LncRNA CCAT1通过TGF-β/Smad信号通路对子宫内膜癌细胞增殖、侵袭和迁移的影响

doi:10.13481/j.1671-587x.20200519

摘要/Abstract

摘要： 目的：探讨长链非编码核糖核酸（LncRNA）CCAT1对子宫内膜癌细胞增殖、侵袭和迁移及转化生长因子β（TGF-β）/Smad信号通路的影响，阐明LncRNA CCAT1在子宫内膜癌发生发展中的作用及可能机制。方法：将人子宫内膜癌Ishiwaka细胞分为空白对照组、阴性对照组、CCAT1-siRNA组和CCAT1-siRNA+LY364947组，阴性对照组细胞转染阴性对照siRNA，CCAT1-siRNA组转染CCAT1-siRNA，CCAT1-siRNA+LY364947组细胞转染CCAT1-siRNA同时加入TGF-β/Smad抑制剂LY364947（3 μL），空白对照组细胞不转染。采用RT-PCR法检测各组细胞中CCAT1 mRNA表达水平，CCK8法检测各组细胞增殖能力，Transwell小室实验检测细胞侵袭和迁移能力，Western blotting法检测各组Ishiwaka细胞中增殖细胞核抗原（PCNA）、E-钙黏蛋白（E-cadherin）、波形蛋白（vimentin）、锌指转录因子（snail）、凋亡抑制蛋白（Twist）、白细胞抑制因子2/3（Smad2/3）、磷酸化Smad（p-Smad2/3）和转化生长因子β1（TGF-β1）蛋白表达水平。结果：Ishiwaka细胞中CCAT1 mRNA表达水平明显高于人子宫内膜基质T-HESC细胞（t=12.929，P<0.01）；与空白对照组和阴性对照组比较，CCAT1-siRNA组和CCAT1-siRNA+LY364947组Ishiwaka细胞中CCAT1 mRNA表达水平、细胞增殖能力、侵袭细胞数和迁移细胞数均明显降低（P<0.05），PCNA、vimentin、snail、Twist、p-Smad2/3和TGF-β1蛋白表达水平均明显降低（P<0.05），E-cadherin蛋白表达水平明显升高（P<0.05）；与CCAT1-siRNA组比较，CCAT1-siRNA+LY364947组Ishiwaka细胞增殖能力、侵袭细胞数和迁移细胞数均明显降低（P<0.05），PCNA、vimentin、snail、Twist、p-Smad2/3和TGF-β1蛋白表达水平降低（P<0.05），E-cadherin蛋白表达水平明显升高（P<0.05）；空白对照组与阴性对照组Ishiwaka细胞中各指标比较差异无统计学意义（P>0.05）。结论：沉默LncRNA CCAT1可通过抑制TGF-β/Smad信号通路抑制子宫内膜癌细胞增殖、侵袭和迁移。

关键词: 长链非编码RNA, 子宫内膜癌, 细胞增殖, 细胞侵袭, 细胞迁移, 上皮间质转化, 转化生长因子-β/Smad信号通路

Abstract: Objective: To investigate the effects of long-chain non-coding RNA (LncRNA) CCAT1 on the proliferation, invasion, migration and transforming growth factor-β (TGF-β)/Smad signaling pathway of the endometrial cancer cells,and to elucidate the role of CCAT1 in the occurrence and development of endometrial cancer and its possible mechanism. Methods: The human endometrial cancer Ishiwaka cells were divided into blank control group, negative control group, CCAT1-siRNA group and CCAT1-siRNA+LY364947 group.The cells in negative control group were transfected with negative control siRNA,the cells in CCAT1-siRNA group were transfected with CCAT1-siRNA; the cells in CCAT1-siRNA+LY364947 group were transfected with CCAT1-siRNA and added with LY364947 (3μL),and the cells in blank control group were not transfected.RT-PCR method was used to determine the CCAT1 miRNA expression levels.CCK8 method was used to measure proliferation abilities of cells.Transwell chamber experiment was used to measure the abilities of invasion and migration of cells.Western blotting method was used to measure the expression levels of proliferating cell nuclear antigen (PCNA), E-cadherin, vimentin, snail, Twist, Smad2/3, phosphorylated Smad2/3 (p-Smad2/3) and TGF-β1 proteins in the Ishiwaka cells in various groups. Results: The expression level of CCAT1 mRNA in the Ishiwaka cells was very obvious higher than that in the human endometrial matrix T-HESC cells (t=12.929, P<0.01).Compared with blank control group and negative control group,the CCAT1 miRNA expression levels, the proliferation abilities of cells the number of invasion cells and the number migration cells in CCAT1-siRNA group and CCAT1-siRNA+LY36494 group were obviously decreased (P<0.05),the expression levels of PCNA, vimentin, snail, Twist, p-Smad2/3 and TGF-β1 proteins were obviously decreased (P<0.05),and the expression levels of E-cadherin protein were obviously increased (P<0.05). Compared with CCAT1-siRNA group, the proliferation ability of cells, the number of invasion cells and the number of migration cells in CCAT1-siRNA+LY364947 group were obviously decreased (P<0.05),the expression levels of PCNA, vimentin, snail, Twist, p-Smad2/3 and TGF-β1 proteins were obviously decreased (P<0.05),and the expression level of E-cadherin protein was obviously increased (P<0.05).There were no significant differences in the indexes mentioned above in the Ishiwaka cells between blank control group and negative control group (P>0.05). Conclusion: Silencing LncRNA CCAT1 can inhibit the proliferation, invasion and migration of endometrial cancer cells by inhibiting the TGF-β/Smad signaling pathway.

Key words: long non-coding RNA, endometrial cancer, cell proliferation, cell invasion, cell migration, epithelial-mesenchymal transition, transforming growth factor-β/Smad signaling pathway

中图分类号:

R737.33

魏旭静, 李林, 张红真, 王景, 徐静. LncRNA CCAT1通过TGF-β/Smad信号通路对子宫内膜癌细胞增殖、侵袭和迁移的影响[J]. 吉林大学学报(医学版), 2020, 46(05): 1016-1022.

WEI Xujing, LI Lin, ZHANG Hongzhen, WANG Jing, XU Jing. Effects of LncRNA CCAT1 on proliferation,invasion and migration of endometrial cancer cells through TGF-β1/smad signaling pathway[J]. Journal of Jilin University(Medicine Edition), 2020, 46(05): 1016-1022.

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