三叶因子3/磷脂酰肌醇-3激酶/蛋白激酶B/核因子-κB在人甲状腺乳头状癌细胞系TPC-1增殖和凋亡中的作用

doi:10.16098/j.issn.0529-1356.2020.04.009

摘要/Abstract

摘要：

目的探讨三叶因子3（TFF3）对人甲状腺乳头状癌细胞系TPC-1细胞增殖和凋亡的影响及分子机制。方法构建TFF3基因过表达和敲低表达的慢病毒表达载体，包装293T细胞，产生慢病毒颗粒，病毒液转染TPC-1细胞，构建稳定增强TFF3表达的TFF3-TPC-1细胞系以及增强对照组细胞系ConTFF3-TPC-1；构建抑制TFF3表达的shRNA-TFF3-TPC-1细胞系以及沉默对照组细胞系shCon-TPC-1；利用Western blotting实验与Real-time PCR验证TFF3过表达和沉默效率；生长曲线和集落形成实验检测4组细胞增殖状况；流式细胞术检测4组细胞凋亡情况；Western blotting和免疫细胞化学染色检测凋亡相关蛋白和磷脂酰肌醇-3激酶/蛋白激酶B（PI3K/Akt）、核因子-κB (NF-κB)通路蛋白表达水平。结果 1. 成功构建TFF3过表达和抑制表达稳转细胞系TFF3-TPC-1及细胞系shRNA-TFF3-TPC-1；2. TFF3-TPC-1细胞增殖能力和集落形成能力明显高于ConTFF3-TPC-1组（P＜0.05或 P＜0.01），shRNA-TFF3-TPC-1细胞增殖能力和集落形成能力明显低于shCon-TPC-1组（P＜0.01）；3. TFF3-TPC-1细胞凋亡率低于ConTFF3-TPC-1（0.75%±0.08% vs 5.62%±0.3%，P＜0.01），shRNA-TFF3-TPC-1凋亡率高于shCon TPC-1（22.2%±1.2 % vs 5.34%±0.4%，P＜0.01）； 4. 沉默TFF3基因后，Bax、细胞色素C（Cyt-C）、cleaved-Caspase-9、cleaved-Caspase-3表达升高，Bcl-2、通路蛋白Akt、p-Akt、NF-κB-P65表达降低（P＜0.05或 P＜0.01）。结论 TFF3可能通过影响PI3K/Akt/NF-κB信号通路调节TPC-1细胞的增殖和凋亡。

关键词: 三叶因子3, 磷脂酰肌醇3-激酶/蛋白激酶B/核因子κB, 甲状腺乳头癌, 慢病毒转染, 免疫印迹法, 流式细胞术, 实时定量聚合酶链反应, 人

Abstract:

Objective To investigate the effects of trefoil factor 3 (TFF3) on the proliferation and apoptosis of human thyroid papillary carcinoma cell line (TPC-1) and its molecular mechanism. Methods The lentiviral expression vector of overexpression and knockdown of TFF3 gene were constructed，293T cell was packaged to produce lentiviral particles, virus solution was collected and transfected into TPC-1 cells, enhanced cell TFF3-TPC-1 and enhanced control group ConTFF3-TPC-1; silencing cell shRNA-TFF3-TPC-1 and silencing control cells shCon -TPC-1. Western blotting, and Real-time PCR were used to detect the expression of TFF3 protein and mRNA of four groups. Growth curve and colony formation assay were used to detect the proliferation.Flow cytometry was used to analyze the apoptosis level of the four groups; Western blotting and immunocytochemistry were used to detect apoptosis-related protein and pathway protein phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), nuclear factor-κB（NF-κB）expression. Results 1. Overexpression and inhibition of expression of TFF3 stable cell TFF3-TPC-1 and shRNA-TFF3-TPC-1 were constructed suscessfully. 2. The proliferation and cloning ability of TFF3-TPC-1 cells were significantly higher than those of ConTFF3-TPC-1 cells（P＜0.05 or P＜0.01）, the proliferation and cloning ability of shRNA-TFF3-TPC-1 cells were significantly lower than those of shCon-TPC-1 cells(P<0.01); 3. The apoptosis rate of TFF3-TPC-1 cells was lower than that of ConTFF3-TPC-1 (0.75%±0.08% vs 5.62%±0.3%, P<0.01),and the apoptosis rate of shRNA-TFF3-TPC-1 was higher than that of shConTPC-1 (22.2% ± 1.2% vs 5.34% ± 0.4%, P<0.01); 4. After silencing TFF3 gene, the expressions of Bax, cytochrome C (Cyt-C), cleavedCaspase-9, cleaved-Caspase-3 were up-regulated, and the expressions of Bcl-2, Akt, p-Akt and NF-κB-P65 were downregulated (P<0.05 or P<0.01). Conclusion TFF3 may regulate the proliferation and apoptosis of TPC-1 cells by affecting the PI3K/Akt/NF-κB signaling pathway.

Key words: Trefoil factor 3, phosphatidylinositol 3-kinase/protein kinase B/nuclear factor kappa B, Papillary thyroid carcinoma, Lentiviral transfection, Western blotting, Flow cytometry, Real-time PCR, Human

中图分类号:

R739.6

郭梦姚孙琳林旭张文静张静吴靖芳薛刚. 三叶因子3/磷脂酰肌醇-3激酶/蛋白激酶B/核因子-κB在人甲状腺乳头状癌细胞系TPC-1增殖和凋亡中的作用[J]. 解剖学报, 2020, 51(4): 528-535.

GUO Meng-yao SUN Lin LIN Xu ZHANG Wen-jing ZHANG Jing WU Jing-fang XUE Gang. Role of trefoil factor3/phosphatidylinositl 3-kinase/protein kinase B/nuclear factorκB in the proliferation and apoptosis of human papillary thyroid carcinoma cell line TPC-1[J]. Acta Anatomica Sinica, 2020, 51(4): 528-535.

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