circGLS抑制非小细胞肺癌细胞恶性表型的作用研究  

circGLS inhibits the malignant phenotype of non small cell lung cancer cells

作　　者：龙开君 赵鹏飞 王凯 宋永祥 陈成 柯希贤 Long Kaijun;Zhao Pengfei;Wang Kai;Song Yongxiang;Chen Cheng;Ke Xixian(Department of Thoracic Surgery,Affiliated Hospital of Zunyi Medical University,Zunyi,Guizhou,563000,China)

机构地区：[1]遵义医科大学附属医院胸外科,贵州遵义563000

出　　处：《遵义医科大学学报》2024年第4期324-331,323,共9页Journal of Zunyi Medical University

基　　金：国家自然科学基金资助项目(NO:81960532);贵州省科技计划资助项目[NO:黔科合基础-ZK(2022)一般644];遵义医科大学附属医院博士科研启动基金[NO:院字(2019)05号]。

摘　　要：目的明确circGLS的环状RNA特性及其环化位点,研究circGLS对NSCLC细胞恶性表型的影响。方法采用Northern-Blot、RNase R耐受实验检测circGLS的耐RNase R消化特性,使用Sanger测序明确其环化位点。分别使用Vector、OE-circGLS慢病毒感染H460和H1299细胞,使用sh-NC及sh-circGLS感染A549和H1975细胞,分组为Control组、Vector组、OE-circGLS组、sh-NC组和sh-circGLS组。采用CCK-8、流式细胞术、划痕和Transwell实验检测上述分组中细胞增殖、凋亡、迁移和侵袭情况。结果Northern-Blot、RNase R消化和Sanger测序实验证实了circGLS耐受RNase R消化,且环化位点为“GA”。RT-qPCR结果证实细胞模型构建成功。CCK-8结果显示,H1299和H460细胞72 h OE-circGLS组细胞活力分别为0.88和0.9;A549和H1975细胞72 h sh-circGLS组活力均为1.13(Vector组和sh-NC组细胞活力均一化为1)。划痕实验结果显示,H460和H1299细胞愈合率分别为:OE-circGLS组vs Vector组=2.42%vs 5.57%和12.09%vs 25.07%;A549和H1975细胞愈合率分别为:sh-circGLS组vs sh-NC组=19.00%vs 13.81%和30.69%vs 21.53%。Transwell实验结果显示,H460细胞和H1299细胞迁移率(OD)分别为:OE-circGLS组vs Vector组=0.94 vs 1.01和0.57 vs 0.92;H1975细胞和A549细胞迁移率(OD)分别为:sh-circGLS组vs sh-NC组=1.044 vs 0.95和1.29 vs 1.09。流式细胞术结果显示,H1299细胞和H460细胞凋亡率分别为:OE-circGLS组vs Vector组=14.45%vs 7.54%和10.74%vs 1.79%;A549细胞和H1975细胞凋亡率分别为:sh-circGLS组vs sh-NC组=8.57%vs 9.52%和2.47%vs 4.95%。上述结果表明,过表达circGLS可以显著抑制NSCLC细胞增殖、迁移和侵袭,且促进其凋亡,而敲低circGLS得到相反的结果。结论circGLS是一个真实存在的环状RNA,具有抑制NSCLC细胞恶性表型的功能,是潜在的非小细胞肺癌治疗靶点。Objective To elucidate the circular RNA characteristics of circGLS,determine its circularization site,and investigate the impact of circGLS on the malignant phenotype of non-small cell lung cancer(NSCLC)cells.Methods Northern blot and RNase R resistance experiments were employed to assess the resistance of circGLS to RNase R digestion,and Sanger sequencing determined its circularization site.H460 and H1299 cells were infected with Vector and OE-circGLS lentivirus,while A549 and H1975 cells were infected with sh-NC and sh-circGLC,resulting in Control(without infection of virus),Vector,OE-circGLS,sh-NC,and sh-circGLS groups.CCK-8,flow cytometry,scratch,and Transwell assays were conducted to investigate cell proliferation,apoptosis,migration,and invasion in the aforementioned groups.Results Northern blot,RNase R digestion experiments and Sanger sequencing confirmed the resistance of circGLS to RNase R digestion,with the circularization site identified as"GA".RT-qPCR results indicated successful construction of the cell models.CCK-8 experiments showed that the cell viability of H1299 and H460 cells in OE-circGLS group at 72 h was 0.88 and 0.9,respectively,while the viability of A549 and H1975 cells in sh-circGLS at 72 h was 1.13(normalized to 1 for Vector and sh-NC groups).Scratch experiments revealed healing rates of H460 and H1299 cells in the OE-circGLS group vs Vector group=2.42%vs 5.57%and 12.09%vs 25.07%,respectively.Healing rates for A549 and H1975 cells in the sh-circGLS group vs sh-NC group=19.00%vs 13.81%and 30.69%vs 21.53%,respectively.Transwell experiments showed migration rates(OD)of H460 and H1299 cells in the OE-circGLS group vs Vector group=0.94 vs 1.01 and 0.57 vs 0.92,respectively.Migration rates for H1975 and A549 cells in the sh-circGLS group vs sh-NC group=1.044 vs 0.95 and 1.29 vs 1.09,respectively.Flow cytometry results indicated apoptosis rates of H1299 and H460 cells in the OE-circGLS group vs Vector group=14.45%vs 7.54%and 10.74%vs 1.79%,respectively.Apoptosis rates of A549 and H1975 cells i

关 键 词：环状RNA circGLS 非小细胞肺癌 恶性表型 

分 类 号：R734.2[医药卫生—肿瘤]