miR-135a-5p增强舌鳞状细胞癌细胞对奥沙利铂敏感性研究

Influence of miR-135a-5p on the sensitivity of tongue squamous cell carcinoma cells to oxaliplatin

目的探讨miR-135a-5p增强舌鳞状细胞癌细胞对奥沙利铂(oxaliplatin,L-OHP)敏感性的作用及其可能机制。方法采用实时荧光定量PCR法检测人口腔角质形成细胞(human oral keratinocyte,HOK)和舌鳞状细胞癌细胞系CAL-27、SCC9、SCC25细胞中miR-135a-5p和B淋巴瘤莫洛尼鼠白血病病毒插入区1(B-lymphoma Moloney murine leukemia virus insertion region-1,BMI1)mRNA相对表达量,采用Western blot法检测4种细胞BMI1蛋白相对表达量。对数生长期SCC25细胞分为miR-NC组(转染miR-NC)、miR-135a-5p模拟物组(miR-135a-5p模拟物)和空白对照组(不进行转染操作),采用实时荧光定量PCR法检测3组细胞miR-135a-5p和BMI1 mRNA相对表达量,采用Western blot法检测3组细胞BMI1蛋白相对表达量,采用荧光素酶报告实验检测miR-135a-5p与BMI1的靶向关系。SCC25细胞加入不同浓度L-OHP,采用CCK-8实验检测L-OHP对细胞的半数抑制浓度,确定L-OHP在后续实验中的浓度。对数生长期SCC25细胞再分为miR-NC+pcDNA-NC组(转染miR-NC和pcDNA-NC)、miR-135a-5p模拟物+pcDNA-NC组(转染miR-135a-5p模拟物和pcDNA-NC)、miR-135a-5p模拟物+pcDNA-BMI1组(转染miR-135a-5p模拟物和pcDNA-BMI1)、对照组(不进行转染操作),采用实时荧光定量PCR法检测细胞miR-135a-5p和BMI1 mRNA相对表达量,采用Western blot法检测细胞BMI1蛋白相对表达量,采用EdU增殖实验检测细胞增殖情况,采用Transwell实验检测迁移细胞数和侵袭细胞数,采用流式细胞术检测细胞凋亡率。结果CAL-27、SCC9、SCC25细胞miR-135a-5p相对表达量(0.43±0.04、0.62±0.09、0.41±0.07)均低于HOK细胞(1.00±0.15)(P<0.05),BMI1 mRNA(4.57±0.67、2.64±0.59、5.82±0.87)和蛋白(0.61±0.22、0.34±0.28、1.00±0.24)相对表达量均高于HOK细胞(1.00±0.31、0.21±0.14)(P<0.05);与HOK细胞比较,SCC25细胞miR-135a-5p、BMI1 mRNA和蛋白相对表达量差异最大,选用SCC25细胞进行后续实验。miR-135a-5p模拟物组细胞miR-135a-5p相对表达量高于miR-NC组、空白对照组(P<0.05),BMI1 mRNA和蛋白相对表达量均低于miR-NC组、空白对照组(P<0.05);miR-NC组细胞miR-135a-5p、BMI1 mRNA和蛋白相对表达量与空白对照组比较差异均无统计学意义(P>0.05)。miR-135a-5p靶向BMI1。miR-135a-5p模拟物+pcDNA-BMI1组、miR-135a-5p模拟物+pcDNA-NC组miR-135a-5p相对表达量均高于对照组、miR-NC+pcDNA-NC组(P<0.05),miR-135a-5p模拟物+pcDNA-BMI1组与miR-135a-5p模拟物+pcDNA-NC组比较差异无统计学意义(P>0.05)。miR-135a-5p模拟物+pcDNA-BMI1组、miR-135a-5p模拟物+pcDNA-NC组BMI1 mRNA和蛋白相对表达量、L-OHP对细胞的半数抑制浓度、EdU阳性细胞数、迁移细胞数、侵袭细胞数均低于对照组、miR-NC+pcDNA-NC组(P<0.05),miR-135a-5p模拟物+pcDNA-BMI1组均高于miR-135a-5p模拟物+pcDNA-NC组(P<0.05)。miR-135a-5p模拟物+pcDNA-BMI1组、miR-135a-5p模拟物+pcDNA-NC组细胞凋亡率高于miR-NC+pcDNA-NC组、对照组(P<0.05),miR-135a-5p模拟物+pcDNA-BMI1组低于miR-135a-5p模拟物+pcDNA-NC组(P<0.05);以上指标miR-NC+pcDNA-NC组与对照组比较差异均无统计学意义(P>0.05)。结论miR-135a-5p通过靶向抑制BMI1表达,增强舌鳞状细胞癌细胞对L-OHP的敏感性。

