Based On Network Pharmacology And Whole-genome Methylation Sequencing To Explore The Molecular Mechanism Of Ganji Fang’s Intervention In Hbv-related Hepatocellular Carcinoma

ObjectiveGanji Fang(GIF)is an empirical remedy developed by my tutor,Professor Xiongwen Wang.Early clinical and animal studies suggested that GJF has the effects of inhibiting tumors and protecting the liver and promoting apoptosis.Based on previous research,combining cell experiments,network pharmacology analysis,and whole-genome DNA methylation sequencing(WGBS)to further explore the molecular mechanism of GJF on hepatitis B-related hepatocellular carcinoma(HBV-HCC),thus providing a scientific basis for the follow-up mechanism research and clinical application of it.Methods(1)In cell experiments,the MTT method was used to study the inhibitory effect of GJF on the proliferation of Hep G2 and Hep G2.2.15 cell lines,and calculated the half inhibition rate(IC50).The cell scratch test was used to analyze the effect of GJF on cell migration.The Annexin V/PI double staining method was used to analyze the regulation effect of GJF on the cell cycle and apoptosis of Hep G2 and Hep G2.2.15 cell lines.(2)To further explore the molecular mechanism of GJF in the treatment of HCC,network pharmacology is used to analyze the possible molecular mechanism and key targets of GJF on HCC.Firstly,the potential active ingredients of GJF were screened from the TCMSP database;the TCMSP and Swiss Target Prediction databases were used to obtain the corresponding protein targets of each compound;the differentially expressed m RNA and differential methylation sites in the TCGA database and a series of key targets obtained by the public database are regarded as the key targets in HCC.Map the protein targets predicted by the potential active ingredients of the GJF to the key targets in HCC,and the key targets of the GJF on HCC can be obtained.The String database is used to construct a protein-protein interaction network,and the Cyto Hubba plug-in in Cytoscape performs network topology analysis to screen key targets.The “cluster Profiler” package in R was used to perform GO and KEGG enrichment analysis on these key targets.The Cytoscape software was used to construct a network diagram of “active ingredients-targets-pathways-disease”,and the molecular docking technology was used to explore the binding ability of key active ingredients and targets in GJF,q RT-PCR is used to verify pathways and phenotype-related molecules m RNA expression level.(3)WGBS is regarded as the “gold standard” for DNA methylation research.It combines bisulfite treatment and high-throughput sequencing to analyze the epigenetic characteristics of HBV-HCC from the perspective of whole-genome methylation.In our study,we selected 12 HCC tissues and matched normal liver tissues(APTs)for WGBS analysis.According to the difference of HBV DNA viral load,we divided 12 groups of patients into high-load HBV DNA expression group,low-load HBV DNA expression group,and HBV DNA expression negative group.Differentially methylated regions(DMRs)and differentially methylated genes(DMGs)between HBV tissues and APTs among the three groups were obtained.DMGs were used for further GO and KEGG enrichment analysis.The TIMER database was used to verify the differential expression of hub targets in various cancer species,the GEPIA database was used to verify the survival differences between different populations with high and low gene expression,and the CBio Portal database was used to analyze the genetic mutations of the hub targets.Results(1)In cell experiments,MTT results suggested that GJF can inhibit the proliferation of Hep G2.2.15 and Hep G2 cell lines,and its inhibitory effect was concentration-dependent.The IC50 of GJF on 24 H,48H and 72 H of Hep G2.2.15 cell line was 1.25mg/ml,0.90mg/ml,0.68mg/ml;GJF on 24 H,48H and 72 H of Hep G2 cell line IC50 is 1.91mg/ml,1.56mg/ml,1.12mg/ml.The results of the cell scratch test showed that GJF can inhibit the migration of Hep G2.2.15 and Hep G2 cell lines.The higher the drug concentration,the stronger the ability to inhibit migration.Annexin V/PI double staining method showed that GJF can block Hep G2.2.15 and Hep G2 cell lines in G0/G1 phase,and its blocking effect is concentration-dependent.GJF can inhibit the total apoptosis rate of Hep G2.2.15 and Hep G2 cell lines,and its inhibitory effect is concentration-dependent.Compared with the control group,the difference between the different concentrations of the GJF group and the control group was statistically significant(P<0.05).