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.
Abstract
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 cir-cGLS 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 ex-periments and Sanger sequencing confirmed the resistance of circGLS to RNase R digestion,with the circulariza-tion site identified as"GA".RT-qPCR results indicated successful construction of the cell models.CCK-8 ex-periments 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 H1 299 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 in the sh-circGLS group vs sh-NC group=8.57％vs 9.52％and 2.47％vs 4.95％,respectively.These results suggest that overexpression of circGLS significantly inhibits cell proliferation,migration,and invasion in NSCLC,while its overexpression promotes apoptosis.Knockdown of circGLS presented the opposite results.Conclusion It was determined that circGLS is a circular RNA which could suppress the malignant phenotype of NSCLC cells.Con-sequently,circGLS emerges as a promising candidate for therapy of NSCLC.
基金项目
国家自然科学基金(81960532)
贵州省科技计划(黔科合基础-ZK2022一般644)
遵义医科大学附属医院博士科研启动基金(院字201905号)
国家科技期刊平台
NSTL
维普
万方数据