| Objective: The mechanism of microRNA-593-5p(miR-593-5p)targeting Paul-like kinase1(PLK1)in regulating the occurrence and development of gastric cancer cells is not clear.The purpose of this study is to observe the expression of miR-593-5p and PLK1 in gastric cancer cells in vitro,and the effect of targeted PLK1 on the biological function of human gastric cancer cells after overexpression and inhibition of miR-593-5p,respectively,so as to provide new insights and ideas for revealing the molecular pathogenesis of gastric cancer,and provide a theoretical basis for exploring a potential tumor marker and a new tumor therapeutic target.Methods:(1)Four human gastric cancer cell lines(MGC-803,AGS,HGC27,MKN-45)and a normal human gastric mucosal epithelial cell line(GES-1)were cultured.(2)Reverse Transcription-Polymerase ChainReaction(RT-PCR)was used to detect theRNA expression of miR-593-5p and PLK1 in the above cell lines,and a cell line with high expression of miR-593-5p and a low expression of miR-593-5p was selected as tool cells for transfection and functional experiments.(3)The bioinformatics website Target Scans Human7.2 was used to detect whether there were binding sites between miR-593-5p and PLK1 genes.(4)Gastric cancer tool cells were transfected with liposomes,and the constructed miR-593-5p mimic and its miR-593-5p mimic negative control(mimic NC)were transiently transfected into miR-593-5p respectively In the cell line MGC-803 with low expression,the miR-593-5p inhibitor and its inhibitor negative control(inhibitor NC)were transiently transfected into the cell line MKN-45 with high miR-593-5p expression In this study,gastric cancer cells overexpressing and inhibiting miR-593-5p were constructed.The fluorescent effect of transfection was observed under a fluorescent microscope,andRT-PCR technology was used to verify and detect whether miR-593-5p was successfully transfected.(5)Western blotting(WB)was used to detect the expression of PLK1 protein in gastric cancer cell lines MGC-803 and MKN-45 after overexpression and inhibition of miR-593-5p,respectively.(6)Scratch test,Transwell test,CCK-8 test and flow cytometry were used to detect the effects of successful overexpression and inhibition of miR-593-5p on the migration,invasion,proliferation and apoptosis of gastric cancer cells.Results:(1)RT-PCR results showed that the expression of PLK1 mRNA in HGC27,MGC-803,AGS and MKN-45 gastric cancer cell lines was significantly higher than that in normal gastric mucosal cells(GES-1).The expression of miR-593-5p in the above four gastric cancer cell lines was significantly lower than that in GES-1,in which the expression of miR-593-5p in MGC-803 cell line was the lowest and that in MKN-45 cell line was the highest.These two cell lines were used as our tool cells for transfection and a series of functional experiments.(2)The corresponding 3’UTRs binding site of miR-593-5p and PLK1 gene was detected by bioinformatics website Target Scans Human 7.2.(3)After miR-593-5p mimic and mimic NC were transfected into gastric cancer cell line MGC-803 by liposome,RT-PCR detection showed that the expression of miR-593-5p in mimic group was significantly higher than that in blank control group and mimic NC group,indicating that the transfection was successful.After miR-593-5p inhibitor and inhibitor NC were transfected into gastric cancer cell line MKN-45,the results ofRT-PCR detection showed that the expression of miR-593-5p in inhibitor group was significantly lower than that in blank control group and inhibitor negative control group,indicating that the transfection was successful.(4)WB results showed that the expression of PLK1 protein in MGC-803 cell line mimic decreased significantly after overexpression of miR-593-5p in gastric cancer cell line MGC-803,while the expression of PLK1 protein in MKN-45 cell line inhibitor increased significantly after inhibition of miR-593-5p in gastric cancer cell line MKN-45.(5)The results of cell scratch assay showed that after miR-593-5p mimic was transfected into gastric cancer cell line MGC-803,the migration ability of gastric cancer cells in MGC-803 cell line mimic group was lower than that in control group,on the contrary,after transfection of miR-593-5p inhibitor into gastric cancer cell line MKN-45,the migration ability of gastric cancer cells in MKN-45 cell line miR-593-5p inhibitor group was enhanced compared with the control group.(6)The results of Transwell assay showed that after successfully transfected miR-593-5p mimic in gastric cancer cell line MGC-803,the invasive ability of gastric cancer cells in MGC-803 cell line decreased compared with the control group,on the contrary,after successful transfection of miR-593-5p inhibitor in gastric cancer cell line MKN-45,the invasive ability in MKN-45 cell line inhibitor group was enhanced compared with the control group.(7)The results of CCk-8 assay showed that after successful transfection of miR-593-5p mimic in gastric cancer cell line MGC-803,compared with the control group,the proliferation ability of mimic group in MGC-803 cell line decreased,on the contrary,after successful transfection of miR-593-5p inhibitor in gastric cancer cell line MKN-45,the proliferation ability of MKN-45 cell line inhibitor group was enhanced compared with the control group.(8)The results of flow cytometry showed that after miR-593-5p mimic was successfully transfected into gastric cancer cell line MGC-803,compared with the control group,the apoptosis rate of gastric cancer cells in MGC-803 cell line mimic group increased,on the contrary,after successful transfection of miR-593-5p inhibitor in gastric cancer cell line MKN-45,the apoptosis rate of gastric cancer cells in MKN-45 cell line inhibitor group decreased compared with the control group.Conclusion:(1)Compared with normal gastric mucosal epithelial cells GES-1,the expression of PLK1 mRNA is higher in gastric cancer cell lines,while the expression of miR-593-5p is lower in gastric cancer cell lines.(2)In MGC-803 gastric cancer cell line transfected with miR-593-5p mimic,the expression of miR-593-5p increased,the expression of PLK1 protein decreased,the ability of cell migration,invasion and proliferation decreased,and the apoptosis rate increased.(3)In MKN-45 gastric cancer cell line transfected with miR-593-5p inhibitor,the expression of miR-593-5p decreased,the expression of PLK1 protein increased,the ability of cell migration,invasion and proliferation enhanced,and the apoptosis rate decreased.(4)There may be a targeted relationship between miR-593-5p and PLK1,and miR-593-5p may regulate a series of biological functions in gastric cancer cells by targeting inhibition of PLK1 expression. |
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