Target Genes And Mechanism Of Aidi Injection For Lung Squamous Cell Cancer

Background Lung cancer is one of the most epidemic and deadly cancer leading to great burden all over the world.Among them,30% to 40% are squamous cell lung carcinoma(LUSC)with poor prognosis and without efficient targeted drugs.Objectives To analyze the research status and research focus of traditional Chinese medicine in the treatment of lung cancer;to identify the prognosis related and immune related gene markers of patients with LUSC;to filter the possible targets of Aidi Injection in the treatment of LUSC,and to explain the mechanism of Aidi Injection in the treatment of LUSC.Methods 1)Bibliometrics analysis: firstly,systematically search three Chinese databases(CBM,CNKI,Wanfang)and one English database PubMed based on the search strategy from starting to September 23,2019.Then,select studies according to the inclusion and exclusion criteria via Endnote 7.At last,BICOMS and related auxiliary software such as gCluto,Ucient and Netdraw are applied to analyze the research status and research hotspot of traditional Chinese medicine in the treatment of lung cancer;2)bioinformatics analysis: firstly,obtain the RNA-seq,phenotype and survival data of patients with LUSC from TCGA and conduct differential expression analysis,single variable Cox risk ratio regression and Lasso to screen the prognosis related genes.Then,the prognostic index(PI)was obtained.Next,the prediction effect of PI was verified in 7 independent GEO data sets and pooled by meta-analysis.At the same time,the prediction ability of PI was compared with the gene markers in the existing literature.Finally,CIBERSORT and Immport was introduced to explore immune related gene markers and immune cell infiltration;3)network pharmacology research: firstly,through literature review,study the effective components of Aidi injection;secondly,explore the target of the effective components of Aidi injection by network pharmacology to get the "drug-target" network;thirdly,by combining data mining and bioinformatics,get the potential target of Aidi Injection related to prognosis of LUSC,and the "drug-target" sub network related to LUSC was obtained.Finally,through the enrichment analysis of GO and KEGG,as well as the analysis of the function of the core target genes,the biological function of Aidi Injection's target genes was studied,and the potential mechanism of Aidi Injection in the treatment of LUSC was speculated.4)Basic experiment: firstly,the inhibition of Aidi Injection on the growth of NCI-H226 cells was observed by MTT method,and then Annexin V/PI double staining and flow cytometry were used to detect the effects of Aidi Injection on the apoptosis and cell cycle of H226 cells.Finally,the genes and proteins related to p53 pathway,apoptosis pathway and cell cycle pathway,such as p53,Bax,Bcl-2,CASP3,CDK1 and cyclinB1,were detected by qRT-PCR and WB to verify the mechanism of Aidi Injection in the treatment of LUSC.Results 1)Research status and hot spots of traditional Chinese medicine for lung cancer: 6308 studies published in 687 Chinese journals and 370 studies published in 128 English journals were included in the analysis.The first Chinese and English literatures were published in 1977 and 1986,respectively.21 countries and regions participated in the study,but the research center is still in mainland China(accounting for 67.69% of all countries and regions)and the domestic research centers are in Jiangsu,Shanghai and other eastern regions.Although the number of related studies is growing rapidly,the quality is generally not ideal.Chinese studies were mainly published in non CSCD journals,while the influence factors of English journals were generally low.The research focus of traditional Chinese medicine in the treatment of lung cancer are mainly clinical efficacy and basic experimental research.The research objects are mainly advanced non-small cell lung cancer,especially adenocarcinoma of lung(LUAD).2)Gene markers,immune cells and immune related genes for prognosis of LUSC: the phenotype,prognosis and expression data of 20530 mRNA and 1046 miRNA in 533 LUSC samples from TCGA were analyzed by bioinformatics,and 18 RNAs(SCRIB,PLIN2,COBL,TRIB3,RHOF,PAQR5,TGM2,STC2,RPH3 AL,MYEOV,TREM1,TMEM99,S1PR5,STAR,GALNT14,AP3B2,DQX1 and hsa-mir-101-2)were obtained to construct PI,and the hazard ratio(HR)of PI was 3.40(2.33,4.96).And the combined HR was still statistically significant when verified in 7 independent GEO datasets.Then,monocytes and eosinophils were associated with the overall survival(OS)of LUSC and plasma cells,positive natural killer cells and eosinophils were associated with the progression free survival(PFS).Positive natural killer cells may be the protective factors of disease progression(HR = 0.03 < 1,P = 0.0472),and the rest immune cells were prognostic risk factors.Moreover,because of the correlation between A2 M and monocytes(r = 0.212,P < 0.05)and B2 M and positive natural killer cells(r = 0.199,P < 0.05),A2 M and B2 M may be related to the prognosis of LUSC.3)There are 22 effective components in Aidi injection,13 of which and 102 corresponding target genes constitute the "drug-target" interaction network of Aidi Injection.By means of bioinformatics and data mining,the interactive network of 8 active components(cantharidin,coniferoside,promethidine,anthocyanin,syringin,ginsenoside-Rd,ginsenoside-Re and Ginsenoside-Rg3)and 7 core genes(CASP3,chrm3,lta4 h,PDE3A,DPP4,star and HTR3A)were obtained.Finally,because the target genes of Aidi Injection contain TP53,BAX,BCL-2 and CASP3,and their functions are enriched in most cancer-related pathways such as p53 pathway,apoptosis pathway and cell cycle pathway.Thus,it is speculated that one of the possible mechanisms of Aidi Injection in the treatment of LUSC is to activate these pathways.4)First of all,Aidi Injection inhibited the growth of H226 cells,with a positive correlation with the concentration and time of administration.The IC50 of 24 h,48h and 72 h was 101.29mg/ml,47.13mg/ml and 26.09mg/ml,respectively.Then flow cytometry showed that Aidi Injection could induce H226 cell apoptosis and G2/M phase arrest,and showed a positive correlation with the concentration and time of administration.Finally,by qRT-PCR and WB technology,48 hours after administration,the high and low concentrations of Aidi Injection could both upregulate the expression of p53,Bax,Caspase-3 and downregulate the expression of Bcl-2,CDK1 and cyclinB1.Conclusions 1)At present,the research on the treatment of lung cancer with traditional Chinese medicine has been paid more and more attention and the core authors cooperated closely,but the quality of the existing both Chinese and English research is generally low.2)In this study,18 RNAs prognostic gene markers of LUSC were obtained,which could forecast the prognosis of LUSC efficiently.Secondly,eosinophils,A2 M and B2 M might be potential immune related prognostic markers in patients with LUSC.3)Aidi Injection has 22 active components and 102 target genes,among which 8 active components and 7 core genes are related to the prognosis of patients with LUSC.Finally,through the functional analysis of genes,it is speculated that one of the potential mechanisms of Aidi Injection in the treatment of LUSC is to activate the p53 pathway,apoptosis pathway and cell cycle pathway.4)It was confirmed that Aidi injection can inhibit the growth of H226 cells,induce H226 cells apoptosis and G2/M phase arrest.One of the mechanisms of Aidi Injection inhibiting H226 cells was to activate p53 pathway,apoptosis pathway and cell cycle pathway,leading to H226 cells apoptosis and G2/M phase arrest,which played an anti-cancer role.