siRNA 靶向沉默TAK1基因对甲状腺癌细胞增殖、迁移和p38 MAPK信号通路的抑制作用

doi:10.13481/j.1671-587x.20210115

摘要/Abstract

摘要： 目的

探讨小干扰RNA（siRNA）靶向沉默转化生长因子β激活激酶1（TAK1）基因对甲状腺癌细胞增殖、迁移和p38 丝裂原活化蛋白激酶（MAPK）信号通路的影响，阐明沉默TAK1基因对甲状腺癌的可能作用机制。

方法

采用实时荧光定量聚合酶链式反应（qRT-PCR）和Western blotting法检测甲状腺癌8505C、NPA、BCPAP和KMH-2细胞中TAK1 mRNA和蛋白表达水平。将KMH-2细胞随机分为空白对照组、阴性对照组和siRNA-TAK1组。对照组细胞不转染，阴性对照组细胞转染阴性对照siRNA， siRNA-TAK1组细胞转染TAK1 siRNA。采用qRT-PCR和Western blotting法检测各组细胞转染效率（即细胞中TAK1 mRNA和蛋白表达水平），MTT法检测细胞增殖活性，Transwell小室实验检测各组细胞侵袭和迁移能力，Western blotting法检测各组细胞中增殖细胞核抗原（PCNA）、基质金属蛋白酶2（MMP-2）、基质金属蛋白酶9（MMP-9）、p38和磷酸化p38（p-p38）蛋白表达水平。

结果

与正常甲状腺上皮Nthyori3-1细胞比较，甲状腺癌8505C、NPA、BCPAP及KMH-2细胞中TAK1 mRNA和蛋白表达水平均明显升高（P<0.05）。与空白对照组和阴性对照组比较，siRNA-TAK1组KMH-2细胞中TAK1 mRNA和蛋白表达水平明显降低（P<0.05），细胞增殖活性、侵袭细胞数和迁移细胞数均明显降低（P<0.05），细胞中PCNA、MMP-2、MMP-9和p-p38蛋白水平明显降低（P<0.05）；与空白对照组比较，阴性对照组KMH-2细胞中上述各指标差异均无统计学意义（P>0.05）。

结论

siRNA 靶向沉默TAK1基因可通过抑制p38 MAPK信号通路的激活进而抑制甲状腺癌细胞的增殖、侵袭和迁移过程。

关键词: 小干扰RNA, 转化生长因子β激活激酶1, 甲状腺肿瘤, 细胞增殖, 细胞迁移, p38丝裂原活化蛋白激酶

Abstract: Objective

To investigate the effects of siRNA targeting silencing transforming growth factor β-activated kinase 1 （TAK1） gene on the proliferation，migration and p38 mitogen-activated protein kinase （MAPK） signaling pathway of thyroid cancer cells，and to clarify the possible mechanism of silencing TAK1 gene in thyroid cancer.

Methods

Quantitative real-time polymerase chain reaction （qRT-PCR） and Western blotting method were used to determine the expression levels of TAK1 mRNA and proteins in the thyroid cancer 8505C， NPA， BCPAP and KMH-2 cells. The KMH-2 cells were randomly divided into blank control group， negative control group and siRNA-TAK1 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 siRNA-TAK1 group were transfected with TAK1 siRNA. MTT method was used to measure the cell proliferation activity. Transwell assay was used to measure the invasion and migration abilities of cells. Western blotting method was used to determine the expression levels of proliferating cell nuclear antigen （PCNA）， matrix metalloproteinase-2 （MMP-2）， matrix metalloproteinase-9（MMP-9），p38 and phosphorylated p38 （p-p38） proteins in the cells in various groups.

Results

Compared with the normal thyroid epithelial cells Nthyori3-1， the expression levels of TAK1 mRNA and protein in thyroid cancer 8505C， NPA， BCPAP and KMH-2 cells were significantly increased（P<0.05）. Compared with blank control group and negative control group， the expression levels of TAK1 mRNA and protein in the KMH-2 cells in siRNA-TAK1 group were reduced （P<0.05）， the cell proliferation activities were reduced （P<0.05）， the number of invasion cells and migration cells was reduced （P<0.05），and the expression levels of PCNA， MMP-2， MMP-9 and p-p38 proteins in the cells were decreased （P<0.05）. There were no significant differences in the indexes mentioned above of the KMH-2 cells between blank control group and negative control group（P>0.05）.

Conclusion

SiRNA targeting silencing TAK1 gene can inhibit the proliferation，invasion and migration of thyroid cancer cells through inhibiting the activation of p38 MAPK signaling pathway.

Key words: siRNA, transforming growth factor β-activated kinase 1, thyroid neoplasms, cell proliferation, cell migration, p38 MAPK

中图分类号:

R736.1

张春英,阴广维,尤鸣达,陈红,胡耀杰,李岩冰,陈春悠. siRNA 靶向沉默TAK1基因对甲状腺癌细胞增殖、迁移和p38 MAPK信号通路的抑制作用[J]. 吉林大学学报(医学版), 2021, 47(1): 110-117.

Chunying ZHANG,Guangwei YIN,Mingda YOU,Hong CHEN,Yaojie HU,Yanbing LI,Chunyou CHEN. Inhibitory effects of siRNA targeting silencing TAK1 gene on proliferation and migration of thyroid cancer cells and p38 MAPK signaling pathway[J]. Journal of Jilin University(Medicine Edition), 2021, 47(1): 110-117.

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Cell	TAK1 mRNA	TAK1 protein
Nthyori3-1	1.00±0.12	0.12±0.04
8505C	1.68±0.11 ^*	0.48±0.06 ^*
NPA	1.97±0.13 ^*	0.82±0.08 ^*
BCPAP	1.84±0.15 ^*	0.78±0.07 ^*
KMH-2	2.32±0.16 ^*	0.94±0.10 ^*
F	90.663	144.385
P	<0.01	<0.01

Group	TAK1 mRNA	TAK1 protein
Black control	1.00±0.08	0.92±0.13
Negative control	0.98±0.09	0.94±0.12
siRNA-TAK1	0.41±0.06^*△	0.26±0.07^*△
F	130.215	86.862
P	<0.01	<0.01

Group	Proliferation activity
Group	(t/h) 24	48	72
Blank control	0.412±0.033	0.784±0.041	1.273±0.044
Negative control	0.415±0.030	0.779±0.043	1.268±0.046
siRNA-TAK1	0.327±0.025^*△	0.461±0.038^*△	0.691±0.038^*△
F	20.055	144.586	427.743
P	<0.01	<0.01	<0.01

Group	Number of invasion cells	Number of migration cells
Blank control	378.46±37.35	215.68±28.49
Negative control	382.19±41.26	214.94±31.02
siRNA-TAK1	153.26±35.49^*△	92.13±25.18^*△
F	82.852	44.111
P	<0.01	<0.01

Group	PCNA	MMP-2	MMP-9
Blank control	0.43±0.08	0.28±0.06	0.33±0.05
Negative control	0.45±0.09	0.30±0.07	0.34±0.06
siRNA-TAK1	0.21±0.05^*△	0.13±0.03^*△	0.15±0.04^*△
F	21.906	19.287	31.182
P	<0.01	<0.01	<0.01