Discussion On The Potential Mechanism Of Shentao Ruangan Prescription(STR)in The Treatment Of Hepatitis B Associated Liver Cancer Based On High-throughput Sequencing And Network Pharmacology

Backgrounds Liver cancer is the world’s one of the most common malignant tumor.As its high malignant degree,poor prognosis,the diagnosis and treatment of liver cancer is still a huge challenge.With the progress of the molecular immunology researches,the treatment of liver cancer had been renewed.However,the embarrassing situation of the low response rates,poor adverse reactions still have not been removed.As the Traditional Chinese medicine in the treatment of liver cancer show certain advantages,which is becoming more and more prominent,however there still are series of questions,such as the complex compositons and unclear mechanisms.Based on this,it is urgent to search for biomarkers with high sensitivity and high specificity,which are closely related to the incidence of liver cancer.Objects Shentaoruangan formula(STR),an empirical formula of professor Zhou daihan,has the effect of strengthening spleen,smoothing liver and dissipating mass.It has been used in clinical practice for decades and has achieved good curative effect.However,due to the complexity of traditional Chinese medicine herbs,efficacy mechanisms of which still unclear,which limit the promotion and application in clinical practice.This study aims to screen the components of STR and targets of hepatitis B virus associated hepatocellular carcinoma(HBV-HCC)by network pharmacologyobserve and GEO databases to establish "drug-the active ingredient-key targets-channel" network diagram.Then observing efficacy of STR by cell experienments and establishing a subcutaneous tumor model in nude mice,and further to find differentially expressed genes(DEGs)through high-throughput sequencing technology(NGS)and bioinformatics comprehensive analysis,so as to identify the potential targets and possible mechanism of action of STR in the treatment of HBV-HCC.Methods 1.Through retrievaling TCMSP database to screen the chemical compounds of STR,according to the oral bioavailability(OB)≥30% and drug likeness(DL)≥0.18,meanwhile combined the results of mass spectrometry of STR.The key targets obtained by STR and the corresponding target genes HBV-related HCC from GEO database.The "cluster Profiler" installation package in R software was used to carry out GO function and KEGG pathway enrichment analysis,and the network diagram of "Traditional Chinese medicine-chemical composition-Key target-pathway" was constructed.2.MTT method was used to observe the effect of STR on the proliferation of human liver cancer cell lines Hep G2.2.15,Hep G2 and MHCC97-H,and cell scratch and Transwell experiment were used to observe the effect of STR on the migration and invasion ability of the three human liver cancer cell lines.3.To establish a human liver cell line Hep G2.2.15 nude mice Subcutaneous neoplasia model,the mice were randomly divided into 5 groups,namely STR low,medium and high dose groups,negative control group,positive control group respectively when volumes of tumors were 100-200mm3,and started to intragastric administration for 25 days in a row by once a day.Observing the growth situation,measuring tumors weight had accumulated and nude mice.At the end of the last day of administration,blood was taken from the eyeball,tumor body was removed,tumor weight was measured for statistical analysis,liver and kidney tissues were taken to observe morphological changes by HE staining.4.Three tumor tissue samples from STR groups and the negative control group respectively were selected to proceed transcriptome sequencing(RNA-seq).Then differentially expressed m RNAs were screened through library construction,data filtration and gene differentially expressed analysis,which were expressed in the form of volcano map and clustering heat map,and enriched by GO and KEGG signaling pathways.Meanwhile,three tumor tissue samples were taken from STR group and the negative control group for whole-genome bisulfite methylation sequencing(WGBS).Through library construction,data filtration and differential methylation expression analysis,differential methylation regions(DMRs)and their corresponding genes were screened out.Then overlapping the DEGs and DMRs related genes.5.Eight patients with HBV-related HCC were selected for WGBS to screen the DMRs related genes,and correlation analysis was conducted with sequencing results of animalsamples to find common differential methylation genes,and GO and KEGG analysis was conducted.6.Building visual PPI network from STRING database through Cytoscape software and screen core genes using topological analysis.Through c Bio Portal database related TCGA database of core gene mutation spectrum analysis,verify the core genes m RNA expression level and the correlation of methylation.And testing core genes m RNA expression level through GEPIA database,as well as evaluating the overall servival(OS)and desease free servival(DFS)of core genes in liver cancer prognosis.Results1.Six drugs including American ginseng,peach seed,rhubarb,Salvia miltiorrhiza,Angelica sinensis and Herba cepacia were retrieved from the TCMSP platform database.125 potential compounds were obtained by further screening under the conditions of OB≥30%and DL≥0.18,and 437 protein target genes were obtained after deletion of repeated targets.HBV-related HCC database GSE121248 gene chip was screened out by GEO database,and 888 HBV-HCC genes were screened out.51 common target genes were obtained by matching STR drug targets and GEO database of DEGs,the GO enrichment in 971 entries,KGEE pathway enrichment analysis showed that target genes were enriched in 33 pathway.The metabolic associated pathways were