,the low expression of TERT indicates a poor prognosis of HCC(P=0.034);in contrast,in the case of non-infected HBV high expression of TERT in the population indicates a poor prognosis of HCC(P=0.0069).The telomere length of cancer and cancer adjacent samples was significantly longer than that of tumor samples(P < 0.05).Conclusion1.Gender difference and AR/ER expression level can affect the prognosis of HCC patients.Male indicates poor prognosis;the influence of AR on prognosis is affected by gender,HBV infection and other factors,such as AR is a risk factor in the non-HBV infected population in the TCGA-LIHC cohort,and a protective factor in the HBV infected population in the TCGA-LIHC cohort and women in the GSE14520 cohort;low expression of ER indicates poor prognosis.The effect of AR/ER on prognosis may be related to hormone level.2.Over expression of AR and addition of androgen can promote the proliferation of liver cancer cells,and maintain a certain level of AR is a necessary condition for the proliferation of liver cancer cells;estrogen alone can promote the proliferation of liver cells,suggesting that the mechanism of hormone promoting the proliferation of cancer and non-cancer is different.3.AR can regulate the gene expression of APOBEC3 family,and only APOBEC3 D overlaps in hepatoma cells and hepatocytes,suggesting that there are differences in the expression of APOBEC3 subtypes in different tissues;AR combined with androgen can further promote the expression of APOBEC3 A,while ER combined with estrogen can down regulate the expression of UNG,indicating that ar promotes cancer and ER The effect of inhibiting cancer was different.4.We found the correlation between APOBEC3 family gene and AR expression,and preliminarily confirmed the close relationship between them.The expression level of APOBEC3 family gene can affect the prognosis of HCC patients,especially APOBEC3 A high expression and APOBEC3 G low expression in HCC caused by HBV infection and poor prognosis of patients,indicating that the interaction between APOBEC3 family gene and HBV leads to cancer.5.TERT is the high frequency integration site of HCC.The insertion of P-region of promoter in the integration fragment of HBV inserted into TERT plays the most significant role.AR combined with androgen has a regulatory effect on some integration fragments,which is one of the reasons that AR promotes integration mutation and carcinogenesis. |
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