| Objective: Micro RNAs(miRNAs),single-stranded small molecule RNAs composed of 20–25 nucleotides,regulate post-transcriptional gene expression via binding to the 3' untranslated region(3'-UTR)of a target gene.Moreover,they can also act as oncogenes or tumor suppressor genes,participating in biological processes,such as embryonic development,cell differentiation,apoptosis,drug resistance,angiogenesis,and tumor metastasis,through specific targets or signaling pathways.Using miRNA high-throughput sequencing and Real Time PCR detection,we found that miR-195-5p expression was significantly down-regulated in cervical cancer tissues compared to normal cervical tissues,and miR-195-5p inhibits cell proliferation and induces cell cycle arrest by targeting cyclin D1 a.Therefore,miR-195-5p is an important entry point for studying the malignant biological behavior of cervical cancer.The purpose of this study was to investigate the effects of miR-195-5p on the malignant biological progress of cervical cancer cells He La and SiHa,and to identify downstream target genes of miR-195-5p,thereby further elucidating the molecular regulatory mechanism of miR-195-5p during cervical cancer invasion and metastasis.Methods: After transfection with miR-195-5p mimics and inhibitor in cervical cancer cells He La and SiHa,their transfection efficiency was detected by Real Time PCR.MTT was used to detect the effect on cancer cell proliferation,scratch effect was used to observe the effect on cancer cell migration ability,Transwell was used to detect its effect on cancer cell invasion ability,TUNEL apoptosis experiment was used to detect cancer cell apoptosis,and Western blot was to detect the expression of EMT-related proteins,such as E-cadhern,Snail,Vimentin,MMP-2,MMP-9 and vascular endothelial growth factor VEGFA,and to detect the apoptosis proteins caspase-3,Bax and Bcl-2,and autophagy related proteins LC3-II and P62.Bioinformatics analysis revealed that YAP1 and ATG9 A were the target genes of miR-195-5p,and double luciferase experiments proved that miR-195-5p binds to YAP1 and ATG9 A 3' UTR,respectively.Subsequently,we constructed YAP1 and ATG9 A overexpression plasmids to test whether their inhibitory effect on miR-195-5p on cervical cancer cells was reversed.Results: In cervical cancer cells HeLa and SiHa,after overexpression of miR-195-5p,the cell proliferation,migration,invasion,and epithelial-mesenchymal transition ability were significantly reduced,but apoptosis and autophagy were increased.In contrast,after inhibiting the expression of miR-195-5p,the cell proliferation,migration,invasion,and epithelial-mesenchymal transition ability were significantly increased,but apoptosis and autophagy were inhibited.The double luciferase reporter vector experiment proved that miR-195-5p can bind to specific sites of 3'UTR of YAP1 and ATG9 A,respectively,and negatively regulate them to inhibit its transcription or translation.After miR-195-5p overexpression,YAP1 mRNA did not change significantly,but the protein level was significantly reduced.In addition,after miR-195-5p overexpression,ATG9 A mRNA and protein levels were significantly reduced.After constructing the YAP1 overexpression plasmid and transfecting He La and SiHa,the cell proliferation viability,migration,and invasion ability were significantly restored.At the same time,after constructing the ATG9 A overexpression plasmid and transfecting HeLa and SiHa,the cell proliferation,migration,apoptosis and autophagy ability were significantly increase.Conclusion: MiR-195-5p inhibits cell proliferation,migration,invasion,epithelial-mesenchymal transition,and induces cell apoptosis and autophagy by targeting YAP1 and ATG9 A.Overexpression of YAP1 and ATG9 A can partially restore the inhibitory effect of miR-195-5p on cervical cancer cells.Therefore,it is clarified that miR-195-5p plays a role as a tumor suppressor gene in the malignant progression of cervical cancer cells,and also provides new ideas and insights for targeted treatment of cervical cancer. |
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