The Study Of Compound Phyllanthus Urinaria Inhibits HBV Related HCC Through Inactivation Hedgehog Pathway

Liver cancer is a malignant tumor with high morbidity and mortality,which poses a serious threat to human health.There are about 700,000 people who are diagnosed with liver cancer every year.Alcoholic fatty liver diseas is the main cause for liver cancer in European and American countries.But in China,hepatitis B virus(HBV)infection is the main reason.China is a high incidence of HBV infection.Among the patients,more than 90 million people are tested positive for HBVsAg,and about 420,000 people died of liver cancer every year,90%havea of HBV infection history.HBV infects through mother-to-child transmission,sexual transmission and blood transmission.Although with be popularized of HBV vaccine,the probability of HBV infection is greatly reduced.However,due to the large number of patients infected with HBV in China,it is still a great challenge to control the chronic hepatitis,liver fibrosis and even liver cancer caused by HBV infection.Sorafenib is the only anticancer drug with FDA approved can be used to treat patients who were diagnosed with liver cancer.However,for patients with HBV related liver cancer,the therapeutic effect is far lower than other patients who with non-HBV related liver cancer.In addition,long-term use of sorafenib may lead to side effects such as drug resistance.Surgery and chemotherapy are the main treatments for liver cancer.However,for HBV-related liver cancer,chronic hepatitis caused by HBV infection and high titer of HBV DNA are still risk factors for recurrence of liver cancer.HBV x gene(HBx)is the smallest of the four open coding frames of HBV and plays an important role in HBV-related HCC(hepatocellularcarcinoma).HBx can activate multiple signaling pathways and oncogenes through directly?epigenetic changes and inactivate tumor suppressor genes.Hedgehog signaling pathway plays an important role in t issue damage and repair,and over-expressed in various tumor tissues,including liver cancer.Inhibiting its expression can delay the proliferation of tumor cells.Ferroptosis is a newly discovered pattern of programmed cell death.This is mainly due to the iron production in cells and that induces Fenton reaction and lead to a large of toxic lipid reactive oxygen species and caused the cells dead.One of the main causes of resistance to sorafenib is its inhibition of ferroptosis in hepatocellular carcinoma cells.Iron accumulation is one of the main characteristics of ferroptosis.In patients with HBV infection,abnormal expression of iron metabolism indicates that HBV infection may involve iron death.Although long non-coding RNA(LncRNA)does not have the function of encoding protein,it can regulate miRNA or affect protein expression through other ways,thus change the normal physiological function of cells and promote cell lesions.Long non-coding RNA can regulate multiple signaling pathways and related factors involved in cell phenotypic changes.Compared with proteins or miRNA,the researchs targeted on long-strand non-coding may have more advantages and prospects.As an oncogene,HBx can mediate the multiple long non-coding RNA to involve the development of HBV-related HCC.Because of its tissue specific and space-time specific,LncRNA studies about as biological markers and drug intervention targets will provide a theoretical basis for basic research and drug research on HBV-related HCC.Compound Phyllanthus urinaria L(CP)was created by the Hepatology department of Shenzhen hospital of traditional Chinese medicine for the treatment of liver cancer,especially HBV-related HCC.The patent application number is 201610517509.2.CP is made of Phyllanthus urinaria L,Astragalus membranaceus(Fisch.)Bunge,Scutellaria barbata D.Don,Curcuma zedoaria(Christm.)Rosc and Cremastra appendiculata(D.Don)Makino.In clinical,CP can reduce serum virological markers(HBVsAg,HBVcAg and HBV DNA)in HBV-related HCC patients and delay the development of HBV cirrhosis into HCC,which shows CP has a better anti-HBV-related HCC effect.It was found that CP could inhibit HBx gene expression.However,the specific mechanism of CP is still unclear and needs further