长基因间非编码RNA 842对非小细胞肺癌细胞侵袭迁移和Wnt/β-catenin信号通路的影响

Effects of long intergenic non-coding RNA 842 on invasion,migration and Wnt/β-catenin signaling pathway of non-small cell lung cancer cells

目的探讨长基因间非编码RNA 842(LINC00842)对非小细胞肺癌(NSCLC)细胞侵袭迁移和Wnt/β-连环蛋白(β-catenin)信号通路的影响。方法从美国国家生物技术信息中心的GEO数据库中下载NSCLC数据集GSE30219的series matrix数据用于分析NSCLC组织的LINC00842水平。采用实时荧光定量PCR(qPCR)检测正常肺上皮细胞BEAS-2B及NSCLC细胞系(NCI-H1299、SPC-A-1、A549和NCI-H1975)的LINC00842水平。脂质体法向NCI-H1975细胞转染LINC00842过表达质粒pcDNA3.1-LINC00842(过表达组)和pcDNA3.1空质粒(空载体组)并设未转染的细胞为对照组。细胞计数试剂盒8(CCK-8)、划痕实验和Transwell小室实验检测细胞增殖活力、划痕愈合率和穿膜细胞数,qPCR和Western blotting检测wnt1和β-catenin水平。结果GSE30219数据集中的基因表达数据显示272例NSCLC组织的LINC00842水平为3.385±0.218,低于14例正常组织的3.538±0.137(P<0.05)。NSCLC细胞的LINC00842水平均低于BEAS-2B细胞(P<0.05),选取LINC00842水平最低的NCI-H1975细胞进行后续的功能验证实验。过表达组NCI-H1975细胞转染48 h后的LINC00842水平高于其余两组(P<0.05)。与对照组和空载体组相比,在转染处理后48、72 h,过表达组NCI-H1975细胞的增殖活力降低(P<0.05)。过表达组NCI-H1975细胞的划痕愈合率和穿膜细胞数分别为(25.764±2.584)%和(159.987±13.203)个,低于对照组的(78.551±6.726)%和(279.464±20.427)个及空载体组的(76.945±4.370)%和(265.823±21.246)个(P<0.05)。与对照组和空载体组相比,过表达组NCI-H1975细胞的wnt1和β-catenin水平均降低(P<0.05)。对照组和空载体组上述指标的差异无统计学意义(P>0.05)。结论LINC00842在NSCLC组织和细胞中表达均下调,增强LINC00842表达可抑制细胞的增殖和侵袭迁移能力及Wnt/β-catenin信号通路的激活,可能通过调控Wnt/β-catenin信号通路来发挥抑癌作用。

Objective To investigate the effect of long intergenic non-coding RNA 842(LINC00842)on invasion,migration and Wnt/β-catenin signaling pathway of non-small cell lung cancer(NSCLC)cells.Methods The series matrix data of NSCLC dataset GSE30219 was downloaded from the GEO database of National Center for Biotechnology Information to analyze LINC00842 levels of NSCLC tissues.Levels of LINC00842 in normal lung epithelial cell BEAS-2B and NSCLC cells(NCI-H1299,SPC-A-1,A549 and NCI-H1975)were detected by real-time quantitative PCR(qPCR).NCI-H1975 cells were transfected with LINC00842 overexpression plasmid pcDNA3.1-LINC00842(Overexpression group)and pcDNA3.1 empty plasmid(Empty vector group)by liposome method,and untransfected cells were set as Control group.Proliferating vitality,wound healing rate and number of cells penetrating the membrane were detected by cell counting kit 8(CCK-8),scratch test and Transwell chamber test.Levels of wnt1 andβ-catenin were detected by qPCR and Western blotting.Results Gene expression data from GSE30219 data set showed that the LINC00842 level in 272 cases of NSCLC tissues was 3.385±0.218,lower than 3.538±0.137 of 14 cases of normal tissues(P<0.05).The LINC00842 levels in NSCLC cells were lower than that in BEAS-2B cells(P<0.05),and NCI-H1975 cells with the lowest LINC00842 level were selected for subsequent functional verification test.LINC00842 level of NCI-H1975 cells in the Overexpression group was higher than that in other two groups at 48 h after transfection(P<0.05).Compared with Control group and Empty vector group,the proliferation activity of NCI-H1975 cells in the Overexpression group decreased at 48 and 72 h after transfection(P<0.05).The wound healing rate and number of penetrating cells in Overexpression group were(25.764±2.584)%and 159.987±13.203,lower than(78.551±6.726)%and 279.464±20.427 in Control group and(76.945±4.370)%and 265.823±21.246 in Empty vector group(P<0.05).Compared with Control group and Empty vector group,levels of wnt1 andβ-catenin of NCI-H1975 cells in Overexpression group were significantly lower than those in Control group and Empty vector group(P<0.05).There was no significant difference on the above indexes between Control group and Empty vector group(P>0.05).Conclusion LINC00842 was down-regulated in both NSCLC tissues and cells.Enforced expression of LINC00842 repressed the abilities of proliferation,migration and invasion of NSCLC cells,and inhibited Wnt/β-catenin pathway.LINC00842 served a tumor suppressor role in NSCLC cells possibly by regulating Wnt/β-catenin axis.