Study On The Expression And Regulation Mechanism Of Autophagy Mediated By MiRNA-155-3p In Colon Cancer Resistant Cells

Objectives To study the differential expression of miRNA-155-3p mediated autophagy in human colon cancer cells(SW480,HCT-15,HT-29,HCT-116)and colon cancer cells resistant to oxaliplatin(SW480/OXA,HCT-15/OXA,HT29/OXA,HCT116/OXA),and to study the proliferation,migration,invasion biological behavior and autophagy regulation drug resistance mechanism in cell lines with significant miRNA differential expression,so as to explore new fields for further effective methods to reverse tumor drug resistance.Methods 1 Human colon cancer cell lines(SW480/OXA,HCT-15/OXA,HT29/OXA,HCT116/OXA)resistant to oxaliplatin(OXA)were established by conventional culture of human colon cancer cells(SW 480,HCT-15,HT-29,HCT-116/OXA)using a combination of sustained action of drugs and intermittent induction of large doses of drugs.The cell resistance index was detected by CCK-8 method.2 Real-time fluorescence quantitative PCR was used to confirm the expression difference of miRNA-155-3p between the parent and drug-resistant colon cancer cells,and the cell line with the greatest expression difference was selected.3 Select the cell line SW480 with the greatest expression difference to carry out the following experiments.Transfect antisense nucleotides with different expression miRNA into drug-resistant cells SW480/OXA(knockout)by using RNA interference technology.According to different transfection substances,divide them into interference group,negative control group and blank control group.Cells in interference group are transfected with miRNA-155-3p inhibitor recombinant plasmid vector,cells in negative control group are transfected with meaningless sequence plasmid vector,and cells in blank control group are added with the same amount of PBS solution.4 RT-q PCR method was used to detect the transcription level of miRNA-155-3p,Western blot method was used to detect the expression level of autophagy-related proteins,and Transwell experiment was used to detect the cell migration and invasion ability.Results 1 Oxaliplatin-resistant colon cancer cell lines were successfully established over a period of three months.According to the drug resistance index,the drug-resistant cell lines were moderately drug-resistant,which were respectively named as cell lines SW480/OXA,HCT-15/OXA,HT29/OXA,HCT116/OXA.2 According to the results of real-time fluorescence quantitative polymerase chain reaction,miRNA-155-3p is highly expressed in colon cancer resistant cell lines.3 WB method was used to detect the expression level of autophagic protein in drug-resistant cell lines.Compared with the parent strain,the expression level of autophagic protein in drug-resistant cell lines increased(p<0.05),with statistically significant difference.4 after silencing miRNA-155-3p,compared with negative control group and blank control group,the expression levels of miRNA-155-3p and autophagy protein in cells of interference group are lower,and the difference is statistically significant(p<0.05).5 after silencing mi R-155-3p,compared with negative control group and blank control group,the number of cells migrating and invading in interference group was lower,and the difference was statistically significant(p<0.05).Conclusions 1 miRNA-155-3p reduces the resistance of colon cancer cells to oxaliplatin by inhibiting the activation of autophagy.2 Silencing miRNA-155-3p can inhibit the migration and invasion of colon cancer drug-resistant cells.Figure9;Table3;Reference 129...