Objective To investigate the role of miR-135 a-5 p in increasing the sensitivity of tongue squamous cell carcinoma to oxaliplatin(L-OHP)and its possible mechanism.Methods The relative expressions of miR-135 a-5 p and B lymphoma Moloney murine leukemia virus insertion region-1(BMI1)mRNA in human oral keratinocyte HOK and tongue squamous cell carcinoma cell line(CAL-27,SCC9,SCC25)were detected by real-time fluorescence quantitative PCR(qRT-PCR).Western blot was used to detect the relative expression of BMI1 protein in those four kinds of cells.SCC25 cells in logarithmic growth phase were divided into miR-NC group(transfected with miR-NC),miR-135 a-5 p mimics group(transfected with miR-135 a-5 p mimics)and blank control group(without transfection).The relative expressions of miR-135 a-5 p and BMI1 mRNA were detected by qRT-PCR,the relative expression of BMI1 protein was detected by Western blot in three groups,and the targeting relationship between miR-135 a-5 p and BMI1 was detected by luciferase reporting assay.SCC25 cells were added with different concentrations of L-OHP,and the half maximal inhibitory concentration(IC50)of L-OHP on cells was detected by CCK-8 assay to determine the concentration of L-OHP in the subsequent experiments.SCC25 cells in logarithmic growth phase were subdivided into miR-NC+pcDNA-NC group(transfected with miR-NC and pcDNA-NC),miR-135 a-5 p mimics+pcDNA-NC group(transfected with miR-135 a-5 p mimics and pcDNA-NC),miR-135 a-5 p mimics+pcDNA-BMI1 group(transfected with miR-135 a-5 p mimics+pcDNA-BMI1)and control group(without transfection).The relative expressions of miR-135 a-5 p and BMI1 mRNA were detected by qRT-PCR,the relative expression of BMI1 protein was detected by Western blot,the cell proliferation rate was detected by EdU proliferation assay,the number of migrating cells and the number of invading cells were detected by Transwell assay,and the apoptosis rate was detected by flow cytometry.Results The relative expression of miR-135 a-5 p was lower in CAL-27(0.43±0.04),SCC9(0.62±0.09)and SCC25 cells(0.41±0.07)than that in HOK cells(1.00±0.15)(P<0.05).The relative expressions of MNI1 mRNA and protein were higher in CAL-27 cells(4.57±0.67,0.61±0.22),SCC9 cells(2.64±0.59,0.34±0.28)and SCC25 cells(5.82±0.87,1.00±0.24)than those in HOK cells(1.00±0.31,0.21±0.14)(P<0.05).Compared with HOK cells,SCC25 cells showed the largest difference in the relative expression levels of miR-135 a-5 p and BMI1 mRNA and protein,therefore,SCC25 cells were selected for subsequent experiments.The relative expression of miR-135 a-5 p was higher in miR-135 a-5 p mimics group than that in miR-NC group and blank control group(P<0.05),and the relative expressions of BMI1 mRNA and protein were lower in miR-135 a-5 p mimics group than those in miR-NC group and blank control group(P<0.05),which showed no significant differences between miR-NC group and blank control group(P>0.05).MiR-135 a-5 p targetedly inhibited BMI1 expression.The relative expression of miR-135 a-5 p was higher in miR-135 a-5 p mimics+pcDNA-BMI1 group and miR-135 a-5 p mimics+pcDNA-NC group than that in control group and miR-NC+pcDNA-NC group(P<0.05),and showed no significant difference between miR-135 a-5 p mimics+pcDNA-BMI1 group and miR-135 a-5 p mimics+pcDNA-NC group(P>0.05).The relative expressions of BMI1 mRNA and protein,IC50,and the numbers of EdU positive cells,migrating cells and invading cells were lower in miR-135 a-5 p mimics+pcDNA-BMI1 group and miR-135 a-5 p mimics+pcDNA-NC group than those in control group and miR-NC+pcDNA-NC group(P<0.05),and higher in miR-135 a-5 p mimics+pcDNA-BMI1 group than those in miR-135 a-5 p mimics+pcDNA-NC group(P<0.05).The apoptosis rate was higher in miR-135 a-5 p mimics+pcDNA-BMI1 group and miR-135 a-5 p mimics+pcDNA-NC group than that in miR-NC+pcDNA-NC group and control group(P<0.05),and lower in miR-135 a-5 p mimics+pcDNA-BMI1 group than those in miR-135 a-5 p mimics+pcDNA-NC group(P<0.05).All the above indexes showed no significant differences between miR-NC+pcDNA-NC group and control group(P>0.05).Conclusion MiR-135 a-5 p enhances the sensitivity of tongue squamous cell carcinoma cells to L-OHP by targetedly inhibiting BMI1 expression.