LINC02532靶向miR-145影响胰腺癌干细胞的增殖、迁移、侵袭和凋亡

Effects of LINC02532 targeting miR-145 on proliferation,migration,invasion and apoptosis of pancreatic cancer stem cells

背景:长链非编码RNA(lncRNA)和微小RNA(miRNA)是两类参与调控细胞增殖、凋亡和迁移等生命活动的小分子非编码RNA,且lncRNA还可作为竞争性内源性RNA与miRNA靶向结合,调控miRNA靶基因的表达,在肿瘤的发生发展中起重要作用。LINC02532可靶向结合miR-129-5p和miR-490-5p促进胃癌细胞增殖、迁移和侵袭,但对胰腺癌干细胞生物学行为的影响及作用机制还未知。目的:探讨LINC02532对胰腺癌干细胞增殖、迁移、侵袭和凋亡的影响及作用机制。方法:①选取2017年1月至2018年6月于华北理工大学附属医院经病理检测证实为胰腺癌且经手术切除的56例患者胰腺癌组织及癌旁组织,实时荧光定量PCR(RT-qPCR)检测LINC02532和miR-145的表达水平;②以CD24+CD44+ESA+为表面标记,流式细胞术在人胰腺癌细胞PANC-1中分选胰腺癌干细胞,分为正常组、si-LINC02532组(转染LINC02532小干扰RNA)、si-NC组(转染乱序无意义阴性序列)、si-LINC02532+anti-miR-145组(共转染LINC02532小干扰RNA和miR-145抑制剂)和si-LINC02532+anti-miR-NC组(共转染LINC02532小干扰RNA和miR-145抑制剂阴性对照序列)。转染12 h后,RT-qPCR检测各组细胞中LINC02532和miR-145表达水平,CCK-8法检测细胞存活率,克隆形成实验检测细胞克隆形成数,Transwell小室检测细胞迁移和侵袭能力,流式细胞术检测细胞凋亡率,Western blot法检测细胞中P21、Bax、Caspases-3、E-钙黏附素和基质金属蛋白酶2蛋白表达水平。双荧光素酶报告基因实验验证miR-145与LINC02532调控关系。结果与结论:①与癌旁组织比较,胰腺癌组织中LINC02532表达水平升高(P<0.05),miR-145表达水平降低(P<0.05);②与si-NC组比较,si-LINC02532组胰腺癌干细胞存活率、克隆形成数、细胞迁移和侵袭数及基质金属蛋白酶2蛋白表达水平降低(P<0.05),细胞凋亡率及P21、Bax、Caspase-3和E-钙黏附素蛋白表达水平升高(P<0.05),而si-NC组与正常组各检测指标比较差异无显著性意义(P>0.05);③LINC02532在胰腺癌干细胞中靶向负调控miR-145表达。与si-LINC02532+anti-miR-NC组比较,si-LINC02532+anti-miR-145组胰腺癌干细胞存活率、克隆形成数、细胞迁移和侵袭数及基质金属蛋白酶2蛋白表达水平升高(P<0.05),细胞凋亡率及P21、Bax、Caspase-3和E-钙黏附素蛋白表达水平降低(P<0.05);④结果表明,LINC02532在胰腺癌组织中表达上调,下调其表达可能通过靶向上调miR-145表达进而抑制胰腺癌干细胞增殖、迁移和侵袭,并诱导细胞凋亡。

BACKGROUND:Long non coding RNA(lncRNA)and microRNA(miRNA)are two types of small non-coding RNA that are involved in regulating cell proliferation,apoptosis and migration and other life activities,and lncRNA can also be used as a competitive endogenous RNA and miRNA target binding to regulate the expression of miRNA target genes and play an important role in the occurrence and development of tumors.LINC02532 is an lncRNA that can target miR-129-5p and miR-490-5p to promote the proliferation,migration and invasion of gastric cancer cells,but its effect on the biological behavior of pancreatic cancer stem cells and the action mechanism are still unknown.OBJECTIVE:To investigate the effect and action mechanism of LINC02532 on the proliferation,migration,invasion and apoptosis of pancreatic cancer stem cells.METHODS:(1)Fifty-six patients with pancreatic cancer confirmed by pathological examination in North China University of Science and Technology Affiliated Hospital from January 2017 to June 2018 were selected as the research subjects.Real time fluorescent quantitative PCR was used to detect the expression levels of LINC02532 and miR-145 in pancreatic cancer tissues and adjacent tissues.(2)Using CD24+CD44+ESA+as surface markers,pancreatic cancer stem cells were sorted from human pancreatic cancer cells PANC-1 by flow cytometry.Pancreatic cancer stem cells were divided into normal group,si-LINC02532 group(transfected with LINC02532 small interfering RNA),si-NC group(transfected with disorderly negative sequence),si-LINC02532+anti-miR-145 group(co-transfected with LINC02532 small interfering RNA and miR-145 inhibitor)and si-LINC02532+anti-miR-NC group(co-transfected with LINC02532 small interfering RNA and miR-145 inhibitor negative control sequence).At 12 hours after transfection,real time fluorescent quantitative PCR was used to detect the expression levels of LINC02532 and miR-145 in each group of cells.Cell counting kit-8(CCK-8)assay was utilized to detect the cell survival rate.The colony formation test was applied to detect the number of cell clones formed.Transwell chamber was used to detect cell migration and invasion.Flow cytometry was utilized to detect cell apoptosis rate.Western blot assay was used to detect the expression levels of P21,Bax,Caspase-3,E-cadherin and MMP-2 protein.The double luciferase reporter gene experiment was utilized to verify the regulatory relationship between miR-145 and LINC02532.RESULTS AND CONCLUSION:(1)Compared with adjacent tissues,the expression level of LINC02532 increased in pancreatic cancer tissues(P<0.05),while the expression level of miR-145 decreased(P<0.05).(2)Compared with the si-NC group,the survival rate,colony formation,cell migration and invasion number,and MMP-2 protein expression level of pancreatic cancer stem cells reduced in the si-LINC02532 group(P<0.05),while the apoptotic rate and the expression levels of P21 and Bax,Caspase-3 and E-cadherin protein increased(P<0.05),but there was no significant difference between the si-NC group and the normal group(P>0.05).(3)LINC02532 targeted negatively regulated miR-145 expression in pancreatic cancer stem cells.Compared with the si-LINC02532+antimiR-NC group,the survival rate,colony formation,cell migration and invasion number,and MMP-2 protein expression level of pancreatic cancer stem cells increased in the si-LINC02532+anti-miR-145 group(P<0.05),while the apoptotic rate and the expression levels of P21 and Bax,Caspase-3 and E-cadherin protein reduced(P<0.05).(4)The results show that LINC02532 was up-regulated in pancreatic cancer tissues,and down-regulating its expression may inhibit the proliferation,migration and invasion of pancreatic cancer stem cells by targeting up-regulation of miR-145 expression,and induce apoptosis.