长链非编码RNA肿瘤蛋白翻译控制反义RNA 1对结肠癌细胞增殖和侵袭能力的影响

Effects of long non coding RNA tumor protein translation controlled antisense RNA 1 on proliferation and invasion of colon cancer cells

目的探讨长链非编码RNA(lncRNA)肿瘤蛋白翻译控制反义RNA 1(TPT1-AS1)在结肠癌组织中的表达和临床意义,及其对结肠癌细胞增殖、细胞周期、迁移和侵袭的影响。方法收集2016年7月至2017年9月于天津医科大学总医院胃肠外科进行结肠癌根治术的72例结肠癌患者的癌组织标本及相应的癌旁组织,采用实时定量反转录聚合酶链反应(RT-qPCR)检测lncRNA TPT1-AS1在结肠癌组织的相对表达量,并分析其表达与结肠癌临床病理特征的相关性。构建lncRNA TPT1-AS1敲低和对照载体(si-TPT1-AS1、si-NC),分别设为敲低组和对照组,采用细胞计数试剂盒(CCK-8)细胞增殖实验、细胞周期分析实验、Transwell迁移侵袭实验检测结肠癌细胞SW620的增殖、细胞周期阻滞、迁移和侵袭能力。计量资料组间比较采用t检验,计数资料采用χ^(2)检验。结果LncRNATPT1-AS1在结肠癌组织中的相对表达量显著高于对应的癌旁组织(3.99±0.21比1.78±0.09,t=35.433,P<0.05),且其表达与TNM分期(χ^(2)=9.000,P<0.05)和远处转移(χ^(2)=4.600,P<0.05)呈正相关,差异均有统计学意义。CCK-8实验结果显示,敲低TPT1-AS148 h(0.47±0.02比0.83±0.05,t=10.543,P<0.05)、72 h后(0.69±0.02比1.23±0.09,t=10.145,P<0.05),SW620细胞的增殖能力均显著低于对照组,差异均有统计学意义。细胞周期分析显示,敲低TPT1-AS1可使G0/G1期细胞比例高于对照组[(68.47±2.79)%比(44.69±1.26)%,t=14.672,P<0.05],S期细胞比例低于对照组[(22.87±1.96)%比(37.48±1.57)%,t=17.246,P<0.05],G2/M期细胞比例低于对照组[(8.22±0.87)%比(20.78±0.45)%,t=18.772,P<0.05],差异均有统计学意义。Transwell迁移侵袭实验结果表明,敲低TPT1-AS1可使SW620细胞的迁移能力显著低于对照组[[(87±8)个比(177±10)个,t=15.237,P<0.05],侵袭能力显著低于对照组[(47±5)个比(122±8)个,t=19.578,P<0.05],差异均有统计学意义。结论lncRNA TPT1-AS1在结肠癌组织中表达升高,敲低TPT1-AS1可抑制结肠癌细胞增殖、细胞周期G1/S期转换、迁移和侵袭。

Objective To investigate the expression and clinical significance of long non-coding RNA(lncRNA)tumor protein translation controlled antisense RNA 1(TPT1-AS1)in colon cancer and explore its effects on proliferation,cell cycle,migration and invasion of colon cancer cells.Methods Cancer tissue specimens and normal adjacent tissue specimens of 72 patients with colon cancer who underwent radical surgery for colon cancer in the Department of Gastrointestinal Surgery of Tianjin Medical University General Hospital from July 2016 to September 2017 were collected.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the relative expression of lncRNA TPT1-AS1 in colon cancer tissues,and the correlation between lncRNA TPT1-AS1 expression and clinicopathological characteristics of colon cancer was analyzed.lncRNA TPT1-AS1 knockdown vector and control vector(si-TP1-AS1 and si-NC)were constructed and set as knockdown group and control group respectively.Proliferation,cell cycle arrest,migration and invasion ability of colon cancer cells(SW620)were detected by cell counting kit-8(CCK-8)cell proliferation assay,cell cycle analysis and Transwell assay,respectively.T-test was used for comparison between metrology data groups,andχ^(2) test was used for enumulation data.Results The relative expression of lncRNA TP1-AS1 in colon cancer tissues was significantly higher than that in corresponding adjacent tissues(3.99±0.21 vs.1.78±0.09,t=35.433,P<0.05),and its expression was positively correlated with TNM staging(χ^(2)==9.000,P<0.05)and distant metastasis(χ^(2)=4.600,P<0.05).The results of CCK-8 assay showed that the proliferation ability of SW620 cells was significantly reduced after TPT1-AS1 knockdown for 48 h(0.47±0.02 vs.0.83±0.05,t=10.543,P<0.05)and 72 h(0.69±0.02 vs.1.23±0.09,t=10.145,P<0.05).Flow cytometry showed that after TPT1-AS1 knockdown,the proportion of cells in G0/G1 phase was significantly increased[(68.47±2.79)%vs.(44.69±1.26)%,t=14.672,P<0.05],and that in S phase[(22.87±1.96)%vs.(37.48±1.57)%,t=17.246,P<0.05]and G2/M phase[(8.22±0.87)%vs.(20.78±0.45)%,t=18.772,P<0.05]was dramatically decreased.Transwell assay showed that the migration and invasion ability of SW620 cells was significantly reduced after TPT1-AS1 was knocked down as compared with the control group(47±5 vs.122±8,t=19.578,P<0.05).Conclusion This study confirmed that lncRNA TPT1-AS1 was up-regulated in colon cancer.Knockdown of TPT1-AS1 can inhibit the proliferation,G1/S phase transition,migration and invasion of colon cancer cells.