Identification Of The Molecular Targets And Mechanisms Of Compound Mylabris Capsules For Hepatocellular Carcinoma Treatment Through Network Pharmacology And Bioinformatics Analysis

Hepatocellular carcinoma(HCC)is the main primary liver tumor and the4 th leading cause of cancer-related deaths around the word.Although there are many options for HCC therapy at present,such as surgical resection,liver transplantation,radiofrequency ablation,transcatheter arterial chemoembolization,and tyrosine kinase inhibitor therapy,the vast majority of HCC patients are diagnosed at an advanced stage and therefore treatment options are limited.The latest edition of The Guidelines for the Diagnosis and Treatment of Primary Liver Cancer in China clearly recommends the use of traditional Chinese medicine(TCM)and western medicine in different periods of liver cancer,such as perioperative period,postoperative comprehensive treatment period,palliative period and rehabilitation period,to control symptoms,prevent recurrence and metastasis and prolong survival time.Compound mylabris capsules(CMC),a classical TCM formula,is mainly composed of a variety of traditional Chinese medicine components.Modern pharmacological studies have shown that cantharidin,the extract of Mylabris phalerata,and cantharidin analog can inhibit tumor cell growth.However,its clinical application as a monomer drug is limited due to the occurrence of severe side effects.Correspondingly,with the strict compatibility principle and the concept of focusing on the whole,CMC has been proven to have good clinical efficacy and low toxicity in treating HCC patients.However,owing to the complexity of CMC composition and the inadequacy of research methods,the action mechanisms and molecular targets of CMC for treating HCC require further investigation.Similar to other TCM formulas,the therapeutic action of CMC on HCC may involve multi-components,-targets,and-pathways.Network pharmacology is a prominent tool for further revealing the therapeutic mechanisms of TCM formulas,which synthetically explores interrelatedness of drug,targets and disease and displays visually the network of drug-targets-disease.This approach characterizes the detailed mechanisms of a complex drug action,and is compliance with the holistic perspective of TCM.It has demonstrated that the validation steps of the therapeutic mechanism of TCM need to be carried out through cell and molecular biology experiments.Previous studies have suggested that serum pharmacology is more appropriate and scientific enough for studying TCM in cytological experiment.This method not only reduces the interferences of the physicochemical properties of crude drugs on experimental results,but also is closer to the native in vivo environment.In this research,the approaches of network pharmacology and bioinformatics were combined to explore the therapeutic mechanism and molecular targets of CMC on HCC.Moreover,the method of serum pharmacology was used to validate the core molecular targets through cell experiments.Part One Prediction of core molecular targets and mechanisms of compound mylabris capsule for hepatocellular carcinoma treatment based on network pharmacologyObjective: CMC is widely used to treat HCC in clinical practice of traditional Chinese medicine.However,its molecular targets for treatment of HCC are not clear.This research aimed to elucidate the molecular targets and mechanisms of CMC for treating HCC.Methods: First,the bioactive ingredients and potential targets of CMC,as well as HCC-related targets were retrieved from publicly available databases.Next,the overlapped genes between potential targets of CMC and HCC-related targets were determined using bioinformatics analysis.Then,networks of ingredient-target and gene-pathway were constructed.Results: In total,151 bioactive ingredients and 255 potential targets of CMC were selected,982 differentially expressed genes of HCC were identified.Among them,34 overlapped genes were finally selected.In addition,20 pathways and 429 GO terms were significantly enriched.Protein-protein interaction and gene-pathway networks indicated that Cyclin B1(CCNB1)and Cyclin Dependent Kinase 1(CDK1)were the core gene targets for the treatment of CMC on HCC.Conclusions: CMC plays a therapeutic role in HCC via multi-component,-target and-pathway mechanisms,in which CCNB1 and CDK1 may be the core molecular targets.Part Two Analysis of the expression and prognostic significance of core target genes in hepatocellular carcinoma based on bioinformaticsObjective: CCNB1 and CDK1 may be the core molecular targets of CMC for HCC treatment based on network pharmacology.This research aimed to analysis the expression and prognostic significance of CCNB1 and CDK1 in HCC based on bioinformatics.Methods: We mainly used two independent patient cohorts,namely The Cancer Genome Atlas(TCGA)cohort and the Clinical Proteomic Tumor Analysis Consortium(CPTAC)cohort,to analyze the roles of CCNB1 and CDK1 in HCC at the m RNA and protein levels,respectively.Initially the differential expressions of CCNB1 and CDK1 between non-tumor and HCC tissues were validated using a number of publicly accessible databases.Then,the Kruskal-Wallis rank sum test or the Wilcoxon rank sum test as well as logistic regression were employed to analyze the association of CCNB1 and CDK1 expressions with clinical characteristics of HCC.The Cox regression and Kaplan-Meier analyses were conducted to assess the prognostic significance.Results: CCNB1 and CDK1 m RNA and protein expression levels were higher in HCC tissues than in adjacent non-tumor tissues(all P<0.05).BothCCNB1 and CDK1 m RNA and protein expressions were positively associated with clinicopathological characteristics(clinical stage,T stage,pathological grade,tumor size,tumor thrombus)and negatively associated with the survival of HCC patients.Univariate and multivariate Cox regression analysis showed that the expression levels of CCNB1 and CDK1 were two independent prognostic factors in HCC patients.Conclusions: The upregulated expressions of CCNB1 and CDK1 were related to an unfavorable prognosis in HCC.Therefor CCNB1 and CDK1 could be used as therapeutic targets for HCC.Part Three Validation of the core molecular targets of compound cantharidin capsule for hepatocellular carcinoma treatment based on serum pharmacologyObjective: CCNB1 and CDK1 could be used as therapeutic targets for HCC,and they also may be the core molecular targets of CMC for HCC treatment.This research aimed to validate them as the core molecular targets with cell experiments.Methods: Firstly,CMC-medicated serum was prepared based on serum pharmacology and was used to treat human HCC cell line Hep G2.Then,CCK-8 assay was carried to assess the effect of CMC on the proliferation of Hep G2 cells.Quantitative reverse transcription-polymerase chain reaction(q RT-PCR)and western blotting(WB)were performed to detect the expressions of CCNB1 and CDK1.Lastly,to explore whether CMC can affect cell cycle progression in HCC,we examined the cell cycle of Hep G2 cell after treatment with different concentrations of CMC-medicated serum using flow cytometry.Results: in vitro studies showed that CMC-medicated serum significantly inhibited the viability of Hep G2 cells.Furthermore,CMC downregulated CCNB1 and CDK1 expressions and induced G2/M phase cell cycle arrest.Conclusions: CMC plays a therapeutic role in HCC via multi-component,-target and-pathway mechanisms,in which CCNB1 and CDK1 may be the core molecular targets.This study indicates that the integration of network pharmacology and bioinformatics analysis,followed by experimental validation,can serves as an effective tool for studying the therapeutic mechanisms of traditional Chinese medicine.