research.In this study,HepG2-HBx over-expression stable cell line was constructed and was treat with CP to measure the proliferation,migration and cloning to evaluate the inhibition effect of CP.Meanwhile,the mRNA and protein expressions level of Hedgehog signaling factors to explore the mechanism of CP about inhibition HepG2-HBx cells.Mice experiments were conducted to further clarify the anti-HBV-related HCC effect of CP;at the same time,the frroptosis related factors were detected to evaluate the effect of CP on ferroptosis.Meanwhile,the targets LncRNA of CP was screened by high-throughput sequencing,which provided a preliminary research basis for further research on the anticancer mechanism of CP.ObjectiveHepG2-HBx over-expression cell was treated with CP to measure the proliferation,migration and cloning to evaluate the inhibition effect of CP.Meanwhile,the mRNA and protein expression levels of HBx and Hedgehog signaling factors SHH(Sonic Hedgehog),PTCH1(Patched-1),GLI-1(glioma-associated oncogene homolog 1),GLI-2(glioma-associated oncogene homolog 2)and SMO(Smoothene)in HepG2-HBx cells were detected to explore the mechanism of CP about inhibition HepG2-HBx cells.Mice experiments were conducted to further clarify the anti-HBV-related HCC effect of CP;iron concentration in tumor tissues as well as the genes expressions of CISD1(CDGSH domain-containing family 1),CISD2(CDGSH domain-containing family 2)and GPX4(Glutathione peroxidase 4)were detected to evaluate the effect of CP on ferroptosis.Meanwhile,the targets LncRNA of CP was sereened by high-throughput sequencing and preliminarily measure by real-time PCR,which can provided a basis for further research on the anticancer mechanism of CP.Methods1.To evaluate the anti-HBV-related HCC of CP in vivo:over-expressing HBx gene HepG2-HBx cell was constructed,and different concentrations of CP were used to treat the HepG2-HBx cell.The inhibition effect of CP on HBV-related HCC was evaluated by observing the proliferation,migration and cloning of HepG2-HBx cells.The expressions of HBx and Hedgehog signaling pathway related factors SHH,PTCH-1,SMO,GLI-1 and GLI-2 at mRNA and protein levels were detected.2.To evaluate the anti-HBV-related HCC of CP in vitro:HepG2-HBx cells were transplant to nude mice and CP was given to mice.The inhibition effect of CP on HBV-related HCC was evaluated by body weight,tumor size and tumor inhibition rate in nude mice.Moreover,by detecting the mRNA and protein expression levels of HBx and Hedgehog signaling pathway related factors SHH,PTCH-1,SMO,GLI-1 and GLI-2 in tumor tissues;as well as using immunohistochemical techniques to located and analyze SHH,PTCH-1,SMO,GLI-1 and HBx,thus to further confirmed that inhibition of Hedgehog signaling pathway activation is one of the anti-HBV-related HCC mechanisms of CP.3.The effect of CP on ferroptosis:by using Prussian blue staining technique to detect the expression of iron in tumor tissue,and by real-time PCR to measure the mRNA expression of FTH and ferroptosis related genes CISD1,CISD2,GPX4,and ACSL4,to confirm that induce the ferroptosis is one of the mechanisms of CP on HBV related HCC.4.LncRNA high-throughput sequencing:HepG2-HBx cells(HBx group)and HepG2-HBx cells treated by CP(treat group)were used to screened LincRNA,and the LincRNAs with statistical differences were verified by real-time PCR.Which will provide ideas for further research on the mechanism of CP on HBV-related HCC.Results1.CP could inhibit the proliferation and migration and clone of HepG2-HBx cells,compared with HepG2-HBx cells,which had statistical significance(p<0.001).At the mRNA level,CP could significantly inhibit the expression of HBx,SHH,PTCH-1 and GLI-1,compared with HepG2-HBx cells,which had statistically significant(P<0.001).CP had no influence on SMO and GLI-2 mRNA expression.At the protein level,CP could inhibit the expression of HBx,SHH,PTCH-1 and GLI-1 in HepG2-HBx cells treated with CP,compared with HepG2-HBx cells,which had statistically significant(P<0.001).The protein results were same as the mRNA,CP had no influence on the SMO and GLI-2 at the protein expression.The results of confocal laser showed that CP could inhibit the expression