miR-217靶向调控HMGA 2基因对内质网应激介导的口腔癌细胞自噬与凋亡的影响  

The effect of miR-217 targeting and regulating HMGA2 gene on endoplasmic reticulum stress-mediated autophagy and apoptosis in oral cancer cells

作　　者：宿伟鹏[1] 赵化荣[1] 李晨曦[2] 刘攀[1] 张洋[1] 龚忠诚[2] SU Weipeng;ZHAO Huarong;LI Chenxi;LIU Pan;ZHANG Yang;GONG Zhongcheng(Cancer Center,The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,China;Maxillofacial Tumor Surgery Department,The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,China)

机构地区：[1]新疆医科大学第一附属医院肿瘤中心,新疆乌鲁木齐830011 [2]新疆医科大学第一附属医院颌面肿瘤外科,新疆乌鲁木齐830011

出　　处：《西部医学》2023年第9期1256-1263,共8页Medical Journal of West China

基　　金：新疆维吾尔自治区卫生计生委青年医学科技人才专项科研项目(WJWY-202017)。

摘　　要：目的探究微小RNA 217(miRNA-217)对口腔癌细胞中内质网应激介导的自噬与凋亡的影响及其分子机制。方法培养人口腔癌细胞和人正常口腔角质形成细胞,实时荧光定量聚合酶链式反应(RT-qPCR)检测细胞中miR-217与高迁移率族蛋白A2(HMGA2)表达水平;将人口腔癌细胞系SAS分为对照组、miR-NC组、miR-217 mimic组和miR-217 mimic+4-PBA组4组,脂质体法进行转染和内质网应激抑制剂4-苯丁酸处理后,流式细胞术检测细胞凋亡率,Hoechst33342染色观察细胞凋亡情况,细胞免疫荧光染色检测细胞中微管相关蛋白1轻链3(LC3)表达,蛋白质免疫印迹(Western Blot)测定细胞中自噬相关蛋白LC3I、LC3II及Beclin-1的表达水平,RT-qPCR和Western Blot测定细胞内质网应激相关分子CCAAT/增强予结合蛋白同源蛋白(CHOP)、葡萄糖调节蛋白78(GRP78)及天冬氨酸蛋白水解酶-12(Caspase-12)的表达水平;双荧光素酶报告基因检测实验和Western Blot验证miR-217对HGMA2的靶向作用。结果与人正常口腔角质形成细胞HNOK比较,人口腔癌细胞HSC3、SAS、SCC15、FaDu中miR-217相对表达量下调而HMGA2相对表达量上调(P<0.05);与对照组比较,miR-217 mimic组SAS细胞凋亡率升高,有大量呈亮蓝色高荧光的凋亡细胞,LC3荧光强度升高,LC3-Ⅱ/LC3-Ⅰ比值与Beclin-1蛋白相对表达量均上调,GRP78、CHOP、Caspase-12 mRNA相对表达量与蛋白相对表达量均上调(P<0.05);而与miR-217 mimic组比较,miR-217 mimic+4-PBA组SAS细胞凋亡率下降,呈亮蓝色高荧光的凋亡细胞明显减少,LC3荧光强度降低,LC3-Ⅱ/LC3-Ⅰ比值与Beclin-1蛋白相对表达量均下调,GRP78、CHOP、Caspase-12 mRNA相对表达量与蛋白相对表达量也均下调(P<0.05);HMGA2与miR-217在特定区域存在碱基互补现象,且miR-217靶向负调控HGMA2表达。结论miR-217可能通过促进内质网应激途径诱导口腔癌细胞发生自噬与死亡,其机制可能与靶向负调控HMGA2表达相关。Objective To investigate the effect of microRNA(miRNA)217 on endoplasmic reticulum stress-mediated autophagy and apoptosis in oral cancer cells and its molecular mechanism.Methods Human oral cancer cells and human normal oral keratinocytes were cultured.The expression levels of miR-217 and high mobility group A2(HMGA2)in cells were detected by real-time quantitative polymerase chain reaction(RT-qPCR).The human oral cancer cell line SAS was divided into 4 groups:control group,miR-NC group,miR-217 mimic group and miR-217 mimic+4-PBA group,after transfection by liposome method and treatment with endoplasmic reticulum stress inhibitor 4-phenylbutyric acid.The flow cytometry was used to detect cell apoptosis rate.The Hoechst33342 staining was used to observe cell apoptosis.The cell immunofluorescence staining was used to detect the expression of microtubule-associated protein 1 light chain 3(LC3)in cells.Western blot was used to determine the expression levels of autophagy-related proteins LC3I,LC3II and Beclin-1 in cells.RT-qPCR and Western Blot were used to determine the expression levels of intracellular endoplasmic reticulum stress-related molecules CCAAT/enhancer-binding protein homologous protein(CHOP),glucose-regulated protein 78(GRP78)and aspartate proteolytic enzyme-12(Caspase-12).The targeting effect of miR-217 on HGMA2 was verified by dual-luciferase reporter gene detection experiments and Western Blot.Results Compared with human normal oral keratinocytes HNOK,the relative expression of miR-217 was down-regulated and the relative expression of HMGA2 was up-regulated in human oral cancer cells HSC3,SAS,SCC15 and FaDu(P<0.05).Compared with the control group,the apoptosis rate of SAS cells in the miR-217 mimic group increased,there were a large number of apoptotic cells with bright blue and high fluorescence,the fluorescence intensity of LC3 increased,the ratio of LC3-II/LC3-I and Beclin-1 protein increased,and the relative expression levels of GRP78,CHOP,Caspase-12 mRNA and protein were up-regulated(P<0.05).C

关 键 词：口腔癌 miR-217 HMGA2 内质网应激 自噬 凋亡 

分 类 号：R739.8[医药卫生—肿瘤]