(2)In the TCMSP and the Swiss Target Prediction database,combined with literature reports,a total of 162 active compounds and 826 corresponding protein targets were screened from 12 traditional Chinese medicines in GJF.We took the intersection of the differential m RNA and differential methylation sites screened in the TCGA database with a series of targets screened in the public database.A total of 611 HCC-related targets were screened,and finally 63 liver product formulas were used to act on the key target of HCC.Through GO and KEGG enrichment analysis of these 63 targets,the biological processes,cell components and molecular functions that these targets participate in were the top-ranked epithelial cell proliferation,cell-substrate connection,and cell adhesion.Attached to molecule binding.The top five pathways are cell cycle,cell senescence,p53 signaling pathway,PI3K-Akt signaling pathway,and progesterone-mediated oocyte maturation.To explore the molecular mechanism of GJF’s intervention in HCC,we further verified the PI3K-Akt signaling pathway.The Cytohubba plug-in screened and found that the top 10 potential hub targets of GJF for HCC are HSP90AA1,SRC,CDK1,CCNA2,CDK4,PLK1,CDK2,TOP2 A,AURKB,and CDK6;CCNE1 and CCNE1 can be seen in the PI3K-AKT pathway.PKN1,CCND2,CDK4,EPHA2,FGFR3,CDK6,CDK2 and HSP90AA1 were enriched.We intersect these 10 targets with the targets enriched in the PI3K-AKT pathway.We speculate that HSP90AA1,CDK2,CDK4,and CDK6 may be the key targets of GJF on HCC.To explore the main active substances that play an anti-cancer effect in GJF,molecular docking technology was used to analyze the binding ability of targets and compounds.The eight active ingredients screened in GJF(Anhydroicaritin,Cubebin,(2R)-7-hydroxy-5-methoxy-2-phenylchroman-4-one,paeoniflorin,albiflorin＿qt,bisdemethoxycurcumin,pachymic acid,7,9(11)-dehydropachymic acid)and the two potential targets(HSP90AA1,EPHA2)docking scores are all less than-4.0,indicating that they have a certain binding activity.Among them,paeoniflorin and HSP90AA1 have the strongest binding energy of-7.3,and pachymic acid and EPHA2 have the strongest binding energy of-8.8.Therefore,paeoniflorin and pachymic acid may have higher drug activity in the process of HCC intervention by GJF.Finally,we verified the relevant molecules in the PI3K-Akt signaling pathway in Hep G2 and Hep G2.2.15 cell lines.The results of q RT-PCR showed that GJF can improve the effects of PIK3R1 and AKT1 in Hep G2 and Hep G2.2.15 cell lines.GJF can reduce the expression level of cycle-related proteins(CCND1,CDK2,CDK4,CDK6)in liver cancer cell lines,and increase the expression level of cycle-related protein CDKN2A;for apoptosis-related proteins,GJF can promote the expression level of Bax and Bim,while the expression level of Bcl-2 was reduced.In the Hep G2 cell line,GJF can reduce the expression level of EPHA2 and HSP90AA1,while in the Hep G2.2.15 cell line,GJF cannot effectively inhibit the expression level of EPHA2.(3)There were 311,901 DMRs with high methylation levels and 13,734 DMRs with low methylation levels between HCC tissues with high HBV DNA loads and APTs.There were 376,068 DMRs with low methylation levels and 12,083 DMRs with low methylation levels between HCC tissues with low HBV DNA loads and APTs.There were 85,412 DMRs with high methylation levels and 8,316 DMRs with low methylation levels between HBV DNA-negative HCC tissues and APTs.DMGs annotated by DMRs mainly played a role in metabolism-related pathways,cancer-related pathways,PI3K-Akt signaling pathways,neuroactive ligand-receptor interactions,focal adhesions,and Rap1 signaling pathways.By combining the DMGs between the three groups,we obtained the differentially methylated sites in HBV-HCC.Combining the above analysis results of network pharmacology,we have obtained five key targets of GJF that may act on HBV-HCC,including EPHA2,HSP90AA1,CDK6,PKN1 and FGFR3.We verified the PI3K-Akt pathway and EPHA2 and HSP90AA1 that may be upstream of this pathway in the TCGA database,and the results suggested that EPHA2 and HSP90AA1 are differentially expressed in a variety of cancer types.Among them,EPHA2 has low expression in HCC,which was not consistent with our conjecture and may require further verification.HSP90AA1 is highly expressed in HCC,and its low expression is associated with a better prognosis.EPHA2 and HSP90AA1 m RNA expression levels are negatively correlated with DNA methylation levels.However,our WGBS sequencing data showed that EPHA2 is hypomethylated in HCC,and HSP90AA1 is hypermethylated.In the CBio Portal data,we further explored the overall mutation rate of EPHA2 and HSP90AA1,and the results showed that the mutation rate of EPHA2 was 1.9%,and the mutation rate of HSP90AA1 was 1.4%.Based on the above results,the PI3K-Akt pathway plays a key role in the process of GJF’s intervention in HBV-HCC.The clinical application value of EPHA2 and HSP90AA1 still needs further verification.ConclusionThe results of network pharmacology suggested that GJF can inhibit the proliferation of liver cancer cells through the PI3K-Akt signaling pathway,induce the G0/G1 phase cycle block of HCC cells,and promote the apoptosis of liver cancer cells.We suspect that EPHA2 and HSP90AA1 may be the key targets upstream of PIK3-Akt,as well as the key targets of GJF.The WGBS further verified the important role of the PI3K-Akt signaling pathway and EPHA2 and HSP90AA1 in HBV-HCC.The conclusions of this study can provide a scientific basis for the in-depth molecular mechanism exploration and clinical application of GJF.