The Study On The Effect Of MiR-632 By The Regulation Of GSK3? Expression In Laryngeal Carcinoma Hep-2 Cells

Part 1 The expression of miR-632 in laryngeal cancer tissues and cells,and its effect on hep-2 biological characteristicsObjective:This section was aimed to study the expression of miR-632 in laryngeal cancer tissues and cells,and its effects on Hep-2 cells.Methods:After screening,tumor tissues and adjacent tissues of 10 patients with laryngeal cancer were collected.Normal human bronchial epithelial cell lines(BEAS-2B)and laryngeal cancer cell lines(SNU899,TU212,and Hep-2)were cultured.miR-632 mimics,miR-632 mimics negative control,miR-632 inhibitor and miR-632 inhibitor negative control were used to transfect Hep-2 cells in the logarithmic phase,respectively.According to the different transfection sequences,Hep-2 cells were named as NC-mimics group,miR-632 mimics group,NC-inhibitor group and miR-632 inhibitor group,respectively.qRT-PCR technology was used to detect the expression of miR-632.MTT assay was performed to measure cells proliferation ability.Plate cloning experiment was conducted to test cells cloning ability.The expression of cyclin D1 and c-myc protein in cells was detected by Western blot analysis.Cells migration ability was performed using cell scratch assays.Transwell assay was used to test cells invasive ability.Results:The relative expression of miR-632 in laryngeal cancer tissues was significantly higher than that in adjacent tissues(P<0.01).Compared with BEAS-2B cells,the relative expression of miR-632 was significantly up-regulated in SNU899,TU212 and Hep-2 cells(P<0.01).The relative expression of miR-632 in Hep-2 cells was the highest.When compared with NC-mimics group,Hep-2 cells of miR-632 mimics group was with significantly higher OD560 value(P<0.01),colony formation numbers(P<0.01),cyclin D1 and c-myc protein relative expression(P<0.01),percentage of migrating cells(P<0.01)and invasion cells(P<0.01).Conclusions:miR-632 was up-regulated in laryngeal cancer tissues and cells,and its up-regulation promoted the proliferation,clone formation,migration and invasion of Hep-2 cells.Part 2 The expression of GSK3? in laryngeal carcinoma tissues and cells,and its effect on hep-2 biological characteristicsObjective:This section was aimed to study the expression of GSK3? in laryngeal cancer tissues and cells,and its effect on laryngeal cancer Hep-2 cells.Methods:Hep-2 cells in the logarithmic growth phase were transfected with GSK3?siRNA and its negative control strand,and the transfected cells were named as si-NC group and si-GSK3? group,respectively.The relative expression of GSK3? mRNA in cells was detected using qRT-PCR.The relative expression of GSK3? protein in cells was detected by Western blot analysis.Cells proliferation ability was measured using MTT assay.Cells cloning ability was tested by plate cloning experiments.Cell migration detection was performed using cell scratch assay.Transwell assay was used to test cells invasive ability.Results:The relative expression of GSK3(3 mRNA was significantly decreased in laryngeal cancer tissues compared with adjacent tissues(P<0.01).Compared with BEAS-2B cells,laryngeal cancer cell lines(SNU899,TU212,and Hep-2)GSK3?mRNA and protein relative expression was significantly decreased(P<0.01).The relative expression of GSK3? mRNA and protein in Hep-2 cells was the lowest.Compared with si-NC group,Hep-2 cells of Hep-2 cells had significantly higher OD560 value(P<0.01),higher colony formation numbers(P<0.01),higher percentage of migrating cells(P<0.01)and invasion cells(P<0.01).Conclusions:GSK3? was down-regulated in laryngeal carcinoma tissues and cells,and its down-regulation promoted the proliferation,clone formation,migration and invasion of Hep-2 cells.Part 3 miR-632 influenced the biological characteristics of Hep-2 cells through targeted regulation of GSK3?Objective:This section was aimed to study the targeting relationship between miR-632 and GSK3?,and the mechanism of action that affected the proliferation,migration and invasion of Hep-2 cells.Methods:The targeted relationship between miR-632 and GSK3? was predicted online by Target Scan.Luciferase reporter assays were used to verify the targeting between miR-632 and GSK3?.Hep-2 cells were co-transfected with miR-632 inhibitor negative control and GSK3? siRNA negative control strand,miR-632 inhibitor and GSK3? siRNA negative control strand,miR-632 inhibitor and GSK3?siRNA,respectively.They were grouped as follows:NC-inhibitor + si-NC group,miR-632 inhibitor + si-NC group,miR-632 inhibitor + si-GSK3? group.The relative expression of GSK3? mRNA in cells was detected using qRT-PCR.Western blot analysis was performed to research the relative expression of GSK3? protein.Proliferation ability of cells was measured using MTT assay.Cloning ability of cells was tested using plate cloning experiments.Cells migration detection was performed using cell scratch assay.Transwell assay was used to test the invasive ability of cells.Results:Target Scan online predictions showed that GSK3? was the target gene of miR-632,and the binding site of them was located in the 3'UTR region.Luciferase reporter assay demonstrated that GSK3? was a downstream target gene for miR-632.Compared with NC-mimics group,the relative expression of GSK3? mRNA and protein in Hep-2 cells of miR-632 mimics group was significantly decreased(P<0.01).Compared with NC-inhibitor group,the relative expression of GSK3? mRNA and protein in Hep-2 cells of miR-632 inhibitor group significantly increased(P<0.01).Pearson correlation analysis showed that there was a negative correlation between the relative expression of miR-632 and GSK3? mRNA in laryngeal cancer.Compared with NC-inhibitor + si-NC group,Hep-2 cells of miR-632 inhibitor +si-NC group had significantly lower OD560 value(P<0.01),lower colony formation numbers(P<0.01),lower percentage of migrating cells(P<0.01)and invasion cells(P<0.01).The OD560 value,colony formation numbers,percentage of migrating cells and invasion cells of miR-632 inhibitor + si-GSK3? group was not statistically significant different from NC-inhibitor + si-NC group.However,they were significantly higher than those of miR-632 inhibitor + si-NC group(P<0.01).Conclusions:miR-632 affected the proliferation,clone formation,migration and invasion of Hep-2 cells by negatively regulating the expression of GSK3?.