沉默赖氨酸乙酰基转移酶基因对人甲状腺乳头状癌细胞增殖和凋亡的影响

doi:10.13481/j.1671-587x.20200319

摘要/Abstract

摘要： 目的：探讨赖氨酸乙酰基转移酶（Kat5）对甲状腺乳头状癌细胞增殖和凋亡的影响及磷脂酰肌醇3-激酶（PI3K）/丝氨酸/苏氨酸激酶（AKT）信号通路的作用，阐明Kat5在甲状腺乳头状癌细胞增殖和凋亡中的可能作用机制。方法：RT-PCR和Western blotting法检测甲状腺乳头状癌BHP10-3、TPC-1、K1细胞和甲状腺上皮Nthy-ori3-1细胞中Kat5 mRNA和蛋白表达水平。将对数生长期甲状腺乳头状癌TPC-1细胞分为空白对照组、阴性对照组和沉默Kat5组（si-Kat5组）。空白对照组甲状腺乳头状癌TPC-1细胞不转染，阴性对照组甲状腺乳头状癌TPC-1细胞转染阴性对照siRNA，si-Kat5组甲状腺乳头状癌TPC-1细胞转染Kat5 siRNA。RT-PCR和Western blotting法检测各组甲状腺乳头状癌TPC-1细胞中Kat5 mRNA和蛋白表达水平，CCK-8法检测各组TPC-1细胞增殖活性，克隆形成实验检测各组TPC-1细胞克隆形成率，流式细胞术检测各组TPC-1细胞凋亡率，Western blotting法检测各组TPC-1细胞中周期蛋白依赖性激酶2（CDK2）、P21、P53、活化的含半胱氨酸的天冬氨酸蛋白水解酶3（cleaved caspase-3）、PI3K、磷酸化PI3K （p-PI3K）、AKT和磷酸化AKT （p-AKT）蛋白水平。结果：甲状腺乳头状癌BHP10-3、TPC-1和K1细胞中Kat5 mRNA和蛋白表达水平均高于甲状腺上皮Nthy-ori3-1细胞（P<0.05）。各组TPC-1细胞中Kat5 mRNA和蛋白水平、细胞增殖活性、细胞克隆形成率、细胞凋亡率以及细胞中CDK2、P21、P53、cleaved caspase-3、PI3K、p-PI3K、AKT及p-AKT蛋白表达水平比较差异均有统计学意义（P<0.05）；与空白对照组和阴性对照组比较，si-Kat5组TPC-1细胞中Kat5 mRNA和蛋白表达水平降低（P<0.05），细胞增殖活性降低（P<0.05），细胞克隆形成率降低（P<0.05），细胞凋亡率升高（P<0.05），细胞中CDK2、p-PI3K和p-AKT蛋白表达水平降低（P<0.05），P21、P53和cleaved caspase-3蛋白表达水平升高（P<0.05）。结论：甲状腺乳头状癌细胞中Kat5mRNA和蛋白表达水平升高，沉默Kat5基因可抑制甲状腺乳头状癌细胞增殖并促进其凋亡，其机制可能与其抑制PI3K/AKT信号通路有关。

关键词: 赖氨酸乙酰基转移酶, 甲状腺乳头状癌, 细胞增殖, 细胞凋亡, 磷脂酰肌醇3-激酶/丝氨酸/苏氨酸激酶

Abstract: Objective: To investigate the effects of lysine acetyltransferase (Kat5) on the proliferation and apoptosis of the thyroid papillary carcinoma cells and the role of phosphatidylinositol 3-kinase (PI3K)/serine/threonine kinase (AKT) signaling pathway,and to elucidate the possible mechanism of Kat5 in the proliferation and apoptosis of thyroid papillarycarcinoma cells. Methods: RT-PCR and Western blotting methods were used to determine the expression levels of Kat5 mRNA and protein in the thyroid papillary carcinoma BHP10-3, TPC-1, K1 cells and thyroid epithelial Nthy-ori3-1 cells.The thyroid papillary carcinoma TPC-1 cells were divided into blank control group, negative control group and silencing Kat5 group (si-Kat5 group).The cells in blank control group were not transfected,the cells in negative control group were transfected with negative control siRNA,and the cells in si-Kat5 group were transfected with Kat5 siRNA.The expression levels of Kat5 mRNA and protein in the thyroid papillary carcinoma TPC-1 cells in various groups were detected by RT-PCR and Western blotting methods.The proliferation activities of the TPC-1 cells in various groups were measured by CCK-8 method,the colony formation assay was used to measure the clone formation rates of the TPC-1 cells in various groups,the apoptotic rates of the TPC-1 cells in various groups were detected by flow cytometry,and the expression levels of cyclin-dependent kinase 2(CDK2), P21, P53, cleaved caspase-3, PI3K, phosphorylated PI3K (p-PI3K), AKT, and phosphorylated AKT(p-AKT) protein in the TPC-1 cells in various groups were detected by Western blotting method. Results: The expression levels of Kat5 mRNA and protein in the thyroid papillary carcinoma BHP10-3, TPC-1 and K1 cells were higher than those in thyroid epithelial Nthy-ori3-1 cells (P<0.05).The differences in the Kat5 mRNA and protein expression levels,the proliferation activities,the clone formation rates,the apoptotic rates and the expression levels of CDK2, P21, P53, cleaved caspase-3, PI3K, p-PI3K, AKT and p-AKT proteins in the TPC-1 cells between various groups were statistically significant(P<0.05).Compared with blank control group and negative control group, the expression levels of Kat5 mRNA and protein in the TPC-1 cells in si-Kat5 group were decreased(P<0.05), the proliferation activity was decreased(P<0.05),the colony formation rate was decreased(P<0.05),the apoptotic rate was increased(P<0.05), the expression levels of CDK2, p-PI3K and p-AKT proteins were decreased(P<0.05), and the expression levels of P21, P53 and cleaved caspase-3 proteins were increased(P<0.05). Conclusion: The expression levels of Kat5 mRNA and protein in the thyroid papillary carcinoma cells are increased. Silencing Kat5 gene can inhibit the proliferation of the thyroid papillary carcinoma cells and promote their apoptosis,and tis mechanism may be related to the inhibition of PI3K/AKT signaling pathway.

Key words: lysine acetyltransferase, thyroid papillary carcinoma, cell proliferation, apoptosis, phosphatidylinositol 3-kinase/serine/threonine kinase

中图分类号:

R736.1

杜静海, 陈春悠, 郭欣, 张静. 沉默赖氨酸乙酰基转移酶基因对人甲状腺乳头状癌细胞增殖和凋亡的影响[J]. 吉林大学学报(医学版), 2020, 46(03): 543-550.

DU Jinghai, CHEN Chunyou, GUO Xin, ZHANG Jing. Effects of silencing Kat5 gene on proliferation and apoptosis of thyroid papillary carcinoma cells[J]. Journal of Jilin University(Medicine Edition), 2020, 46(03): 543-550.

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