LncRNA PITPNA-AS1靶向miR-491-3p基因调控宫颈癌细胞增殖、迁移和侵袭的分子机制研究

The molecular mechanism of LncRNA PITPNA-AS1 targeting miR-491-3p gene to regulate the proliferation,migration and invasion of cervical cancer cell

目的研究长链非编码RNA(LncRNA)PITPNA反义RNA 1(PITPNA-AS1)是否通过靶向miR-491-3p调控宫颈癌细胞增殖、迁移、侵袭。方法qRT-PCR检测宫颈癌细胞系(HeLa、SiHa、Caski)、宫颈上皮永生化细胞H8中LncRNA PITPNA-AS1和miR-491-3p表达。将未处理的宫颈癌细胞HeLa设为对照(NC组),并在宫颈癌细胞HeLa中转染si-NC(si-NC组)、si-LncRNA PITPNA-AS1(si-LncRNA PITPNA-AS1组)、miR-NC(miR-NC组)、miR-491-3p mimics(miR-491-3p组)、pcDNA(pcDNA组)、pcDNA-LncRNA PITPNA-AS1(pcDNA-LncRNA PITPNA-AS1组)、si-LncRNA PITPNA-AS1和anti-miR-NC(si-LncRNA PITPNA-AS1+anti-miR-NC组)、si-LncRNA PITPNA-AS1和anti-miR-491-3p(si-LncRNA PITPNA-AS1+anti-miR-491-3p组)。Western Blot、细胞计数试剂盒8(CCK-8)法和Transwell法分别检测转染前后细胞周期蛋白D1(CyclinD1)、基质金属蛋白酶(MMP)2、MMP9蛋白表达、细胞增殖、迁移和侵袭变化。LncBase Predicted v.2和双荧光素酶活性检测分析LncRNA PITPNA-AS1和miR-491-3p的靶向结合。结果与宫颈上皮永生化细胞H8比较,宫颈癌细胞系HeLa、SiHa、Caski中LncRNA PITPNA-AS1表达水平升高,miR-491-3p表达水平降低(P<0.05)。选择差异最显著的宫颈癌细胞HeLa进行后续研究。低表达LncRNA PITPNA-AS1或过表达miR-491-3p降低CyclinD1、MMP-2、MMP-9表达水平、细胞增殖、迁移和侵袭数量(P<0.05)。LncRNA PITPNA-AS1靶向miR-491-3p,并调控miR-491-3p的表达。anti-miR-491-3p逆转低表达LncRNA PITPNA-AS1对宫颈癌细胞HeLa增殖、迁移、侵袭的抑制作用。结论低表达LncRNA PITPNA-AS1通过靶向miR-491-3p基因,抑制宫颈癌细胞增殖、迁移、侵袭。

Objective To investigate the molecular mechanism of Long noncoding RNA PITPNA antisense RNA 1(PITPNA-AS1)targeting miR-491-3p gene to regulate the proliferation,migration and invasion of cervical cancer cell.Methods The qRT-PCR was used to detect the expression of LncRNA PITPNA-AS1 and miR-491-3p in cervical cancer cell lines(HeLa,SiHa,Caski)and cervical epithelial immortalized cells H8.Taking untreated cervical cancer cell HeLa as controls(NC group),and si-NC(si-NC group),si-LncRNA PITPNA-AS1(si-LncRNA PITPNA-AS1 group)were transfected into HeLa cells,and miR-NC(miR-NC group),miR-491-3p mimics(miR-491-3p group),pcDNA(pcDNA group),pcDNA-LncRNA PITPNA-AS1(pcDNA-LncRNA PITPNA-AS1 group),si-LncRNA PITPNA-AS1 and anti-miR-NC(si-LncRNA PITPNA-AS1+anti-miR-NC group),si-LncRNA PITPNA-AS1 and anti-miR-491-3p(si-LncRNA PITPNA-AS1+anti-miR-491-3p group)were transfected into HeLa cells.Western Blot,Cell Counting Kit-8(CCK-8)and Transwell method were performed to detect the expression levels of CyclinD1,Matrix metalloprotease(MMP)2,MMP9 protein,and cell proliferation,migration and invasion before and after transfection.Moreover LncBase Predicted v.2 and dual luciferase activity detection were performed to analyze the targeted binding of LncRNA PITPNA-AS1 and miR-491-3p.Results Compared with those in H8 cells,the expression levels of LncRNA PITPNA-AS1 in HeLa cells,SiHa,and Caski were significantly increased,however,the expression levels of miR-491-3p were significantly decreased(P<0.05).The low expression of LncRNA PITPNA-AS1 or overexpression of miR-491-3p decreased the expression levels of CyclinD1,MMP-2,MMP-9,cell proliferation,the number of migration and invasion in HeLa cells with significant differences(P<0.05).Moreovere LncRNA PITPNA-AS1 targeted miR-491-3p and regulated the expression levels of miR-491-3p.In addition anti-miR-491-3p reversed the inhibitory effects of low expression of LncRNA PITPNA-AS1 on the proliferation,migration and invasion of HeLa cells.Conclusion The low expression of LncRNA PITPNA-AS1 can inhibit the proliferation,migration and invasion of cervical cancer cells by targeting the miR-491-3p gene.