lncRNA SMAD5-AS1在肺腺癌细胞中的表达及其通过Wnt/β-catenin通路对癌细胞增殖、侵袭、迁移的影响

Expression of lncRNA SMAD5-AS1 in Lung Adenocarcinoma Cells and Its Effect on Cancer Cell Proliferation,Invasion and Migration Through Regulating Wnt/β-catenin Pathway

目的探讨长链非编码RNA(lncRNA)SMAD家族成员5反义RNA1(SMAD5-AS1)在肺腺癌(LUAD)中的表达及其对LUAD细胞恶性生物学行为的影响,并分析其潜在机制。方法通过生物信息学网站对LUAD组织中lncRNA SMAD5-AS1的表达进行分析;应用实时荧光定量PCR(qRT-PCR)检测72例LUAD组织与邻近正常组织以及人正常支气管上皮细胞(BEAS-2B)与人LUAD细胞(SPC-A-1、A549、H1299、LTEP-a-2)中lncRNA SMAD5-AS1表达水平。体外培养对数生长期的SPC-A-1和H1299细胞,分为SPC-A-1对照组(SPC-A-1-Ctr组)、SPC-A-1+sh-NC组、SPC-A-1+sh-SMAD5-AS1组、H1299对照组(H1299-Ctr组)、H1299+LV5-NC组、H1299+LV5-SMAD5-AS1组。采用携带lncRNA SMAD5-AS1敲除的短发夹RNA(sh-SMAD5-AS1)和lncRNA SMAD5-AS1过表达的重组质粒(LV5-SMAD5-AS1)及相应空载体的重组慢病毒转染细胞,qRT-PCR验证转染效率;5-乙炔基-2′-脱氧尿苷(EdU)测定细胞增殖活性;划痕愈合实验和Transwell小室实验检测细胞迁移、侵袭能力;Western blot检测细胞上皮间质转化(EMT)和Wnt/β-连环蛋白(β-catenin)通路相关蛋白[E-钙粘蛋白(E-cadherin)、N-钙粘蛋白(N-cadherin)、波形蛋白(Vimentin)、糖原合酶激酶-3β(GSK-3β)、β-catenin]的表达。结果lncRNA SMAD5-AS1在LUAD组织和细胞中呈高表达(均P<0.05);敲低lncRNA SMAD5-AS1表达后,SPC-A-1细胞的增殖活性、侵袭和迁移能力及N-cadherin、Vimentin和细胞核β-catenin蛋白水平显著降低(均P<0.05),E-cadherin和GSK-3β蛋白水平显著升高(均P<0.05);过表达lncRNA SMAD5-AS1后,H1299细胞的增殖活性、侵袭和迁移能力及N-cadherin、Vimentin和细胞核β-catenin蛋白水平显著升高(均P<0.05),E-cadherin和GSK-3β蛋白水平显著降低(均P<0.05)。结论lncRNA SMAD5-AS1在LUAD中高表达,其可能通过激活Wnt/β-catenin通路促进LUAD细胞增殖、迁移、侵袭和EMT。

Objective To explore the expression level of long non-coding RNA(lncRNA)SMAD family member 5 antisense RNA1(SMAD5-AS1)in lung adenocarcinoma(LUAD)and its effect on the malignant biological behavior of LUAD cells,and to analyze its underlying mechanism.Methods The expression levels of lncRNA SMAD5-AS1 in LUAD tissues were analyzed through the bioinformatics database;real-time fluorescent quantitative PCR(qRT-PCR)was used to detect the expression levels of lncRNA SMAD5-AS1 in 72 cases of LUAD tissues,adjacent normal tissues,human normal bronchial epithelial cells(BEAS-2B)and human LUAD cells(SPC-A-1,A549,H1299,LTEP-a-2).SPC-A-1 and H1299 cells in logarithmic growth phase were cultured in vitro and divided into SPC-A-1 control group(SPC-A-1-Ctr group),SPC-A-1+sh-NC group,SPC-A-1+sh-SMAD5-AS1 group,H1299 control group(H1299-Ctr group),H1299+LV5-NC group,and H1299+LV5-SMAD5-AS1 group.Cells were transfected with recombinant lentivirus carrying lncRNA SMAD5-AS1 knockout short hairpin RNA(sh-SMAD5-AS1)recombinant plasmids,lncRNA SMAD5-AS1 overexpressed(LV5-SMAD5-AS1)recombinant plasmids and corresponding empty vectors,qRT-PCR was performed to verify the transfection efficiencies;5-ethynyl-2′-deoxyuridine(EdU)was used to detect cell proliferation activity;scratch healing test and Transwell chamber test were performed to measure cell migration and invasion abilities;Western blot was performed to measure the expression levels of cell epithelial-mesenchymal transition(EMT)and Wnt/β-catenin(β-catenin)pathway related proteins[E-cadherin,N-cadherin,Vimentin,glycogen synthase kinase-3β(GSK-3β),andβ-catenin].Results lncRNA SMAD5-AS1 was highly expressed in LUAD tissues and cells(all P<0.05);after knockdown of lncRNA SMAD5-AS1,the proliferation activity,invasion and migration ability of SPC-A-1 cells and levels of N-cadherin,Vimentin and nuclearβ-catenin proteins were significantly reduced(all P<0.05),and the levels of E-cadherin and GSK-3βproteins were also significantly increased(all P<0.05);after upregulation of lncRNA SMAD5-AS1 expression,the proliferation,invasion and migration ability of H1299 cells and the levels of N-cadherin,Vimentin and nuclearβ-catenin proteins were significantly increased(all P<0.05),and the levels of E-cadherin and GSK-3βproteins were also significantly reduced(all P<0.05).Conclusion lncRNA SMAD5-AS1 is upregulated in LUAD,and it can promote cell proliferation,migration,invasion and EMT by activation of Wnt/β-catenin pathway.