长链非编码RNA linc 00261通过竞争性结合微小RNA-182调控RND3基因参与卵巢癌癌细胞的生物学行为研究

Long chain noncoding RNA linc 00261 participates in the biological behavior of ovarian cancer cells by regulating RND3 gene through competitive binding to miR-182

目的探讨长链非编码RNA linc 00261通过竞争性结合微小RNA-182(miR-182)调控RND3基因参与卵巢癌癌细胞生物学行为研究及其作用机制。方法选取人卵巢表面上皮细胞系IOSE80和4株卵巢癌细胞系SKOV3、HO8910、A2780和OVCAR。实时荧光定量聚合酶链式反应(qRT-PCR)检测linc 00261、miR-182和RND3 mRNA的表达;双荧光素酶实验验证linc00261和miR-182结合,miR-182靶向调控RND3;RIP验证linc 00261和AGO2结合;RNA pull down结果证明linc 00261和miR-182结合;CCK8检测细胞的生长能力;Transwell实验检测细胞迁移侵袭能力;细胞流式术检测细胞凋亡率;蛋白质免疫印迹(Western Blot,WB)检测RND3和凋亡相关因子Bax和Bcl-2蛋白的表达。结果与人卵巢表面上皮细胞株IOSE80(1.00±0.11)相比,4株卵巢癌细胞SKOV3(0.25±0.04)、HO8910(0.71±0.08)、A2780(0.53±0.07)和OVCAR(0.36±0.05)中linc 00261的表达显著降低(P<0.05),而miR-182的表达在SKOV3(3.46±0.42)、HO8910(2.07±0.21)、A2780(2.79±0.31)和OVCAR(2.98±0.32)中均显著增加(均P<0.05),其中SKOV3中linc 00261的表达最低。双荧光素酶实验验证linc 00261能与miR-182结合,RIP实验验证AGO2蛋白与linc 00261结合,RNA pull down实验证明linc 00261与miR-182特异性结合,且miR-182能够靶向调控RND3的表达。过表达linc 00261或沉默miR-182可以抑制卵巢癌细胞的增殖、迁移和侵袭能力,并促进其凋亡,凋亡相关因子Bax表达上调,而抗凋亡相关因子Bcl-2表达下降,而过表达miR-182或沉默RND3可以逆转linc 00261过表达对卵巢癌细胞生物学活性的抑制作用。结论长链非编码RNA linc 00261通过竞争性结合miR-182,从而上调RND3基因,进而抑制卵巢癌癌细胞增殖、迁移和侵袭,并促进其凋亡。

Objective To investigate the role of long-chain noncoding RNA linc 00261 in the regulation of RND3 gene by competitive binding to miR-182 in participating the biological behavior of ovarian cancer cells.Methods In this study,human ovarian epithelial cell lines IOSE80 and four ovarian cancer cell lines SKOV3,HO8910,A2780 and OVCAR were selected.Detection of mRNA expression of linc 00261,miR-182 and RND3 by qRT-PCR.The binding of linc 00261 and miR-182 was confirmed by double luciferase experiment,and miR-182 targeted to regulate RND3.RIP verifies the combination of linc 00261 and AGO2.The results of RNA pull down showed that linc 00261 combined with miR-182.CCK8 test cell growth ability.Transwell test was used to detect the ability of cell migration and invasion.Flow cytometry was used to detect the apoptosis rate.WB was used to detect the expression of RND3 and apoptosis related factors Bax and Bcl-2.Results In ovarian cancer cells,Linc 00261 and Rnd3 were low expression[SKOV3(0.25±0.04),HO8910(0.71±0.08),A2780(0.53±0.07)and OVCAR(0.36±0.05)vs.IOSE80(1.00±0.11)],while mir-182 was high expression[SKOV3(3.46±0.42),HO8910(2.07±0.21),A2780(2.79±0.31)and OVCAR(2.98±0.32)](P<0.05).Linc 00261 can bind to miR-182 by double luciferase assay,and AGO2 can bind to linc 00261 by RIP assay,and linc 00261 can specifically bind to miR-182 by RNA pull down assay,and miR-182 can target and regulate the expression of RND3.Overexpression of linc 00261 or silence of miR-182 can inhibit the proliferation,migration and invasion of ovarian cancer cells,and promote their apoptosis.The expression of Bax is up-regulated,while Bcl-2 is down regulated,but overexpression of miR-182 or silencing of RND3 could reverse the inhibition of biological activity of ovarian cancer cells by overexpression of Linc 00261.Conclusion Long-chain noncoding RNA linc 00261 can up regulate RND3 gene by competitive binding miR-182,and then inhibit the proliferation,migration and invasion of ovarian cancer cells,and promote their apoptosis.