The Effects And Mechanisms Of The MiRNA On The Proliferation And Invasion In Cripto-1 High-expressing Tumor Cells

Cancer is a malignant disease with high morbidity and mortality,its mechanism is not yet clear.Cripto-1 is the original member of the epidermal growth factor(EGF)-Cripto-1-FRL-1-Cryptic(CFC)family,also known as teratomas derived growth factor-1(TDGF-1).It has been proved that Cripto-1 is highly expressed at the early stage of embryonic development,not expressed or low expressed in normal tissues,but re-expressed in cancer cells and can promote cell proliferation,migration,invasion,epithelial-mesenchymal transformation and tumor angiogenesis.It indicates that Cripto-1 is closely related to tumor angiogenesis,thus further study of its expression regulating mechanisms is of great significance.miRNA,a group of small non-coding RNA,can specifically bind to 3’UTR of target mRNA to silence gene expression by inhibiting translation or degradation of the mRNA.It plays an important role in the regulation of gene expression and cancer tumorigenesis,but whether miRNA involved in regulating the expression of Cripto-1 protein is unclear.In this study,we used common databases including TargetScan,PicTar,miRBase Targets and other bioinformatics software to forecast potential miRNA binding sites on Cripto-1 3’UTR.We screened the miRNA which can regulate the activity of Cripto-1 mRNA 3’UTR by luciferase activity assay,and explore the possible function and molecular mechanisms of the miRNA on the tumor promoting effect of Cripto-1,following results were obtained.1.Prediction and identification of mi RNA targeting Cripto-1 mRNA 3’UTR.We used bioinformatics software and Cripto-1 mRNA 3’UTR driven luciferase activity reporting system to screen the miRNA that regulating Cripto-1 expression,and three mi RNAs were identified,namely mi R-1285-5p,mi R-4776-3p and miR-3929.Then we confirmed that the three miRNAs can inhibit the expression of Cripto-1 at mRNA and protein levels by the qRT-PCR and western blot analysis.Then we carried out the reverse validation assay of miRNA targeting Cripto-1.The three miRNAs and Cripto-1 expression vectors(not included in the 3’UTR region)were transfected into HeLa cells simultaneously.Result of western blot showed that the over expression of Cripto-1 was not affected by the miRNA.It is further proved that the three miRNAs regulate Cripto-1 expression by interacting with the Cripto-1 mRNA 3’UTR.2.The effects of the miRNA on Cripto-1 high-expressing tumor cells.In order to detect the biological activity of the three miRNAs,we first detected the effect of miRNA on the cell viability of Cripto-1 high-expressing tumor cells by MTT.The results showed that miR-1285-5p,miR-4776-3p and miR-3929 can inhibit the cell activity of human cervical cancer cells(HeLa).BrdU incorporation assay and Ki67 immunofluorescence assay showed that miR-1285-5p,miR-4776-3p and miR-3929 have inhibitory effect on the proliferation of human cervical cancer cells(HeLa).Boyden chamber assay and wound healing test showed that miR-1285-5p,miR-4776-3p and miR-3929 can inhibit cell invasion and migration ability of human cervical cancer cell(HeLa).3.The mechanisms of the miRNA regulating the proliferation and invasion in Cripto-1 high-expressing tumor cells.Results from DAPI staining and AnnexinV-FITC/PI double staining by flow cytometry showed that the three miRNAs can induce apoptosis of Cripto-1 high-expressing tumor cells.Further results from western blot revealed that the three miRNAs can upregulate the levels of cleaved caspase-9 and cleaved caspase-3,and downregulate the expression of Bcl-2/Bax ratio,suggesting that the three mi RNAs can promote the apoptosis of HeLa cells through the mitochondrial pathway.Results from western blot showed that three mi RNAs can increase the expression of CDK2,p27 and decrease the expression of cyclinE in HeLa cells.In order to study the mechanisms of the miRNA inhibiting the migration and invasion,we studied the effect of the miRNA on the expression of EMT related protein by western blot.The results showed that the three miRNAs can promote the expression of E-cadherin,and inhibit the expression of N-cadherin,Vimentin and MMP9,suggesting that the three miRNAs can affect the expression of EMT related protein in HeLa cells.To sum up,our reseach revealed the effects and the mechanisms of the miRNA on the proliferation and invasion in Cripto-1 high-expressing tumor cells,and laid a solid theoretical foundation for tumor diagnosis and treatment targeting Cripto-1.