miRNA-224-5p通过TGF-β/Smad4信号通路调控甲状腺乳头状癌细胞凋亡

MiRNA-224-5p regulates apoptosis of papillary thyroid carcinoma cells via the transforming growth factor beta/Smad4 signaling pathway

目的探讨miRNA-224-5p通过TGF-β/Smad4信号通路对甲状腺乳头状癌细胞凋亡的调控作用。方法选取2015年7月至2017年11月新疆维吾尔自治区人民医院手术切除所得甲状腺乳头状癌组织及相应的癌旁组织冻存标本各60份。通过逆转录聚合酶链反应(RT-PCR)分别检测60例甲状腺乳头状癌组织及60例癌旁组织中miRNA-224-5p的相对表达量,二者比较采用配对资料t检验。将miRNA-224-5p(miRNA-224-5p组)与miRNA-224-5p抑制剂(miRNA-224-5p抑制剂组)分别转染到甲状腺乳头状癌细胞株GLAG-66中,通过RT-PCR检测两组中GLAG-66细胞内miRNA-224-5p的相对表达量,应用流式细胞仪检测两组GLAG-66细胞转染48 h后凋亡率,应用Western blot实验检测两组GLAG-66细胞中Bcl-2、Bax、Cleaved-Caspase3、TGF-β1、Smad4蛋白的相对表达量,并采用两个独立样本t检验进行比较。结果 miRNA-224-5p在甲状腺乳头状癌组织中的相对表达量与癌旁组织比较明显升高,差异具有统计学意义[(4.175±0.523)vs(1.236±0.236),t=9.675,P=0.001)]。miRNA-224-5p抑制剂组GLAG-66细胞中miRNA-224-5p的相对表达量与miRNA-224-5p组比较明显下降,差异具有统计学意义[(0.381±0.078)vs(1.000±0.023),t=19.785,P <0.001)]。miRNA-224-5p抑制剂组GLAG-66细胞转染48 h后的凋亡率与miRNA-224-5p组比较明显增高,差异具有统计学意义[(18.66%±0.98%)vs(3.78%±0.36%),t=17.561,P <0.001]。miRNA-224-5p抑制剂组和miRNA-224-5p组比较,GLAG-66细胞中Bcl-2、TGF-β1和Smad4蛋白的相对表达量明显降低,差异具有统计学意义[(0.197±0.008)vs(0.278±0.013),t=8.259,P=0.001;(0.117±0.013)vs(0.227±0.012),t=13.269,P <0.001;(0.268±0.012)vs(0.439±0.016),t=9.897,P=0.001];Bax、Cleaved-Caspase3蛋白的相对表达量明显升高,差异具有统计学意义[(0.286±0.011)vs(0.159±0.009),t=12.569,P <0.001;(0.128±0.009)vs(0.083±0.010),t=7.693,P=0.002]。结论 miRNA-224-5p在甲状腺乳头状癌组织中表达增高,下调miRNA-224-5p可以促进甲状腺乳头状癌细胞的凋亡,其作用机制可能与TGF-β/Smad4信号通路有关。

Objective To explore the role of miRNA-224-5p in the apoptosis of papillary thyroid carcinoma cells and the underlying mechanism.Methods The expression of miRNA-224-5p in 60 cases of papillary thyroid carcinoma and adjacent tissue was detected by RT-PCR.Human papillary thyroid carcinoma GLAG-66 cells were cultured,miRNA-224-5p and miRNA-224-5p inhibitor were transfected into cells,and the expression of miRNA-224-5p was detected by RT-PCR.Cell apoptosis was detected by flow cytometry 48 h later.The expression of Bcl-2,Bax,Cleaved-Caspase3,TGF-β1,and Smad4 proteins was detected by Western blot.Results The expression of miRNA-224-5p in papillary thyroid carcinoma tissue was significantly higher than that in paracancerous tissue[(4.175±0.523)vs(1.236±0.236),t=9.675,P=0.001],and the expression of miRNA-224-5p was significantly lower in the miRNA-224-5p inhibitor group than in the miRNA-224-5p group[(0.381±0.078)vs(1.000±0.023),t=19.785,P<0.001)].Compared with the miRNA-224-5p group,the apoptosis rate significantly increased in the miRNA-224-5p inhibitor group[(18.66%±0.98%)vs(3.78%±0.36%),t=17.561,P<0.001].Compared with the miRNA-224-5p group,the expression of Bcl-2,TGF-β1 and Smad4 in the miRNA-224-5p inhibitor group was significantly decreased[(0.197±0.008)vs(0.278±0.013),t=8.259,P=0.001;(0.117±0.013)vs(0.227±0.012),t=13.269,P<0.001;(0.268±0.012)vs(0.439±0.016),t=9.897,P=0.001],and the expression of Bax and Cleaved-Caspase3 protein was significantly increased[(0.286±0.011)vs(0.159±0.009),t=12.569,P<0.001;(0.128±0.009)vs(0.083±0.010),t=7.693,P=0.002].Conclusion MiRNA-224-5p is highly expressed in papillary thyroid carcinoma,and down-regulation of miRNA-224-5p promotes cell apoptosis via mechanisms possibly related to regulation of the TGF-β/Smad4 signaling pathway.