| Objective:The incidence and mortality of gastric cancer are still high in our country.It is of great clinical significance to develop anti-tumor drugs with high efficiency and low toxicity from plant.The mechanisms of the inhibition of nevadensin in gastric cancer have been explored based on transcriptomics and miRNAomics in present work.Methods:1.Pharm Mapper was applied to get targets of nevadensin and bioinformatic analysis based on DAVID online tool was used to explore in which biological and metabolic pathways targets were involved.The inhibitory effect of nevadensin in gastric cancer was studied in vitro and vivo.2.Differentially expressed genes generated by nevadensin treated HGC27 cells were analyzed based on the next-generation sequencing.Functional enrichment and GSEA analysis were performed.And differential alternative splicing,fusion genes and differently expressed exons were analyzed.Real-time quantitative PCR(qRT-PCR)and western blot were conducted to verify the possible mechanisms of the effects of nevadensin on apoptosis,cell cycle,invasion,and migration.Furthermore,the effect of nevadensin on PI3K/AKT signaling pathway was verified.Molecular docking was used to analyze the binding ability of nevadensin to key proteins involved in possible mechanisms above.The expression differences of COL1A1,COL1A2 and other collagen genes in gastric cancer in transcriptome sequencing differential expression profiles were analyzed by TCGA database.Survival analysis of ERBB3,PDGFRB,COL1A1 and COL1A2 was conducted by K-M Plotter online analysis.3.Differentially expressed miRNAs(including known and novel miRNAs)were analyzed based on smallRNA sequencing.The most likely novel miRNAs were screened out.To examine the binding ability of the novel miRNA to AGO2 protein,molecular docking was used.The negative miRNA-mRNA relationship was analyzed by combining transcriptomics and miRNAomics.TCGA database was used to find out the miRNAs that may play a role on the mechanisms of nevadensin in gastric cancer.QRT-PCR was used to verify transcriptional expression of these miRNAs.Results:1.195 target genes of nevadensin were collected and GO and KEGG analysis of the target genes based on DAVID online tool showed that nevadensin may be involved in cell proliferation,invasion,cell cycle,and apoptosis and be related to PI3K/AKT,MAPK,and miRNA pathways in tumors.The result of CCK8 assay showed that nevadensin significantly inhibited the viability of HGC27 and AGS.Also,nevadensin could inhibit the proliferation of HGC27 and AGS from the results of cloning formation assay.The flow cytometry and Hoechst staining showed that nevadensin had obvious induction of apoptosis on HGC27 and AGS.The result of cell cycle analysis by flow cytometry showed that nevadensin induced S-phase arrest in HGC27 cells,however nevadensin had different effects on AGS cells at low concentration and high concentration.In the low concentration group,it could arrest G0/G1 phase,while in the high concentration group,it could arrest G2/M phase.Cell scratch assay and tranwell assay showed that nevadensin could inhibit the invasion and migration of HGC27 and AGS.In vivo,nevadensin could inhibit the growth of transplanted tumor,and had no obvious adverse effect on the mental and health status of nude mice.2.A total of 509 genes,including 389 upregulated genes and 120 downregulated genes,were differentially expressed at the transcriptional level by Next-generation sequencing between nevadensin treated group and control group.Differential genes involved in a variety of GO items,such as endogenous immune response,regulation of cell proliferation,antibacterial response,programmed cell death,DNA binding and transcriptional activity were revealed by using GO analysis.KEGG enrichment analysis revealed that differential genes were significantly involved in tumor-related pathways.GSEA analysis also found that these differently expressed genes were involved in PI3K-AKT.The differentially alternative splicing of 69277 genes was found,including30150 upregulated events and 39127 downregulated events.Further analysis of the top10 upregulated and downregulated genes in the variable splice types of A3 SS,A5SS,MXE,RI,and SE showed that the mRNA transcription levels of ITGB7 and other genes were upregulated.In addition,six new fusion genes were found in control group but not in nevadensin treated group.Differentially expressed exons analysis found that at least one exon of eight gene was differentially expressed(| log2 FC | > 1,FDR < 0.01).The results of transcriptome sequencing exhibited that ERN1 was upregulated at the mRNA level,and qRT-PCR and Western blot showed similar results.Molecular docking showed that nevadensin had stable binding affinity to ERN1.The expression levels of CCNA2 and CDK2 mRNA and protein were decreased in HGC27 after treatment with nevadensin compared with the control group,consistenting with S-phase cell cycle arrest induced by nevadensin.Molecular docking showed that nevadensin had stable binding affinity to CCNA2 and CDK2.In terms of cell invasion,the protein level of E-cadherin was up-regulated,while the protein level of vimentin protein was down-regulated.The expression of PI3K(p85)and AKT was downregulated after treatment with nevadensin.The differential expression analysis of the above genes in TCGA database also found that ERBB3 and PDGFRB were upregulated in a variety of tumors including gastric cancer,and survival analysis found that patients with low expression of ERBB3 and PDGFRB had better prognosis.QRT-PCR and western blot verified ERBB3 and PDGFRB were both downregulated.Molecular docking showed that nevadensin had stable binding affinity to ERBB3 and PDGFRB.The expressions of COL1A1 and COL1A2 were upregulated in gastric cancer,and the prognosis of patients with low expression of COL1A1 and COL1A2 was better.Moreover,the expressions of COL1A1 and COL1A2 may be downregulated by nevadensin.3.The expression levels of 120 miRNAs(65 known miRNAs and 55 newly predicted miRNAs)were significantly changed by smallRNA sequencing after treatment of HGC27 cells with nevadension,of which 81 were upregulated and 39 were downregulated.Among 55 novel miRNAs which were differentially expressed,novel＿miR-608,novel＿miR-607,novel＿miR-114,novel＿miR-317 and novel＿miR-735(all provisionally named)were the most likely novel miRNAs based on the scores.The mature sequences of novel＿miR-607,novel＿miR-114 and novel＿miR-317 from different loci and with different precursor sequences and secondary predictive structures were completely consistent.Molecular docking showed that the mature sequence had stable binding affinity to AGO2.A total of 10 upregulated miRNAs and15 downregulated miRNAs may have negative regulatory relationships with differently expressed genes obtained in transcriptome sequencing.And 10 upregulated miRNAs constitute 63 miRNA-mRNA negative regulatory pairs,and 15 downregulated miRNAs constitute 226 miRNA-mRNA pairs.TCGA database analysis found that miR-210-5p expression was upregulated in gastric cancer,which was correlated with the M stage of gastric cancer.QRT-PCR verification found that miR-210-5p was significantly downregulated after nevadension treatment.The analysis of the predicted target genes of miR-210-5p in the TCGA database showed that four of them were downregulated in gastric cancer.Conclusions:1.Nevadension has an anti-gastric cancer activity,which may provide a new idea for the treatment of gastric cancer.2.Nevadension may play an anti-tumor role in gastric cancer by ERBB3 and PDGFRB/PI3K/AKT signaling pathway.3.Nevadension may regulate known and novel miRNAs. |
