Investigation Of The Function Of Differentially Expressed MiR-18a-5p And MiR-802 Target Genes In Colorectal Cancer SW480 Cells

In this study,we used miR-18a-5p and miR-802 as research objects to detect the effect of over-expression of mi R-18a-5p and miR-802 mimics on the proliferation and apoptosis of colon cancer SW480 cells,and to predict miR-18a-5p and miR-802 may regulate target genes.To construct a transient eukaryotic expression vector that over-expresses microRNAs and analyze the effects of over-expression of miR-18a-5p and miR-802 and their regulation of target genes involved in the signaling pathway.and the molecular mechanisms of miR-18a-5p and miR-802 in the development of colon cancer were discussed.1.In this study,Lipofectamin?3000 transfection reagent was used to transfect miR-18a-5p and miR-802 mimics into SW480 cells by liposome transient transfection technique.The transfection efficiency at 24h,48h and 72h after transfection was detected.Real-time fluorescence quantitative qRT-PCR results showed that a colon cancer SW480 cell model with transient over-expression of miR-18a-5p and miR-802was successfully established.At 48 h,the relative expression levels of miR-18a-5p and miR-802 mimics transfected into colon cancer SW480 cells were 57.38±8.38 and24.62±4.22,respectively.UsingMTTcolorimetricmethodtodetectthe over-expression of miR-18a-5p-eGFP group,NC-eGFP group,and blank control group transfected colon cancer SW480 cells at different concentrations of mimic（plasmid DNA）50μg/mL、100μg/mL、150μg/mL and 200μg/mL inhibition of the proliferation of colon cancer SW480 cells,at Different Time 12h,24h,48h and72h,The results of MTT assay showed that the plasmid DNA concentration was200μg/mL,and the inhibitory effect was better.Over-expression of miR-18a-5p mimic significantly inhibited the proliferation of colon cancer SW480 cells and proliferation inhibition was dose and time-dependent manner.The proliferation inhibition rates of plasmid DNA at 200μg/mL for 12h、24h、48h and 72h were26.80%,46.37%,66.02%and 74.66%,respectively.There were significant differences in different concentrations at different times of 12h、24h、48h and 72h（P<0.05）.2.By Annexin V-PE/7-AAD double staining method,flow cytometry was used to detect over-expression of miR-18a-5p-eGFP group and negative control（NC-eGFP group）was transfected into SW480 colon cancer cells at different time 24h、48h and72h,after the rate of apoptosis.The results showed that over-expression of miR-18a-5p mimic（mi R-18a-5p-eGFP）transfected into colon cancer SW480 cells induced time-dependent apoptosis.Finally,using real-time fluorescence quantitative qRT-PCR method,β-actin was used as a reference control transfected SW480 cell total RNA,qRT-PCR assay detected mRNA as a template to detect DUSP5,FZD3 and CCND2 gene mRNA transcription levels.The results showed that the transcription levels of DUSP5 and FZD3 m RNA were up-regulated,with the differential expression multiples of 2.15 and 1.74(2^-△△Ct,P<0.01).Moreover,the CCND2 gene mRNA expression was down-regulated and the differential expression was 0.62(2^-△△Ct,P<0.01).The experimental results suggest that over-expression of miR-18a-5p can inhibit the proliferation of SW480 cells and induces apoptosis.The mechanism may be related to overexpression of miR-18a-5p through MAPK、Wnt and PI3K-AKT signaling pathways that target the regulation of the up-regulated expression of DUSP5and FZD3 genes and CCND2 gene down-regulation.3.miR-802 regulated target genes HSPA6,TCF7L2 and NKD1 m RNA transcription level,qRT-PCR experimental results showed that m RNA transcription levels of NKD1 and HSPA6 target genes were up-regulated,the differential expression folds were 2.15 and 1.74(2^-△△Ct,P<0.01),Moreover,the mRNA level of TCF7L2 gene was down-regulated and the differential expression folds was 0.25(2^-△△Ct,P<0.01).The experimental data is consistent with the results of the target gene chip.the change trend of mRNA expression of each gene conformed to the results of the previous chip,further verifying the accuracy of the chip results.miR-18a-5p and miR-802 play a tumor suppressive role in the development of human colon cancer.Therefore,miR-18a-5p and miR-802 are important molecular targets for the treatment of human colon cancer.