环状RNA-1565对前列腺癌PC-3细胞生物学行为的影响及其机制

Biological role and mechanism of circRNA-1565 in prostate cancer PC3 cells

目的观察环状RNA(circRNA)-1565对前列腺癌PC-3细胞生物学行为的影响,并探讨其可能的机制。方法将PC-3细胞分为观察组和对照组,分别转染circRNA-1565 siRNA(si445)和阴性对照siRNA。两组转染24 h,分别检测其培养0~4 d细胞增殖、克隆形成、凋亡、侵袭及迁移能力。免疫共沉淀实验筛选与circRNA-1565结合的miRNA分别为miR-21和miR-153,采用双荧光素酶报告基因实验观察circRNA-1565对miR-21和miR-153的调控作用。将PC-3细胞分为si445组、si445+miR-21 inhibitor组、si445+miR-153 inhibitor组、阴性对照组,分别转染si445、si445+miR-21 inhibitor、si445+miR-153 inhibitor及阴性对照siRNA,并检测细胞侵袭、迁移能力。采用Western blotting法检测si445组、si445+miR-153 inhibitor组、阴性对照组细胞磷脂酰肌醇3-激酶(PI3K)、磷酸酶和张力蛋白同源物(PTEN)、蜗牛家族转录抑制子1(SNAIL1)蛋白表达。结果观察组与对照组不同时间点细胞增殖OD值、克隆形成个数、细胞凋亡率比较差异均无统计学意义(P均>0.05),观察组进入下室的细胞个数、细胞移动距离均低于对照组(P均<0.05)。双荧光素酶报告基因结果显示,circRNA-1565可以分别与miR-21及miR-153直接结合。与阴性对照组比较,其余三组进入下室的细胞个数、细胞移动距离均降低,且si445组、si445+miR-21 inhibitor组降低更明显(P均<0.05)。si445组、si445+miR-21 inhibitor组进入下室的细胞个数、细胞移动距离比较P均>0.05。si445组、si445+miR-153 inhibitor组、阴性对照组细胞PI3K蛋白相对表达量依次升高(P均<0.05),三组细胞PTEN和SNAIL1蛋白相对表达量比较P均>0.05。结论circRNA-1565可促进前列腺癌PC-3细胞侵袭和迁移,其机制可能与竞争性结合miR-153并沉默其下游靶蛋白PI3K表达有关。

Objective To observe the effect of circular RNA(circRNA)-1565 on the biological behavior of prostate cancer PC-3 cells and to explore its possible mechanism.Methods PC-3 cells were divided into the observation group and control group,which were transfected with circRNA-1565 siRNA(si445)and negative control siRNA,respectively.Both of the two groups were transfected for 24 h,and their cell proliferation,colony formation,apoptosis,invasion,and migration abilities were measured.Co-immunoprecipitation experiments screened microRNA 21(miR-21)and microRNA 153(miR-153)for miRNA binding to circRNA-1565,respectively.Dual-luciferase reporter gene experiments were used to observe the regulatory effects of circRNA-1565 on miR-21 and miR-153.PC-3 cells were divided into the siRNA group,siRNA+miR-21 inhibitor group,siRNA+miR-153 inhibitor group,and negative control group,which were transfected with siRNA,siRNA+miR-21 inhibitor,siRNA+miR-153 inhibitor,and negative control siRNA.The invasion and migration were detected.Western blotting was used to detect phosphatidylinositol 3-kinase(PI3K),phosphatase and tensin homolog(PTEN),snail family transcriptional repressor 1(SNAIL1)in the siRNA group,siRNA+miR-153 inhibitor group,and negative control group.Results There were no significant differences in the cell proliferation,the number of clones or apoptosis rate between the observation group and the control group at different time points(all P>0.05).The number of cells entering the lower chamber and the cell movement distance of the observation group were lower than those of the control group(both P<0.05).The dual luciferase reporter gene results showed that circRNA-1565 could directly bind to miR-21 and miR-153,respectively.Compared with the negative control group,the number of cells entering the lower chamber and the cell migration distance decreased,and the decrease in the si445 group and si445+miR-21 inhibitor group was more significant(P<0.05).No significant differences were found in the number of cells entering the lower chamber or the cell movement distance between the siRNA group and siRNA+miR-21 inhibitor group(all P>0.05).The relative expression levels of PI3K protein in the si445 group,si445+miR-153 inhibitor group,and negative control group increased in turn(P<0.05).No significant difference was found in the relative expression of PTEN and SNAIL1 between the three groups(all P>0.05).Conclusion CircRNA-1565 can promote the invasion and migration of prostate cancer PC-3 cells,which may be related to its competitive binding to miR-153 and silencing its downstream target protein PI3K expression.