| Objective:To elucidate the molecular mechanism of active ingredient sanguinarine inhibits the proliferation of human gastric cancer cell line BGC-823 in vivo and in vitro,through miRNAs post transcriptional regulation mechanis,which provides a certain experimental basis for the research and development of new drugs for the treatment of gastric cancer with miRNAs as the target.Methods:1.The inhibitory effects of different concentrations of SAN on the proliferation of human gastric cancer cell line BGC-823,human breast cancer cell MCF-7,human colon cancer cell LS-180,human cervical cancer cell Hela and human hepatoma cell HepG-2were detected by CCK-8 assay.SAN sensitive tumor cell lines were screened by calculating the inhibition rate and IC50 value,and the effect of SAN on the proliferation of gastric cancer cells in vitro was verified on 3 kinds of gastric cancer cell lines（MGC-803,BGC-823,SGC-7901）.2.The model of subcutaneous transplantation of BGC-823 cells in nude mice was constructed,and 5-fluorouracil（5-FU,50 mg/kg）and SAN（2.5 mg/kg,5 mg/kg,10mg/kg）were given respectively,the intraperitoneal injection for 4 weeks and 1 times for 3days,and the effect of SAN on the volume,weight and histopathological morphology of the transplanted tumor was observed by HE staining and other techniques,to observe the inhibitory effect of SAN on proliferation of BGC-823 cells in vivo.3.The differential expression of miRNAs and mRNAs in normal gastric mucosal epithelial cells GES-1,gastric cancer cells BGC-823 and BGC-823 treated with SAN cells was obtained by high throughput screening by gene chip technology,miRNAs and mRNAs were expressed in BGC-823 cells after 24h,and miRanda database was used to predict the target gene of differential miRNAs.The key differential miRNAs and its target gene were analyzed by signal pathway（pathway）.4.The expression of 8 key differential miRNAs and target mRNAs in normal gastric epithelial cells GES-1 and gastric cancer cell BGC-823,and the change of the expression level of the differential gene before and after the action of SAN were tested by real time fluorescence quantitative RT-PCR.5.The Immunohistochemistry was used to detect the common target gene MAP4K4of key differential miR-96-5p and miR-29c-3p,and its downstream phosphorylated MEK4and JNK1 protein expression level,and the molecular mechanism of SAN inhibition of BGC-823 cell proliferation was preliminarily studiedResults:1.Inhibitory effect of SAN on the proliferation of tumor cells in vitroThe effect of SAN after action 24 h could obviously inhibit the proliferation of 5human tumor cells in a concentration dependence.The IC50 of BGC-823 in human gastric cancer cells was significantly lower than that of the other 4 tumor cells;the IC50 of SAN action 24h and 48h on BGC-823 cells was significantly lower than that of human gastric cancer cells MGC-803 and SGC-7901.2.Effect of SAN on BGC-823 cell xenografts in nude miceThe SAN intervention of different doses（5 mg/kg and 10 mg/kg）for 4 weeks could significantly inhibit the growth of xenografts in nude mice of gastric cancer BGC-823cells,and the tumor tissue in the SAN intervention group had different degrees of necrosis and apoptosis.3.Effect of SAN on miRNAs expression in BGC-823 cells and bioinformatics analysisCompared with GES-1,there were significant differences in the expression level of291 miRNAs in BGC-823 cells.224 miRNAs expression levels,including miR-96-5p and miR-29c-3p,were significantly changed in BGC-823 cells treated with SAN for 24 h,and the bioinformatics analysis showed that the differential genes were mainly involved in Purine metabolism,Calcium,and MAPK signaling pathway,MAP4K4 is the target genes of miR-96-5p and miR-29c-3p.4.RT-PCR validation of key differences miRNAsThe expression of miR-96-5p and miR-29c-3p was highly expressed in BGC-823cells,and the expression was down regulated by SAN.The expression of CPS1 and PDE1A was highly expressed in BGC-823 cells,and the expression was downregulated after SAN,and MAP4K4 was significantly lower in BGC-823 cells.Up regulation after SAN action,which consistented with the Results of chip detection.5.Effect of SAN on the expression of key protein of MAPK signaling pathway in nude mice transplanted tumor tissueSAN significantly increased the expression levels of MAP4K4,P-MEK4 and P-JNK1in a dose-dependent manner.Conclusion:In this study,the inhibitory effects of SAN on the proliferation of human gastric cancer BGC-823 cells were observed at the cell level and animal level,and the mechanism of SAN inhibition of BGC-823 cell proliferation was discussed from the miRNAs angle.The conclusions were summarized as follows:1.SAN can inhibit the proliferation of a variety of human tumor cells in a concentration-dependent in vitro,which is most sensitive to human gastric cancer BGC-823cells,and can inhibit the proliferation of BGC-823 cells in vivo and in vivo.2.SAN may reduce the target inhibition of target gene MAP4K4 by downregulating the expression level of miR-96-5p and miR-29c-3p in BGC-823 cells,and indirectly increase the expression level of MAP4K4.3.SAN can increase the expression of MAP4K4 and its downstream P-MEK4 and P-JNK1 protein in the transplanted tumor tissue of nude mice,and play an antitumor effect by activating the MAPK signaling pathway. |
