miR-135a通过下调SOX2抑制喉癌Hep-2细胞的恶性生物学行为和增强对奥沙利铂的敏感性

miR-135a knockdown inhibits the malignant biological behaviors and promotes oxaliplatin-sensitivity of human laryngeal carcinoma Hep-2 cells by down-regulation of SOX2

目的:探讨敲降miR-135a对人喉癌上皮Hep-2细胞的恶性生物学行为和奥沙利铂敏感性的影响。方法:收集2018年1月至2018年6月郑州大学附属南阳医院南阳市中心医院行喉癌切除术10例患者的喉癌组织和癌旁组织标本。采用qPCR检测喉癌组织和Hep-2细胞中miR-135a的表达水平。miR-135 inhibitor转染喉癌Hep-2细胞后,采用CCK-8检测Hep-2细胞活性,集落形成实验检测Hep-2细胞集落形成能力,Transwell实验检测Hep-2细胞的侵袭及迁移能力,WB实验检测Hep-2细胞中SOX2蛋白的表达水平。0.5、1.0、1.5、2.0μmol/L的奥沙利铂处理已转染miR-135 inhibitor的Hep-2细胞,CCK-8实验检测Hep-2细胞的增殖活性,Annexin-V-FITC/PI染色流式细胞术检测Hep-2细胞的凋亡率。采用miR-135a inhibitor质粒、对照pcDNA空载体(SOX2-Con)质粒、pcDNA-SOX2(SOX2-OE)质粒共转染Hep-2细胞,构建miR-135a inhibitor+SOX2-Con组和miR-135a inhibitor+SOX2-OE组,检测此2组细胞的增殖活性、集落形成能力、侵袭及迁移能力。结果:与癌旁组织比较,喉癌组织中miR-135a表达水平显著升高(P<0.01);与正常NHP细胞比较,miR-135a在Hep-2细胞中表达水平明显上调(P<0.01);转染miR-135a inhibitor导致Hep-2细胞中miR-135a表达水平明显降低(P<0.01)。敲降miR-135a明显降低Hep-2细胞的增殖活性、细胞集落数、迁移、侵袭和SOX2表达(均P<0.01);明显增强细胞对奥沙利铂敏感性(P<0.01);与miR-135a inhibitor+SOX2-Con组比较,miR-135a inhibitor+SOX2-OE组的Hep-2细胞的增殖活性、细胞集落数、迁移与侵袭能力均明显增加(均P<0.01);同时,用不同浓度的奥沙利铂处理此2组细胞,相对于miR-135a inhibitor+SOX2-Con组,miR-135a inhibitor+SOX2-OE组的Hep-2细胞存活率显著升高(P<0.01)。结论:敲降miR-135a可能通过下调转录因子SOX2的表达抑制Hep-2细胞的恶性生物学行为,并增强其对奥沙利铂的敏感性。

Objective:To investigate the effect of miR-135a on the malignant biological behaviors of human laryngeal carcinoma epithelial Hep-2 cells and its sensitivity to oxaliplatin.Methods:Samples of laryngeal carcinoma tissues and para-cancerous tissues were collected from 10 patients who underwent laryngectomy in Nanyang Hospital Affiliated to Zhengzhou University-Nanyang City Center Hospital from January 2018 to June 2018.The expression of miR-135a in laryngeal carcinoma tissues and Hep-2 cells was detected by qPCR.After being transfected with miR-135 inhibitor,cell proliferation viability of Hep-2 cells was measured by CCK-8 assay,cell colony formation ability was detected by colony formation assay,and cell proliferation invasion and migration abilities were detected by Transwell analysis,and the expression of SOX2 protein in Hep-2 cells was detected by WB.Hep-2 cells transfected with miR-135 inhibitor were further treated with various concentrations(0.5,1.0,1.5and 2.0μmol/L)of oxaliplatin,and the cell proliferation viability was detected by CCK-8 while cell apoptosis was detected by Annexin-V-FITC/PI double staining flow cytometry.miR-135a inhibitor plasmid,control pcDNA empty vector(SOX2-Con)plasmid,and pcDNA-SOX2(SOX2-OE)plasmid were transfected into Hep-2 cells to construct the miR-135a inhibitor+SOX2-Con group and miR-135a inhibitor+SOX2-OE group,and the cell viability,cell colony formation ability,cell invasion and migration ability in two groups were detected.Results:Compared with para-cancerous tissues,miR-135a expression in laryngeal cancer tissues was significantly increased(P<0.01).Compared with normal NHP cells,miR-135a expression in Hep-2 cells was significantly increased(P<0.01).miR-135a inhibitor significantly reduced the expression level of miR-135a in Hep-2 cells(P<0.01).miR-135a knockdown significantly reduced the cell proliferation viability,cell colony number,migration,invasion and SOX2 expression in Hep-2 cells(all P<0.01),but significantly enhanced the sensitivity of Hep-2 cells to oxaliplatin(P<0.01).Compared with miR-135a inhibitor+SOX2-Con group,the cell proliferation viability,cell colony number,migration and invasion of Hep-2 cells in miR-135a inhibitor+SOX2-OE group were significantly increased(P<0.01);Meanwhile,the cells of the 2 groups were treated with different concentrations of oxaliplatin,and the results of CCK-8 assay showed that,compared with the miR-135a inhibitor+SOX2-Con group,the cell proliferation viability of Hep-2 cells in miR-135a inhibitor+SOX2-OE group was significantly increased(P<0.01).Conclusion:miR-135a knockdown inhibits the malignant biological behaviors and promotes oxaliplatin-sensitivity of Hep-2 cells possibly by inhibiting the expression of the transcription factor SOX2.