三叶因子3对甲状腺乳头状癌细胞周期的影响及机制

doi:10.16098/j.issn.0529-1356.2019.02.011

摘要/Abstract

摘要：

目的探讨三叶因子3 (TFF3)基因沉默对人甲状腺乳头状癌TPC-1和BCPAP细胞增殖及细胞周期的影响及相关分子机制。方法包装TFF3 shRNA 慢病毒载体，病毒感染获得TPC-1和BCPAP稳转细胞株；生长曲线和集落形成实验检测沉默TFF3后细胞增殖状况；流式细胞术检测TFF3基因对TPC-1和BCPAP细胞周期的影响；实时定量聚合酶链反应(Real-time PCR)检测P27、P21和cyclin D1、周期蛋白依赖性激酶（CDK）4 mRNA的表达情况；免疫印迹法(Western blotting)、免疫细胞化学染色检测周期相关蛋白P27、P21、cyclin D1、CDK4和蛋白激酶B（Akt）、pAkt的蛋白表达水平。结果成功包装TFF3 shRNA 慢病毒载体，病毒液感染获得TPC-1和BCPAP稳转细胞株；生长曲线和集落形成实验结果显示，沉默TFF3后，细胞增殖能力减弱；流式细胞术结果显示，与对照组相比，TFF3基因沉默组G₁期的细胞比例明显增高(*P<0.05)，S和G2期的细胞比例明显下降（*P<0.05)；TFF3沉默组TPC-1和BCPAP细胞中的P27、P21 mRNA和蛋白明显上调（*P<0.05)，而cyclin D1，CDK4 mRNA和蛋白表达降低（*P<0.05)；4株细胞Akt蛋白含量无明显差异，但沉默TFF3组磷酸化蛋白激酶B（pAkt）表达减弱；免疫细胞化学染色显示,cyclinD1阳性蛋白位于癌细胞胞核，P27、P21蛋白表达于胞质和胞核，且沉默TFF3后在细胞核中阳性信号增强，细胞质中阳性表达降低。结论沉默TFF3可明显延长甲状腺癌细胞周期，抑制细胞的增殖，可能与抑制磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/Akt）通路相关蛋白的表达有关。

关键词: 三叶因子3, 细胞周期, 慢病毒转染, 磷脂酰肌醇3-激酶/蛋白激酶B, 流式细胞术, 人

Abstract:

Objective To investigate the effects of trefoil factor family 3(TFF3) gene silencing on proliferation and cell cycle of papillary thyroid carcinoma TPC-1,BCPAP cell lines and related molecular mechanisms. Methods To obtain TPC-1 and BCPAP stable cell lines of thyroid papillary carcinoma by packaging TFF3 shRNA lentiviral vectors and virus infection;Growth curve and colony formation assay were used to detecte cell proliferation after TFF3 silencing.Flow cytometry was used to detect the effects of TFF3 gene on cell cycle of TPC-1 and BCPAP cell; Real-time PCR was used to investigate the mRNA levels of P27,P21,cyclin D1 and cyclin-dependent kinase(CDK)4,in TPC-1 and BCPAP cell. The protein expression levels of P27,P21,cyclin D1 and CDK4,protein kinase B(Akt) and phosphorylated protein kinase B(pAkt) were detected by Western blotting and immunocytochemistry. Results shTFF3-TPC-1 and shTFF3-BCPAP stable cell lines were obtained by TFF3 shRNA lentiviral packaging and virus infection. Growth curve and colony formation assay result showed that cell viability was decreased after silencing TFF3.The result of flow cytometry showed that the proportion of cells in the G₁ phase was significantly increased (*P<0.05), however the proportion of cells in the S and G₂ phase was significantly decreased in TFF3 slienced group compared with the control group (*P<0.05)；The expression of P27 and P21 mRNA and protein in TPC-1 and BCPAP cells was significantly up-regulated(*P<0.05),while the expression of cyclin D1 and CDK4 mRNA and protein was decreased in group TFF3 silenced (*P<0.05).There was no significant difference in the content of Akt protein in the four groups of cells, but the expression of pAkt was weakened after TFF3 silenced;Immunocytochemical staining showed that cyclin D1 positive protein expression was located in the nucleus of cancer cells, P27 and P21 proteins lo ated in cytoplasm and nucleus,and after silencing TFF3, the positive signals were upregulated in the nucleus, but cytoplasmic positive expression was decreased. Conclusion Silencing TFF3 can significantly prolong the cell cycle and inhibit cell proliferation，which might be related to the inhibition of phosphatidylinosital 3-kinase and protein kinase B(PI3K/Akt) pathway-related protein expression.

Key words: Trefoil factorfamily 3, Cell cycle, Lentivirus transfection, Phosphatidylinositol 3-kinase and protein kinase B, Flow cytometry, Human

代金林旭郭梦姚张静张文静薛刚吴靖芳. 三叶因子3对甲状腺乳头状癌细胞周期的影响及机制[J]. 解剖学报, 2019, 50(2): 211-219.

DAI Jin LIN Xu GUO Meng-yao ZHANG Jing ZHANG Wen-jing XUE Gang WU Jing-fang. Effect and molecular mechanism of trefoil factor family 3 on the cell cycle of thyroid papillary carcinoma cell[J]. Acta Anatomica Sinica, 2019, 50(2): 211-219.

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