微小RNA-362-5p通过靶向调控CYLD对胃癌细胞株SGC-7901生物学行为的影响  

Effect of microRNA - 362 - 5p on biological behavior of gastric cancer cell line SGC - 7901 by targeting cylindromatosis

作　　者：宋亚锋 肖飞 李运奇 徐媛 Song Yafeng;Xiao Fei;Li Yunqi;Xu Yuan(Department of Gastrointestinal Surgery, Wuhan Fourth Hospita;Puai Hospital, Tongfi Medical College, Huazhong University of Science and Technology, Wuhan 430033, Chin;Department of Critical Care Medicine, Wuhan Fourth Hospita;Puai Hospital, Tongfi Medical College, Huazhong University of Science and Technology, Wuhan 430033, China)

机构地区：[1]武汉市第四医院华中科技大学同济医学院附属普爱医院(西院)胃肠外科,430030 [2]武汉市第四医院华中科技大学同济医学院附属普爱医院(西院)重症医学科,430030

出　　处：《中华实验外科杂志》2018年第6期1055-1058,共4页Chinese Journal of Experimental Surgery

基　　金：武汉市卫生计生委科医学研资助项目（WX17Q18）;湖北省卫生计生委科研资助项目（WJ2017M191）

摘　　要：目的探讨微小RNA（miRNA，miR）-362-5p对人胃癌细胞株SGC-7901增殖、凋亡的影响及其调控机制。方法通过实时荧光定量聚合酶链反应（FQ-PCR）检测人胃癌细胞株和正常胃黏膜细胞中miR-362-5p的表达；采用脂质体瞬时转染技术转染miR-362-5p抑制物；噻唑蓝（MTT）法检测miR-3626p对胃癌细胞株SGC-7901增殖能力的影响；流式细胞术检测miR-3626p对胃癌细胞周期和凋亡的影响；利用Targetscan信息学预测软件预测miR-362-5p靶向调控Cylindrom．atosis（CYLD）基因，构建携带CYLD野生型及突变型3’非翻译区（3’UTR）野生型和突变型荧光素酶报告载体，并利用双荧光素酶活性分析检测；Westernblot检测miR-362-5p对CYLD蛋白表达的调控作用。结果miR-3626p在胃癌细胞株SGC-7901、AGS、MKN28的表达水平分别为6．274-0．70、4．63±0．35、5．53±0．45，明显高于胃黏膜细胞（P=0．006、0．003、0．003）。转染miR-362-5p抑制物后，胃癌细胞SGC-7901中miR-362-5p表达水平下降[miR-362-5p-IN组（0．41±0．07），miR-362-5pNC组（0．84±0．07），P=0．022]。下调miR-362-5p后显著抑制胃癌细胞SGC-7901的增殖[48h（吸光度值）：miR-362-5p-IN组：0．28±0．02，miR-3626pNC组：0．45±0．04，P=0．028；72h（吸光度值）：miR-362-5p-IN组：0．33±0．02，miR-362-5pNC组：0．61±0．04，P=0．003]；下调miR-362-5p后流式细胞术结果显示胃癌细胞G1／G0期比例增加[miR-362-5p-IN组：（78．04±3．74）％，miR-362-5pNC组：（67．86±3．35）％，P=0．017]，凋亡增加[miR-362-5p-IN组：（33．29±4．86）％，miR-3626pNC组：（18．20±0．80）％，P=0．040]。双荧光素酶报告基因实验验证CYLD是miR-362-5p的潜在靶基因。Westernblot法检测结果显示CYLD蛋白水平与miR-362-5p表达呈负相关。结论miR-362-5p可通过靶向负调控CYLD蛋白，促进胃癌细胞的增殖，抑制细胞凋亡，进而促进胃癌的发生发展。Objective To investigate the effect of microRNA （miRNA, miR） -362 -5p on pro- liferation and apoptosis of human gastric cancer cell line SGC - 7901 and the underlying mechanism. Methods The expression level of miR -362 -5p in human gastric cancer cell lines and normal gastric mucosal cells was detected by real- time fluorescent quantitative polymerase chain reaction （FQ -PCR）. miR -362 -5p inhibitor is transfected by liposomes. The effect of miR -362 -5p on the proliferation abil- ity of gastric cancer cell line SGC -7901 was detected by methyl thiazol tetrazolium （MTF） assay. The effect of miR -362 -5p on the cell cycle and apoptosis of gastric cancer ceils was detected by flow cytome- try. The Targetscan izfformatics prediction software was used to predict the miR- 362 -5p targeting Cylin- dromatosis （CYLD） gene, and the wild type and mutant type 3 ＇ untranslated region （ 3＇ UTR） wild type and mutant luciferase reporter vectors carrying CYLD wild - type and mutant 3 ＇ UTR was constructed and analyzed. Results The expression levels of miR -362 -5p in gastric cancer cell lines SGC -7901, AGS and MKN28 were （6. 27 ±0. 70, 4. 63±0. 35, 5.53 ±0. 45）, which were significantly higher than those in gastric mucosal cells （P =0. 006, 0. 003, 0. 003）. Transfection of miR - 362 - 5p could significantly decrease the level of miR -362 -5p in SGC -7901, in comparison with the contro （miR -362 -5p -IN： 0. 41 _＋ 0. 07, miR - 362 - 5p NC ： 0. 84± 0. 07, P = 0. 022）. Down - regulation of miR - 362 - 5p signif- icantly inhibits the proliferation of gastric cancer cell line SGC -7901 [ 48 h （absorbance value ）： miR-362 -5p - IN： 0. 28 ± 0. 02, miR - 362 - 5p NC： 0. 45 ± 0. 04, P = 0. 028; 72 h （absorbance value） ： miR-362 -5p-IN： 0. 33 ±0. 02, miR-362 -5p NC： 0. 61 ±0. 04, P =0. 0031. The ratio of GI/GO phase in gastric cancer cells increased [ miR - 362 - 5p - IN ： （78.04 -＋ 3.74 ） % , miR - 362 - 5p NC： （67.86-±3.35）%, P=0.0171 and the apoptosi

关 键 词：微小RNA-362-5p 胃癌 Cylindromatosis 

分 类 号：R735.2[医药卫生—肿瘤]