miR-9-5p下调FRMD6促喉癌Hep2细胞存活的机制研究  

Mechanism of miR-9-5p Down Regulating FRMD6 to Promote Survival of Laryngeal Carcinoma Hep2 Cells

作　　者：程秀琴[1] 谭元元[1] 艾力根·阿不都热依木[1] 唐亮[1] Cheng Xiuqin;Tan Yuanyuan;Ailigen·Abudureyimu(Otorhinolaryngology Center,The Xinjiang Uygur Autonomous Region People′s Hospital,Xinjiang 830001,China)

机构地区：[1]新疆维吾尔自治区人民医院耳鼻喉诊疗中心

出　　处：《医学研究杂志》2019年第8期63-69,共7页Journal of Medical Research

基　　金：国家自然科学基金资助项目(81760023)

摘　　要：目的探讨miR-9-5p通过下调FRMD6表达促进喉癌Hep2细胞存活的具体机制。方法通过实时荧光定量PCR检测miR-9-5p、FRMD6在人喉癌组织中的表达情况。Hep2细胞转染对照抑制剂作为对照组,实验组分别转染miR-9-5p抑制剂、FRMD6、miR-9-5p抑制剂+FRMD6,利用MTT、细胞克隆形成实验等观察miR-9-5p及FRMD6对Hep2细胞增殖活力的影响;caspase-3蛋白活性检测以评估miR-9-5p、FRMD6对Hep2细胞的凋亡情况。对照组转染阴性对照,实验组分别转染miR-9-5pmimics,Western blot法检测过表达细胞中FRMD6的表达;Luciferase实验验证miR-9-5p与FRMD63′-UTR的结合。对照组转染阴性对照/抑制剂,实验组分别转染miR-9-5p对照/抑制剂,RT-PCR、Western blot法分别检测转染细胞中miR-9-5p、FRMD6、YAP等表达情况。结果RT-PCR法检测结果显示,喉癌组织中miR-9-5p表达增加而FRMD6表达下降。MTT及细胞克隆形成实验结果显示,Hep2细胞分别瞬转miR-9-5p抑制剂或FRMD6过表达活细胞数量均明显减少,增殖能力显著下降(P<0.05),而两者共瞬转后,细胞增殖能力较单转染组显著降低(P<0.05);caspase-3蛋白活性:Hep2细胞分别瞬转miR-9-5p抑制剂或FRMD6细胞凋亡增加(P<0.05),而两者共瞬转后细胞凋亡较单转染组高(P<0.05)。Westernblot法检测结果显示,Hep2细胞过表达miR-9-5p后FRMD6表达减少;Luciferase实验结果显示,miR-9-5p与FRMD63′-UTR直接结合。RT-PCR及Westernblot法检测结果显示,miR-9-5p阴性组FRMD6转录及翻译显著降低,YAPmRNA及蛋白表达则显著上调(P<0.05);miR-9-5p抑制剂组FRMD6的表达升高,而YAP表达降低(P<0.05)。结论miR-9-5p通过抑制FRMD6基因表达,促进人喉癌组织及Hep2细胞增殖存活,抑制细胞凋亡,这个过程可能与Hippo-YAP通路受抑制有关。Objective To investigate the mechanism of miR-9-5p promoting the survival of laryngeal carcinoma Hep2 cells by down regulating FRMD6 expression.Methods Real-time quantitative PCR was used to detect the expression of miR-9-5p and FRMD6 in human laryngeal carcinoma.Hep2 cells were transfected with control inhibitor as control group.Mi-9-5p inhibitor,FRMD6,Mi-9-5p inhibitor + FRMD6 were transfected into experimental group respectively.The effects of miR-9-5p and FRMD6 on the proliferation of Hep2 cells were observed by MTT and cell clone formation test.Caspase-3 protein activity assay was used to evaluate the apoptosis of Hep2 cells by miR-9-5p and FRMD6.The control group was transfected with control mimics,the experimental group was transfected with microRNAs-9-5p mimics,and the expression of FRMD6 was detected by Western blot.Luciferase experiments verify the combination of miR-9-5p and FRMD6 3′-UTR.The control group was transfected with control mics/inhibitor,and the experimental group was transfected with microRNAs-9-5p mimics/inhibitor,respectively.The expression of microRNAs-9-5p,FRMD6 and YAP in the transfected cells was detected by RT-PCR and Western blot respectively.Results RT-PCR detection showed that miR-9-5p expression increased and FRMD6 expression decreased in laryngeal carcinoma tissues.MTT and cell clone formation assay showed that the number of viable cells transiently overexpressed by Mi-9-5 pinhibitor or FRMD6 in Hep2 cells decreased significantly ( P <0.05),and their proliferation ability decreased significantly ( P <0.05).Caspase-3 protein activity:the apoptosis of transient RNA-9-5p inhibitor or FRMD6 cells was increased in Hep2 cells ( P <0.05),and the apoptosis of the cells co-transfected with transient RNA-9-5p inhibitor and FRMD6 cells was higher than that of the single transfection group ( P <0.05).Western blot results showed that the expression of FRMD6 decreased after overexpression of miR-9-5p in Hep2 cells.Luciferase experiment showed that miR-9-5p was directly combined with FRMD6 3

关 键 词：miR-9-5p FRMD6 人喉癌Hep2细胞存活 Hippo/YAP 

分 类 号：R739-65[医药卫生—肿瘤]