摘要
目的 探究circPRMT5通过靶向miR-98-5p对膀胱癌细胞增殖、迁移侵袭的机制研究.方法 qRT-PCR检测永生化正常尿路上皮细胞SV-HUC-1和膀胱癌细胞(T24、J82、HT-1376)中circPRMT5、miR-98-5p和X染色体连锁的凋亡抑制蛋白(XIAP)mRNA相对表达水平.核糖核酸酶(RNase)R和放线菌素D的消化实验鉴定cir-cPRMT5 的环状结构.将 T24 细胞分为 si-NC 组、si-circPRMT5 组、miR-NC 组、miR-98-5p 组、si-circPRMT5+anti-miR-NC 组、si-circPRMT5+anti-miR-98-5p 组.qRT-PCR 检测 circPRMT5 和 miR-98-5p 表达水平;MTT、克隆实验检测细胞增殖水平;Transwell检测迁移、侵袭能力;Western Blot检测MMP2、MMP9、XIAP蛋白表达;双荧光素酶报告实验检测circPRMT5和miR-98-5p靶向关系.结果 与永生化正常尿路上皮细胞SV-HUC-1相比,膀胱癌细胞T24、J82、HT-1376 中 circPRMT5、XIAP mRNA 相对表达水平增加,miR-98-5p 降低.circPRMT5 比线性 PRMT5 稳定性更强.沉默circPRMT5或过表达miR-98-5p降低膀胱癌细胞存活率,减少克隆细胞数、迁移细胞数、侵袭细胞数,降低MMP2、MMP9、XIAP蛋白表达.circPRMT5靶向负调控miR-98-5p的表达,抑制miR-98-5p可部分回复沉默circPRMT5对膀胱癌细胞增殖、迁移、侵袭及XIAP mRNA和蛋白表达的作用.结论 circPRMT5靶向负调控miR-98-5p抑制膀胱癌细胞增殖、迁移和侵袭能力.
Abstract
Objective To explore the mechanism of circPRMT5 targeting miR-98-5p on the proliferation,migration and invasion of bladder cancer cells.Methods qRT-PCR was used to detect the relative expression levels of cir-cPRMT5,miR-98-5p and X-linked inhibitor of apoptosis(XIAP)mRNA in immortalized normal urothelial cells SV-HUC-1 and bladder cancer cells(T24,J82,HT-1376).The ring structure of circPRMT5 was identified by digestion ex-periments of ribonuclinase(RNase)R and actinomycin D.Divide T24 cells into si-NC group,si-circPRMT5 group,miR-NC group,miR-98-5p group,si-circPRMT5+anti-miR-NC group,si-circPRMT5+anti-miR-98-5p group.qRT-PCR to detect the expression levels of circPRMT5 and miR-98-5p;MTT,cloning experiments to detect cell proliferation;Tran-swell to detect migration and invasion ability;Western Blot to detect MMP2,MMP9 and XIAP protein expression;dual luciferase reporter experiment to detect the targeting relationship between circPRMT5 and miR-98-5p.Results Com-pared with the immortalized normal urothelial cells SV-HUC-1,the relative expression levels of circPRMT5 and XIAP mRNA in bladder cancer cells T24,J82,HT-1376 increased,and miR-98-5p decreased.circPRMT5 is more stable than linear PRMT5.Silencing circPRMT5 or overexpression of miR-98-5p reduces the survival rate of bladder cancer cells,reduces the number of cloned cells,the number of migrating cells,and the number of invasive cells,and reduces the pro-tein expression of MMP2,MMP9 and XIAP.circPRMT5 targets and negatively regulates the expression of miR-98-5p,and inhibition of miR-98-5p can partially restore the effect of silencing circPRMT5 on the proliferation,migration,inva-sion and the expression of XIAP mRNA and protein of bladder cancer cells.Conclusion circPRMT5 targets and nega-tively regulates miR-98-5p to inhibit the proliferation,migration and invasion of bladder cancer cells.
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