Effects Of Lentivirus-mediated RPL31 Gene Silencing On The Proliferation,migration And Apoptosis Of Colon Cancer Cells

Objective:The effect of RPL31 gene on the proliferation,migration and apoptosis of colon cancer cells in vitro was investigated by targeting the silencing of its gene expression through transfection with RPL31-interfering lentiviral vector.Methods:HCT116 and RKO colon cancer cells with high RPL31 expression were selected as the target cells for RPL31 gene silencing experiments.According to the RPL31 gene,two different RNA interference sequences were designed,and two RNA interference lentiviral vectors,LV-sh RPL31-1 and LV-sh RPL31-2,carrying the green fluorescent protein gene and the purinomycin resistance gene,as well as a negative control lentiviral vector,LV-sh Ctrl,were constructed to verify the packaging and infect target colon cancer cell lines.The colon cancer cells infected with two kinds of sh RPL31 lentiviral vectors were taken as the experimental group sh RPL31-1 and sh RPL31-2,and the colon cancer cells infected with negative lentiviral vectors were taken as the negative control group sh Ctrl.Fluorescence microscopy was used to observe the infection status of colon cancer cells in each group,and q RT PCR was used to detect the expression of RPL31 m RNA in colon cancer cells.The MTT method was used to continuously observe the changes in the number of cells in each group for5 days using an enzyme-linked immunosorbent assay(ELISA),in order to evaluate the effect of silencing RPL31 gene expression on the proliferation ability of colon cancer cells.The wound-healing assay and transwell assay were used to observe the changes of cell migration after 48 hours and the number of cells penetrating the membrane at 72 hours,respectively,to evaluate the effect of silencing RPL31 gene expression on the migration ability of colon cancer cell migration.To evaluate the effect of silencing RPL31 gene expression on apoptosis in colon cancer cells,cell apoptosis rate was detected by Annexin V-APC single staining method by flow cytometer.Results:After continuous screening with purithromycin,72 hours later,the infection efficiency of cells in each experimental group was observed under a fluorescence microscope to exceed80%,successfully infecting HCT116 and RKO colon cancer cells.The q RT PCR experiment results showed that compared with the sh Ctrl group,the experimental group sh RPL31-1 had a gene knockdown efficiency of 68.7%,while the experimental group sh RPL31-2 had a gene knockdown efficiency of 83.5%,both of which were used for subsequent experiments.The MTT assay results showed that compared with the control group sh Ctrl,in HCT116 and RKO cells silencing RPL31,the doubling of cells in the experimental group was significantly reduced from day 3 and day 2,respectively.The results of wound-healing assay showed that compared with sh Ctrl in the control group,in HCT116 and RKO cells silencing RPL31,the cell migration rate of the experimental group was reduced.Transwell assay showed that compared with sh Ctrl in the control group,the cell migration rate of the experimental group was significantly reduced in HCT116 and RKO cells that silenced RPL31.The flow cytometry detection results showed that compared with the control group sh Ctrl,the experimental group showed an increase in the number of apoptotic cells in HCT116 and RKO cells silencing RPL31.Conclusions:Silencing the expression of RPL31 gene can inhibit the proliferation and migration ability of HCT116 and RKO colon cancer cells,and promote apoptosis.