TUBA1C促进人肺腺癌细胞系增殖和迁移的机制研究

Study on the Mechanism of TUBA1C Promoting Proliferation and Migration of Lung Adenocarcinoma Cells

目的探究TUBA1C在肺腺癌中的表达特征及其对人肺腺癌细胞系增殖、迁移的影响和相关潜在分子机制。方法通过临床数据库GEPIA检索TUBA1C在肺腺癌中的表达特征以及与临床预后相关性;通过30组临床组织样本探究TUBA1C在肺腺癌及其临近正常组织中的表达特征;通过siRNA介导敲低TUBA1C表达,利用细胞增殖实验、克隆形成实验、划痕迁移实验和细胞周期实验分别检测TUBA1C对肺腺癌细胞生物学行为的影响;通过分析与TUBA1C共表达基因参与的信号通路,探索TUBA1C在肺腺癌发展进程中的潜在分子机制,并进一步通过回补实验进行验证。结果GEPIA数据库检索显示肺腺癌中TUBA1C显著高表达(P<0.05),且高表达的病人具有不良预后(logrank P=9.3E-5)。30组肺腺癌临床样本中TUBA1C表达水平(6.4±1.3)显著高于癌旁正常组织(5.2±0.9),差异有统计学意义(t=4.157,P<0.001),且随着癌症的不断恶化,TUBA1C表达逐渐升高(P=0.00349)。转染培养3,4和5天时siTUBA1CA#1组和siTUBA1CA#2组细胞增殖A值明显低于对照组,差异均有统计学意义(F=7.000~27.780,P<0.05)。siTUBA1CA#1组(41.2±1.5)和siTUBA1CA#2组(40.3±1.3)细胞克隆形成率明显低于对照组(72.4±2.2),差异有统计学意义(F=342.482,P<0.001);两实验组细胞迁移率为73.4±3.2和72.1±2.8,明显低于对照组(98.6±1.7),差异有统计学意义(F=95.778,P<0.001);细胞周期较对照组相比显著阻滞在G1期。siTUBA1CA#1组(0.21±0.02,0.33±0.03)和siTUBA1CA#2组(0.20±0.03,0.36±0.02)细胞中E2F1和MYC mRNA表达水平较对照组(1.01±0.03,1.00±0.02)明显降低,差异有统计学意义(F=883.773,758.294,均P<0.001)。在敲低TUBA1C表达的细胞中分别回补E2F1,MYC以及共回补E2F1和MYC后,细胞增殖速率、迁移速率及细胞周期较单纯敲低TUBA1C组相比均逐渐恢复正常(P<0.01),接近正常水平。结论肺腺癌中TUBA1C高表达,通过激活促癌基因E2F1和MYC的表达促进肺腺癌细胞的增殖、克隆形成及迁移,诱导细胞凋亡,参与促进肺腺癌的发展进程。

Objective To explore the expression characteristics of TubA1c in lung adenocarcinoma and its effect on the proliferation and migration of human lung adenocarcinoma lines,as well as the related potential molecular mechanisms.Methods The expression characteristics of TUBA1C in lung adenocarcinoma and its correlation with clinical prognosis were retrieved through clinical database GEPIA,and the expression characteristics of TUBA1C in lung adenocarcinoma and its adjacent normal tissues were investigated through 30 groups of clinical tissue samples.SiRNA mediated knockdown of TUBA1C expression was used to detect the effect of TUBA1C on the biological behavior of lung adenocarcinoma cells by cell proliferation experiment,clone formation experiment,scratch migration experiment and cell cycle experiment,respectively.By analyzing the signaling pathways involved in TUBA1C co-expressed genes,the potential molecular mechanism of TUBA1C in the development of lung adenocarcinoma was explored and further verified by the complement experiment.Results GEPIA database retrieval showed that the high expression of TUBA1C in lung adenocarcinoma was significant(P<0.05),and the patients with high expression had a poor prognosis(Logrank P=9.3E-5).The expression level of TUBA1c in 30 groups of lung adenocarcinoma clinical samples(6.4±1.3)was significantly higher than that in adjacent normal tissues(5.2±0.9),the difference was statistically significant(t=4.157,P<0.001),and TUBA1C expression gradually increased with the progression of cancer(P=0.00349).The OD value of cell proliferation in Situba1CA#1 and Situba1CA#2 groups was significantly lower than that in control group,the difference were statistically significant(F=7.000~27.780,all P<0.05)after 3,4,and 5 days of transfection culture.The cell clone formation rate in Situba1CA#1 group(41.2±1.5)and Situba1CA#2 group(40.3±1.3)was significantly lower than that in control group(72.4±2.2),the difference was statistically significant(F=342.482,P<0.001).The cell migration rates of the two experimental groups were(73.4±3.2)and(72.1±2.8),respectively,with significantly lower than that of the control group(98.6±1.7),the difference was statistically significant(F=95.778,P<0.001).Cell cycle was significantly arrested in the G1 phase compared with the control group.The mRNA expression levels of E2F1 and MYC in SiTUBA1CA#1 group(0.21±0.02,0.33±0.03)and SITUBA1CA#2 group(0.20±0.03,0.36±0.02)were higher than those in the control group(1.01±0.03,1.00±0.02)significantly decreased,the difference were statistically significant(F=883.773,758.294,all P<0.001).After E2F1 and MYC were supplemented separately and in combination with E2F1 and MYC,the cell proliferation rate,migration rate and cell cycle gradually returned to normal(P<0.01)in the knockdown group compared with that in the knockdown group alone.Conclusion The high expression of TUBA1C in lung adenocarcinoma promotes the proliferation,cloning,formation and migration of lung adenocarcinoma cells,induces cell apoptosis,and participates in the development of lung adenocarcinoma by activating the expression of oncogenic genes E2F1 and MYC.