Effects Of MiR-3162-5p On Proliferation,Migration And Invasion In Cervical Cancer SiHa Cells And Its Target Genes

Objective: To investigate the effects of miR-3162-5p on Proliferation,Migration and invasion in human cervical cancer(Cervical cancer,CC)SiHa cells.Bioinformatics predicted the potential target genes of miR-3162-5p,screened high-score pathways of target genes enrichment,and preliminarily verified the potential target genes of miR-3162-5p in RAS signaling pathway.Methods: First,we constructed recombinant lentiviruses miR-3162-5p mimic(miR-3162-5p-LV),miR-3162-5p mimic negative control(LV-NC),miR-3162-5p inhibitor(miR-3162-5p)-IN),miR-3162-5p inhibitor negative control(IN-NC),then transfected them into cervical cancer SiHa cells,respectively.The difference of proliferation ability of SiHa cells transfected with miR-3162-5p-LV,LV-NC and miR-3162-5p-IN,IN-NC was compared by CCK-8 method.Scratch assay was used to compare the migration and invasion abilities of SiHa cells after transfection of miR-3162-5p-LV,LV-NC and miR-3162-5p-IN and IN-NC.Transwell assay was used to compare the migration and invasion ability of SiHa cells after miR-3162-5p-LV,LV-NC and miR-3162-5p-IN,IN-NC.MicroRNA database such as miRBase,miRWalk,miRTarBase,et al.was searched,and then the online bio-software Targetscanwas used to predict possible target genes of miR-3162-5p.Signal pathway websites,such as KEGG,STRING,COREMINE,were searched,and the threshold of signal pathway was analyzed by bioinformatics software microRNA pathway,and the signal pathway with the highest number of target gene enrichment was selected.Look for all the genes on the RAS signaling pathway on the KEGG.Oncomine microarray database was used to investigate the differential expression of all the genes that could be searched on KEGG in cervical cancer tissues and non-cancer tissues in the signal pathway with the largest number of target genes enriched.The protein-protein interaction network(PPI)and module analysis were constructed by STRING and Cytoscape.According to the clinical analysis of GEPIA online database,the Kaplan-Meier curve of GEPIA was used to search the overall survival rate and disease-free survival rate of the patients with CC one by one,and the key genes with statistical significance were screened out.According to the survival analysis,for statistically hub genes,the expression of these genes in CC cancer tissues and non-cancer tissues was preliminarily verified by immunohistochemical streptomyces antibiotic protein-peroxidase binding method.RASA4 is a member of the RasGAP family that acts on the GDP/GTP cycle in the RAS signaling pathway.The difference of RASA4 protein expression in SiHa cells transfected with miR-3162-5p-LV,LV-NC,miR-3162-5p-IN,IN-NC was verified by Western blot.Results: After transfection with miR-3162-5p-LV,the proliferation,invasion and migration ability of SiHa cells were increased.After transfection with miR-3162-5p-IN,the proliferation and invasion of SiHa cells were decreased.The proliferation ability of SiHa cells transfected with miR-3162-5p-LV was increased by CCK-8 method(p < 0.05),and theproliferation ability of SiHa cells was decreased by miR-3162-5p-IN transfection(p < 0.05).The migration and invasion ability of SiHa cells transfected with miR-3162-5p-LV was increased by scratch assay(p < 0.05),but decreased after transfection with miR-3162-5p-IN(p < 0.05).Integrate each microRNA pathway online database analysis,the results showed that the number of miR-3162-5p target genes enriched in RAS signaling pathway was the largest(34).Oncomine microarray databases screened out differentially expressed genes of RAS signaling pathway between cervical cancer tissues and normal cervical tissues.There were 77 differentially expressed genes screening,including 31 upregulated genes,42 downregulated genes and 4 genes with either upregulation or downregulation in different data sets of Oncomine microarray database.Through(PPI)and module analysis of protein-protein interaction network constructed by STRING and Cytoscape,24 hub genes differentially expressed in the RAS pathway between cervical cancer and non-cancer tissues were obtained.The results showed that the overall survival rate of cervical cancer patients with FGFR2,GNG7,IGF1,GNB5,PTPN11 and VEGFA mutations was poor,while the disease-free survival rates for patients with KITLG and PTPN11 mutations were lower.Immunohistochemical results showed that there was a tendency of high expression of PTPN11 in cervical cancer tissues.Western blot showed that the expression of RASA4 in the SiHa cells transfected with miR-3162-5p-LV was reduced,and the expression of ASA4 in the SiHa cells transfected with miR-3162-5p-IN was increased.Conclusion: MiR-3162-5p can promote the proliferation,invasion and migration of human cervical cancer SiHa cells.MiR-3162 / 5p regulates the RAS signaling pathway to promote the carcinogenesis,development and metastasis of cervical cancer.PTPN11,RASA4 may be the target genes of RASsignaling pathway regulated by miR-3162-5p.The low expression of RASA4 and the high expression of PTPN11 may be one of the mechanisms of miR-3162-5p regulating RAS pathway to promote the carcinogenesis,development and metastasis of CC.