长链非编码RNA LINC00222对肺腺癌细胞增殖、迁移、侵袭和凋亡的影响及作用机制的研究

Effect of Long Non Coding RNA LINC00222 on Proliferation,Migration,Invasion and Apoptosis of Lung Adenocarcinoma Cells and Its Mechanism

目的检测肺腺癌组织及细胞系中LncRNA LINC00222表达,探究其对肺腺癌细胞增殖、迁移、侵袭和凋亡的影响和相关作用机制。方法采用实时荧光定量PCR(qRT-PCR)法和Western blot实验检测肺腺癌组织及细胞中LINC00222表达;构建LINC00222过表达载体,验证其转染效率;采用CCK-8法、Hoechst 33342/PI染色法、划痕实验及Transwell实验分别检测过表达LINC00222对肺腺癌细胞增殖、凋亡、迁移和侵袭的影响;采用Western blot法检测肺腺癌细胞中P-GSK-3β,GSK-3β蛋白及β-catenin核蛋白表达;采用荧光霉素基因实验及RIP实验验证探究LINC00222影响肺腺癌生物学行为的相关作用机制。结果肺腺癌组织中LINC00222 mRNA和蛋白表达明显低于癌旁正常组织,差异有统计学意义(t=7.388,15.100,均P<0.001);肺腺癌细胞系中LINC00222表达明显低于人正常肺胚细胞(F=21.926,P<0.001)。过表达LINC00222后,肺腺癌细胞增殖、迁移、侵袭能力明显抑制,细胞凋亡数目明显增多(P<0.01)。过表达LINC00222后,GSK-3β磷酸化明显降低,GSK-3β催化活性明显增强,β-catenin核转位受到抑制(P<0.01)。LINC00222靶向调控结合GSK-3β和GSK-3β蛋白共沉淀中LINC00222表达显著高于IgG蛋白共沉淀(P<0.05)。结论肺腺癌中LncRNA LINC00222低表达,其过表达可抑制肺腺癌细胞的增殖、迁移及侵袭,促进细胞凋亡,可能与其调控GSK-3β催化活性,抑制β-catenin核转位有关。

Objective The expression of lncRNA LINC00222 in lung adenocarcinoma tissues and cell lines was detected to explore the effects of lncRNA LINC00222 on the proliferation,migration,invasion and apoptosis of lung adenocarcinoma cells and the related mechanisms.Methods The expression of LINC00222 in lung adenocarcinoma tissues and cells was detected by qRT-PCR and Western blot.Overexpression vector LINC00222 was constructed to verify its transfection efficiency.CCK-8 assay,Hoechst 33342/PI staining assay,scratch assay and Transwell assay were used to detect the effects of overexpression of LINC00222 on proliferation,apoptosis,migration and invasion of lung adenocarcinoma cells.Western blot was used to detect the expression of P-GSK-3β,GSK-3βprotein andβ-catenin nuclear protein in lung adenocarcinoma cells.Luciferin gene experiment and RIP experiment were used to investigate the mechanism of LINC00222 affecting the biological behavior of lung adenocarcinoma.Results The mRNA and protein expressions of LINC00222 in lung adenocarcinoma tissues were significantly lower than those in adjacent normal tissues,the difference were statistically significant(t=7.388,15.100,all P<0.001).The expression of LINC00222 in lung adenocarcinoma cell lines was significantly lower than that in normal human lung embryo cell lines(F=21.926,P<0.001).After the overexpression of LINC00222,the proliferation,migration and invasion of lung adenocarcinoma cells were significantly inhibited,and the number of cell apoptosis was significantly increased(P<0.01).After overexpression of LINC00222,GSK-3βphosphorylation was significantly decreased,GSK-3βcatalytic activity was significantly enhanced,andβ-catenin nuclear translocation was inhibited(P<0.01).LINC00222 targeted the binding of GSK-3β,and the expression of LINC00222 in GSK-3βprotein co-precipitation was significantly higher than that in IgG protein coprecipitation(P<0.05).Conclusion LncRNA LINC00222 was low expressed in lung adenocarcinoma,and its overexpression inhibited the proliferation,migration and invasion of lung adenocarcinoma cells,and promoted cell apoptosis,which may be related to its regulation of GSK-3βcatalytic activity and inhibition ofβ-catenin nuclear translocation.