LAMC3在肺腺癌中的表达及对细胞生物学行为的影响

Expression of LAMC3 in lung adenocarcinoma and its influence on cell biological behavior

目的:探讨层粘连蛋白亚基γ3(laminin subunit gamma 3,LAMC3)在肺腺癌(lung adenocarcinoma,LUAD)中的表达及对细胞生物学行为的影响。方法:通过在线生物信息学数据库GEPIA查询LAMC3在LUAD组织中的表达情况;收集2018年1月至2020年12月本院收治的行LUAD手术的48例LUAD患者的LUAD组织及癌旁组织,使用RT-qPCR检测了组织中LAMC3的mRNA表达水平,并分析LAMC3表达水平与LUAD患者临床病理特征的相关性。体外培养人LUAD细胞株(A549、H1299、PC-9)和人正常肺细胞株MRC-5,使用RT-qPCR和Western blot检测了细胞中LAMC3的mRNA和蛋白表达水平。将PC-9细胞分为5组:对照组(control组)、si-NC组、LAMC3沉默组、pcDNA3.1-NC组、LAMC3过表达组,采用小分子RNA干扰技术(siRNA)和pcDNA3.1质粒转染构建LAMC3沉默/过表达细胞,RT-qPCR和Western blot检测各组细胞中LAMC3的mRNA和蛋白表达水平;CCK-8法检测各组细胞增殖活力;流式细胞术检测各组细胞凋亡率;Transwell小室实验检测各组细胞的迁移、侵袭能力;Western blot检测各组细胞中上皮间质转化(epithelial-mesenchymal transition,EMT)相关蛋白[E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin,VIM)、基质金属蛋白9(matrix metalloproteinase 9,MMP-9)]的表达。结果:LAMC3在LUAD组织和细胞中低表达,且LAMC3低表达与肿瘤大小、TNM分期和淋巴结转移显著相关(P<0.05)。沉默LAMC3后,细胞的增殖、迁移和侵袭能力以及N-cadherin、VIM、MMP-9的表达升高,细胞凋亡率和E-cadherin的表达降低(P<0.05);过表达LAMC3后,细胞的增殖、迁移和侵袭能力降低,细胞凋亡率升高(P<0.05),且LAMC3过表达后细胞中E-cadherin的表达升高,N-cadherin、VIM、MMP-9的表达降低(P<0.05)。结论:LAMC3在LUAD的发生发展中发挥着重要作用,过表达LAMC3可抑制LUAD细胞的增殖、迁移和侵袭,并诱导细胞凋亡,其作用机制可能与抑制EMT有关。

Objective:To explore the expression of laminin subunit gamma 3(LAMC3)in lung adenocarcinoma(LUAD)and its influence on cell biological behavior.Methods:The online bioinformatics database GEPIA was used to query the expression of LAMC3 in LUAD tissues.The LUAD tissues and adjacent tissues were collected from 48 LUAD patients who underwent LUAD surgery in our hospital from January 2018 to December 2020,and RT-qPCR was used to detect the mRNA expression level of LAMC3 in the tissues,and the correlation between the expression level of LAMC3 and the clinicopathological characteristics of LUAD patients was analyzed.Human LUAD cell line(A549,H1299,PC-9)and human normal lung cell line MRC-5 were cultured in vitro,and RT-qPCR and Western blot were used to detect the mRNA and protein expression levels of LAMC3 in the cells.PC-9 cells were divided into five groups:Control group,si-NC group,LAMC3 silence group,pcDNA3.1-NC group and LAMC3 overexpression group.The small molecule RNA interference technology(siRNA)and pcDNA3.1 plasmid transfection were used to construct LAMC3 silence/overexpression cells.RT-qPCR and Western blot were used to detect the mRNA and protein expression levels of LAMC3 in each group of cells.CCK-8 method was used to detect cell proliferation activity in each group.Flow cytometry was used to detect the apoptosis rate of each group.Transwell chamber experiment was used to detect the migration and invasion abilities of each group of cells,and Western blot was used to detect the expression of epithelial-mesenchymal transition(EMT)related proteins[E-cadherin,N-cadherin,vimentin(VIM),matrix metalloproteinase 9(MMP-9)]of cells in each group.Results:LAMC3 was lowly expressed in LUAD tissues and cells,and the low expression of LAMC3 was significantly correlated with tumor size,TNM stage and lymph node metastasis(P<0.05).After silencing LAMC3,the abilities of cell proliferation,migration and invasion,and the expression of N-cadherin,VIM and MMP-9 increased,the apoptosis rate and the expression of E-cadherin decreased(P<0.05).After overexpression of LAMC3,the abilities of cell proliferation,migration and invasion decreased,the cell apoptosis rate increased(P<0.05),and the expression of E-cadherin in the cells increased,the expression of N-cadherin,VIM and MMP-9 decreased(P<0.05).Conclusion:LAMC3 plays an important role in the occurrence and development of LUAD.Overexpression of LAMC3 can inhibit the proliferation,migration and invasion of LUAD cells,and induce cell apoptosis.Its mechanism may be related to the inhibition of EMT.