Effects Of Let-7e-5p Targeting CDKN1A Gene On Proliferation, Invasion And Cell Cycle Of Endometrial Cancer Cell

Objective: The previous sequencing results confirmed the low expression of let-7e-5p in endometrial cancer Ishikawa cells,and the dual luciferase experiment proved that let-7e-5p targets and negatively regulates the CDKN1 A gene.This article will study the overexpression The effect of let-7e-5p on the biological function of endometrial cancer.Methods:(1)Synthesize Hsa-let-7e-5p mimics and Hsa-let-7e-5p NC,use Lipofectamine TM 2000 to transfer the two into the endometrial cancer Ishikawa-GFP cell line,and transfect for 24 h,After 48 h,Real-time PCR detects the relative expression of hsa-let-7e-5p and target gene CDKN1 A m RNA to verify the titer of mimics.(2)Take the cells in logarithmic growth phase for cell function experiments.The cells are grouped as follows:(1)ISK-NC group: ISK-GFP cells transfected with hsa-let-7e-5p negative control;(2)ISK-let-7e-5p Group: ISK-GFP cells transfected with hsa-let-7e-5p mimics.Fluorescence microscope was used to observe the ISK-GFP fluorescence of cells at 1-5 days and photographed and recorded;CCK8 experiment was used to detect the absorbance of endometrial cancer cell Ishikawa at OD450 nm at 24 h,48h,72 h,96h,120 h,and draw CCK8 proliferation line graph to reflect proliferation Viability;Transwell invasion assay detects the number of Ishikawa cells transmembrane after overexpression of let-7e-5p to reflect the change of invasion ability;uses flow cytometry to detect the cell cycle distribution of Ishikawa cells after overexpression of let-7e-5p.Results:(1)At 24 h and 48 h,the m RNA expression of let-7e-5p in the ISK-let-7e-5p group increased significantly,and the m RNA of CDKN1 A decreased correspondingly,indicating that overexpression of hsa-let-7e-5p can inhibit the target at the m RNA level The expression of gene CDKN1 A,and the overexpression effect of the 24 h group is better,the best transfection time is selected as 24 h,Real-time PCR identified let-7e-5p mimics successfully transfected.p<0.05,the difference is statistically significant.(2)Observation of cell status and CCK8 proliferation curve under a fluorescence microscope: Compared with the ISK-NC group,the cell proliferation ability of the ISK-let-7e-5p group was significantly improved.p<0.05,the difference is statistically significant.(3)Transwell invasion experiment showed that overexpression of let-7e-5p promoted the invasion of Ishikawa cells,p<0.05.(4)The results of cell cycle detection by flow cytometry: the proportion of cells in the G1 phase of the ISK-let-7e-5p group increased,but the change in the S phase was not significant,and the proportion of cells in the G2 phase was relatively decreased.P>0.05,the difference was not statistically significant.Conclusions:(1)It is further verified that Hsa-let-7e-5p negatively regulates the CDKN1 A gene in Ishikawa cells,which is in line with the results of the previous dual-luciferase experiment.(2)Overexpression of Hsa-let-7e-5p significantly improves the proliferation and invasion of endometrial cancer Ishikawa cells,but the effect on the cell cycle is not statistically significant.(3)The results of cell function experiments provide research ideas for the subsequent exploration of the molecular mechanism through which overexpression of let-7e-5p may affect the occurrence and development of endometrial cancer.Objective: To study the possible mechanisms involved in promoting the proliferation and invasion of endometrial cancer cells after expressing let-7e-5p,and hope to provide new biomarkers for the screening and diagnosis of endometrial cancer.Methods:Lipofectamine TM2000 was used to transfer hsa-let-7e-5p mimics and hsa-let-7e-5p NC into ISK-GFP cells respectively.Experiment grouping:(1)ISK-NC group: Ishikawa-GFP transfected with let-7e-5p negative control;(2)ISK-let-7e-5p group: transfected with let-7e-5p mimics Ishikawa-GFP cells.When the cells of each experimental group were in the logarithmic growth phase,real-time PCR was used to determine the molecules related to proliferation,invasion and and epithelial mesenchymal transformation: bcl-2,p53,CDKN1A/p21,mmp-9,E-cadherin,vimentin,Cyclin The relative expression level of D1 m RNA,and the protein expression levels of bcl-2,p53,CDKN1A/p21,mmp-9,E-cadherin,vimentin,and Cyclin D1 were detected by Western blot.Gapdh and U6 are used as internal controls.Results:(1)Real-time PCR results showed that: Compared with ISK-NC group,ISK-let-7E-5P group was significantly higher than ISK-NC group.Let-7E-5p(7.50±0.62)vs(1.00±0.05),Bcl-2(1.48±0.39)vs(0.99±0.12),The m RNA expression levels of Cyclin D1(2.50±0.28)vs(0.96±0.06),MMP-9(2.93±0.47)vs(1.08±0.23)and Vimentin(2.53±0.07)vs(0.94±0.05)were relatively increased.The decreased indexes were CDKN1A/p21(0.43±0.02)vs(1.00±0.04),p53(0.39±0.03)vs(1.04±0.06),and E-cadherin(0.43±0.03)vs(0.95±0.04).(P< 0.05)(2)Western blot showed that: Compared with ISK-NC group,ISK-let-7E-5P group was significantly higher than ISK-NC group.Anti-apoptotic protein Bcl-2(0.1593±0.000)vs(0.086±0.001),Cyclin D1(0.3691 ± 0.000)vs(0.3814 ± 0.000),MMP-9(0.3515±0.000)vs(0.2308±0.000),vimentin(0.3182±0.000)vs(0.2079±0.000)To rise;Cancer suppressor gene CDKN1A/p21(0.2273±0.000)Vs(0.4095±0.000),p53(0.2640±0.000)vs(0.5538±0.000),epithelial marker E-cadherin(0.3910±0.000)vs(0.4312±0.000),the protein expressions of these three indexes were relatively decreased.(P < 0.05)Conclusions:(1)Over-expression of let-7E-5p may promote distant invasion of endometrial cancer cells by regulating the expression of molecules related to epithelial mesenchymal transformation,such as down-regulation of epithelial marker E-cadherin,which maintains epithelial stability,and matrix metalloproteinase MMP-9,which degrades extracellular matrix,while up-regulation of mesenchymal marker vimentin.(2)let-7e-5p may promote the proliferation of endometrial cancer cells and accelerate tumor progression by inhibiting the cell cycle inhibition and pro-apoptosis effect of cyclin kinase inhibitor CDKN1A/p21,and jointly upregulating the expression levels of cell cycle G1 "promoter molecule" Cyclin D1 and anti-apoptotic protein Bcl-2.