tyrosine metabolism,metabolism of xenobiotics by cytochrome P450,and retinol metabolism pathway.Il-17 signaling pathway,Toll-like receptor signaling pathway,NOD signaling pathway and relaxin signaling pathway were associated with inflammation.Cell cycle related pathways include cell senescence,cell cycle,apoptosis,p53 signaling pathway,etc.Estrogen signaling pathways related to hormone regulation,ovarian steroid generation,etc.VEGF signaling pathway was related to angiogenesis.In addition,the PI3K-Akt signaling pathway and MAPK signaling pathway were widely involved in cell biological behaviors in HCC.2.Cell experiments showed that STR could inhibit the proliferation of human liver cancer cell lines Hep G2.2.15,Hep G2 and MHCC97-H in a time and dose dependent manner.The scratch test showed that the STR could inhibit the migration of Hep G2.2.15,Hep G2 and MHCC97-H cells,Statistical results showed significant difference(12h、24h Hep G2.2.15 scratch test P<0.05,24 h Hep G2 scratch test P<0.05,12h、24h MHCC97-H scratch test P<0.05).Transwell experiments showed that STR could inhibit the invasion of Hep G2.2.15,Hep G2 and MHCC97-H cell lines,compared with the control group,the low-dose、middle-dose and high-dose groups of STR showed significant difference(P<0.05).3.Animal experiments showed that the tumor volume and tumor weight of STR groups were smaller than those of the negative control group,and there were significant differences of medium dose and high dose groups compared with the control group(P <0.05).It indicated that STR could inhibit the tumor growth of Hep G2.2.15 hepatocellular carcinoma in nude mice.4.RNA-seq of tumor tissue samples from tumor-bearing nude mice revealed 157,033 genes,and conditional screening revealed 221 differential expression of m RNA,185 upregulated genes and 36 down-regulated genes.GO functional annotation and KEGG pathways enrichment were performed on 221 differentially expressed genes through STRING online database.GO analysis showed that these genes are mainly involved in cell component movement and cell biological processes.Biological processes(BP)analysis showed that the genetic difference between enrichment in the protein glycosylation,hormone regulation,regulation of hormone secretion,unsaturated fatty acid metabolism,cell matrix adhesion,innate immune response to activate the cell surface receptors signaling pathways,et al.The cell component(CC)results showed that the molecular function of golgi lumen,collagen-containing extracellular matrix,sarcomere,myofibril and other related molecules could be analyzed.Molecular functions(MF)showed that these genes were mainly related to the extracellular matrix structural constituent,calcium-dependent phospholipid binding,lysophospholipase activity and other functions.In addition,enrichment analysis of KEGG pathway found that these differentially expressed genes were related to the PI3K-AKT signaling pathway,IL-17 signaling pathway,TGF-signaling pathway,glutathione metabolism and other pathways.5.WGBS sequencing results of tumor tissue samples from tumor-bearing nude mice detected 80510 DMRs regions with a total length of 8056,280.A total of 8,986 genes were screened out,1,600 hypermethylated genes and 2,160 hypomethylated genes.The DEGs and DMRs related genes were screened by overlapping,and 151 overlapping genes with negative correlation between methylation level and differentially expressed genes were obtained.GO enriched 499 items,and KEGG showed 12 pathways.6.146 common differential methylated genes were obtained by overlapping the WGBS results of clinical HBV-HCC specimens and animal sequencing results,and through topological analysis,10 genes closely related to HBV-HCC were finally screened,namely CD44,LGALS3,ACTA1,LCN2,MUC1,IGFBP3,HAMP,IRS2,PDK4,and BDKRB2.Through external database verification,we found that CD44,LGALS3,LCN2,MUC1,IRS2 and BDKRB2 had carcinogenic effects of Hub genes,while IGFBP3,HAMP and PDK4 were tumor suppressor genes,and ACTA1 was still controversial as a tumor suppressor factor.Clinical samples and animals sequencing common to the difference of gene methylation enrichment analysis showed that the GO enriched 509 items,KEGG enriched 13 signaling pathways.Including closely related with liver fibrosis of the ECM-receptor interaction pathways,IL-17 signaling pathways associated with inflammation,relaxin signal pathway,TGF-beta signaling pathways,complement clotting unicom road,related to the metabolism of lipid regulation and exogenous substances cytochrome P450 way such as metabolism,protein digestion and absorption,and widely involved in the regulation of tumor biology such as PI3K-AKT signaling pathways and c AMP signaling pathways.Conclusion In this study,high-throughput molecular sequencing revealed the differentially expressed genes and pathways of abnormal methylation modification under the action of STR,providing a new idea for the diagnosis and treatment of liver cancer.We found part of oncogenes and tumor suppressor genes,of which CD44,LGALS3,LCN2,MUC1,IRS2,BDKRB2 were supposed to be carcinogenic activity,while IGFBP3,HAMP,PDK4 were regarded as tumor suppressor genes.And genes expression level were in negative correlation relationship between the methylation status.STR may play an anti-liver cancer role by inhibiting the expression of LCN2 and upregulating the expressions of IGFBP3,HAMP and PDK4,which may be potential targets for anti-liver cancer.Compared with the targets and pathways screened by the network pharmacology method,the PI3K-Akt signaling pathway,IL-17 signaling pathway,relaxin signaling pathway,and the metabolism of exogenous cytoplasmic cytoplasmic cytoplasmic P450 metabolized pathway were found,suggesting that STR may play an anti-tumor role by influencing these pathways.