of GLI-1 in cytoplasm.2.CP could inhibit tumor volume growth,reduce ratio of tumor/body weight,and improve the tumor inhibition rate,compared with HepG2-HBx group,it had statistically significant(P<0.01,P<0.001).Consistent with the results of in vitro,CP could inhibit the expression of HBx,SHH,PTCH-1,and GLI-1 at mRNA and protein levels,compared with HepG2-HBx group,which had statistically significant(P<0.05,P<0.01)and it has a dose-effect relationship.However,CP had no inhibition effect on SMO and GLI-2 at mRNA and protein levels(P>0.05).Immunohistochemical results showed that HBx increased the expression of SHH,PTCH-1,GLI-1 and SMO in the cytoplasm,while CP at 625mg/kg could significantly inhibit the expression of HBx,SHH,PTCH-1 and GLI-1 in the cytoplasm,compared with HepG2-HBx group,which has statistically significant(P<0.05,P<0.01).CP had no inhibition effect on SMO(P>0.05).3.As for iron level,there is no difference between HepG2-NC group and HepG2-HBx group.However,CP(625mg/kg)and cyclopamine can increase iron expression in tumor tissue.As for FTH mRNA,HBx can obviously improve the expressin(P<0.05),and the cyclopamine and CP(P<0.05)could decrease the expression.Real time PCR results showed that HBx can decrease the CISD1(P<0.05)and GPX4 mRNA expression,compared with HepG2-NC group,GPX4 result has no statistically significant(P>0.05);HBx also can decrease the mRNA expressionod CISD2(P<0.05),and CP(625mg/kg,300mg/kg)and cyclopamine can increase the expression CISD2 in tumor tissue,compared with HepG2-HBx group,which has statistically significant(P<0.01,P<0.00).As for ACSL4,there is no statistically significant between HepG2-HBx group and CP group(P>0.05).4.High-throughput LncRNA sequencing results showed that HepG2-HBx cells treated by CP[treat group(70?g/mL)]were up regulated 114 and down regulated 65 LncRNAs,respectively,compared with HepG2-HBx cell group(HBx group).LncRNA MALAT1,LncRNA 0023,LncRNA GAS5,LncRNA ROR,LncRNA BISPR,Lnc PSMB8-AS1,LncH19 and Lnc RP11-998D10.4 were further verified by real-time PCR.It was found that CP had an obvious inhibition effect on Lnc BISPR and have a dose-response relationship,which was statistically significant when compared with HepG2-HBx group(P<0.01).The expression of Lnc PSMB8-AS1 and Lnc RP11-998D10.4 were significantly increased at 120mg/ml of CP,which compared with HepG2-HBx group(P<0.05,P<0.001).The LncRNA MALAT1 could significantly reduce at the dosage of 30mg/ml and 60mg/ml,which was statistically significant compared with HepG2-HBx group(P<0.05,P<0.01).While at 120mg/ml,CP could promote its expression but there is no statistically significant.For LncRNA 0023,LncRNA GAS5,LncRNA ROR and LncRNA H19,CP had no obvious inhibition or promotion effects(P>0.05).ConclusionThe studies from the in vivo and in vitro showed that CP could inhibit the proliferation,migration and clone of HepG2-HBx cells.And it also can inhibit the growth of transplanted tumor,reduce the ratio of tumor/body weight,and improve the tumor inhibition rate,which indicated that CP has an anti-HBV-related HCC effect.CP could inhibit the expression of HBx,SHH,PTCH-1 and GLI-1,which indicated that inactivate Hedgehog signaling pathway is one of the mechanisms of CP to exert anti-HBV-related HCC effects.CP could increase the concentration of iron in the tumor tissues,increased CISD2 mRNA expression,which means CP maybe could induce ferroptosis.So,inducing ferroptosis maybe is one of mechanisms for CP to play an anti-cancer role.In addition,the Hedgehog signaling pathway inhibitor cyclopamine can obviously increase iron level and increased the expression of CISD2,decreased CISD1 and GPX4 mRNA expression,which means the Hedgehog signaling pathways likely to be involved in mediate ferroptosis process.Therefore,the relationship between Hedgehog signaling pathways and ferroptosis in HBV associated HCC or HCC need to be further studied.CP could decrease the expression of LincBISPR,but the function of LincBISPR is till unclear.LncRNA MALAT1 as an oncogene,its role in HBV related HCC and if it is a therapeutic target of CP on HCC need to be study.As for,LncPSMB8-AS1 and Lnc RP11-998D10.4,their roles in HBV related HCC need further studied.