| ObjectivesTo study the effect of miR-7-5p gene on the proliferation and migration of gastric cancer cells,and to further explore the role of miR-7-5p in gastric cancer is related to the RAF1/MEK/ERK pathway.Treatment provides new theoretical basis.Methods(1)Use NCBI-pubmed,Targetscan,Star Base and miRBase databases and bioinformatics technology to find relevant miRNAs,predict and screen their target genes.(2)The expression level of miR-7-5p in human gastric cancer SGC-7901 cells was detected by real-time quantitative PCR.(3)SGC-7901 cells were transfected with miR-7-5p mimics,miR-7-5p mimics NC,miR-7-5p inhibitor,miR-7-5p inhibitor NC,and transfection group was detected by Real-time PCR.Whether miR-7-5p transfection was successful,compare the transfection efficiency at 24,36,and 48 h,select the optimal transfection time,and detect the target gene RAF1 expression.(4)The proliferation activity of human gastric cancer cell SGC-7901 was detected by MTT method under different conditions.(5)Hoechst33258 nuclear staining experiment to observe the apoptosis of human gastric cancer cells under different conditions.(6)Flow cytometry was used to detect the cycle changes and apoptosis of human gastric cancer cells.(7)Wounding healing assay to detect the migration of humangastric cancer cells under different conditions.(8)Western-blot detected RAF1,p-RAF1,MEK,p-MEK,ERK,p-ERK,Bax,Bcl-2,caspase3 and caspase9 protein expression levels in gastric cancer cells.Results One.Screening and verification of miRNA and target genes1.Through the NCBI-Pubmed database,27 miRNAs and 66 genes in gastric cancer tissues and cells were found in the MAPK family;Targetscan analysis showed that four pairs of them have good relationship with genetic targets and genetic conservation(Context ++ Score percentile≥80,PCT>0.4).KEGG pathway enrichment showed that RAF1 as a target gene of miR-7-5p affects the occurrence and development of gastric cancer through the MAPK-ERK signal transduction pathway.2.The expression of miR-7-5p in human gastric cancer cell line SGC-7901 was significantly lower(P <0.05).After SGC-7901 cells were transfected with miR-7-5p mimics,the expression level of miR-7-5p was significantly increased,and 48 h transfection efficiency was the highest.3.After treating cells with miR-7-5p mimics and RAF1 inhibitor separately,the RAF1 expression level of over-expressed miR-7-5p gene was significantly down-regulated(P <0.05),and the expression level of silent RAF1 gene miR-7-5p Significantly up-regulated(P <0.05).Prove that there is a cross-target relationship between RAF1 and miR-7-5p.Two.miR-7-5 promotes apoptosis of human gastric cancer cell line SGC-79011.For cells overexpressing miR-7-5p,the percentage of G0/G1 cells was75.94%,and the percentage of G2/M cells was 5.40%.The cell division process was significantly slower than that of the control group(P <0.05).2.The cells overexpressing miR-7-5p group had a slower migration rate,while the miR-7-5p silencing group had a significantly faster migration rate(P<0.05).3.The apoptosis rate of the over-expressed miR-7-5p group was significantly increased,while that of the silenced miR-7-5p group was significantly decreased(P <0.05).4.The expression levels of caspase3 and caspase9 protein in the over-expressed miR-7-5p group were significantly increased,and the ratios of Bcl-2/Bax,P-RAF1/RAF1 were significantly reduced(P <0.05),while the miR-7-5p silent group did the opposite.Three.miR-7-5p induces gastric cancer SGC-7901 cell apoptosis through RAF1/MEK/ERK signaling pathway1.After miR-7-5p mimics and ERK inhibitors interfered with SGC-7901 cells for 24 hours,the apoptosis rates were 18.71% and 27.3%,and the combined apoptosis rate was 30.7%.Apoptosis was significantly accelerated and the combined effect is strongest(P <0.05).2.After 24 hours of transfection and drug administration,the cell migration rate slowed down;miR-7-5p mimics combined with ERK inhibitors had the slowest cell migration rate.3.24 hours after transfection and administration,the expression levels of caspase3 and caspase9 protein in human gastric cancer SGC-7901 cells were significantly increased.The ratios of Bcl-2/Bax,P-RAF1/RAF1,P-MEK/MEK,and P-ERK/ERK were significantly reduced(P <0.05),and the combination intervention group had the strongest effect.Conclusion1.Mi R-7-5p plays an important inhibitor role in the proliferation and migration of human gastric cancer SGC-7901 cells.2.The over-expressing miR-7-5p and after miR-7-5p silencing,the two conditions are completely opposite,indicating that miR-7-5p may inhibit cell proliferation and migration through targeted regulation of RAF1 expression in gastric cancer.3.Both miR-7-5p mimics and ERK inhibitors can inhibit the proliferation and migration of gastric cancer cells.The combined effect is the strongest.In this case,the expression level of apoptosis-related proteins increases,and the expression of negative apoptosis-related proteins is reduced;After miR-7-5p was silent,all indicators were just the opposite.This indicates that miR-7-5p may inhibit the activation of MEK/ERK signal transduction pathway by negatively regulating RAF1,and thus inhibit the proliferation and migration of human gastric cancer SGC-7901 cells. |
