Department of Endocrinology, Second People's Hospital of Guilin, Guilin, Guangxi Zhuang Autonomous Region 541002, P.R. China.
Department of Pathophysiology, Guilin Medical University, Guilin, Guangxi Zhuang Autonomous Region 541001, P.R. China.
Oncol Rep. 2020 Feb;43(2):471-480. doi: 10.3892/or.2019.7443. Epub 2019 Dec 23.
The present study aimed to investigate the effect of miR‑449a‑mediated Notch signaling pathway on the proliferation, apoptosis and invasion of papillary thyroid carcinoma cells. Human papillary thyroid carcinoma cell line TPC‑1 was selected, and cells were grouped and transfected: Control group (without any treatment), negative control (NC) group (transfection with NC plasmid), miR‑449a mimic group (transfection with miR‑449a mimic), miR‑449a inhibitor group (transfection with miR‑449a inhibitor), DAPT group (addition of γ‑secretase inhibitor DAPT to inhibit the Notch signaling pathway), and miR‑449a inhibitor + DAPT group (transfection with miR‑449a inhibitor and addition of DAPT). The target relationship between miR‑449a and Notch1 was detected by dual‑luciferase reporter assay. qRT‑PCR and western blotting were used to assess the expression of miR‑449a, Notch1 and Jagged1 in cells. Cell proliferation was detected using EdU; the cell cycle and apoptosis were detected by flow cytometry; cell invasion ability was detected by Transwell assay. PCNA, MMP‑2, MMP‑9, Bcl‑2 and Bax mRNA and protein expression were assessed by qRT‑PCR and western blotting. The results revealed that miR‑449a negatively regulated Notch1. Compared with the control group, there was significantly increased miR‑449a expression in the miR‑449a mimic group, and there was significantly decreased expression of Notch1, Jagged1, PCNA, MMP‑2, MMP‑9 and Bcl‑2, increased Bax, reduced cell proliferation, increased G1‑phase cell fraction, decreased S‑phase cell fraction, an increased apoptosis rate, and decreased invasion ability in the miR‑449a mimic group and DAPT group (all P<0.05). However, the results in the miR‑449a inhibitor group were the opposite of those in miR‑449a mimic group (all P<0.05). There was no significant difference in cell proliferation, apoptosis and invasion in the NC group and miR‑449a inhibitor + DAPT group compared to the control group (all P>0.05). miR‑449a overexpression can inhibit Notch signaling pathway, thereby inhibiting the proliferation and invasion of papillary thyroid carcinoma cells and promoting cell apoptosis.
本研究旨在探讨 miR-449a 介导的 Notch 信号通路对甲状腺乳头状癌细胞增殖、凋亡和侵袭的影响。选择人甲状腺乳头状癌细胞系 TPC-1，分组并转染：对照组（无任何处理）、阴性对照组（转染 NC 质粒）、miR-449a 模拟组（转染 miR-449a 模拟物）、miR-449a 抑制剂组（转染 miR-449a 抑制剂）、DAPT 组（加入γ-分泌酶抑制剂 DAPT 抑制 Notch 信号通路）和 miR-449a 抑制剂+DAPT 组（转染 miR-449a 抑制剂并加入 DAPT）。双荧光素酶报告实验检测 miR-449a 与 Notch1 的靶关系。qRT-PCR 和 Western blot 检测细胞中 miR-449a、Notch1 和 Jagged1 的表达。EdU 检测细胞增殖；流式细胞术检测细胞周期和凋亡；Transwell 检测细胞侵袭能力。qRT-PCR 和 Western blot 检测 PCNA、MMP-2、MMP-9、Bcl-2 和 Bax mRNA 和蛋白表达。结果显示，miR-449a 负调控 Notch1。与对照组相比，miR-449a 模拟组 miR-449a 表达明显升高，Notch1、Jagged1、PCNA、MMP-2、MMP-9 和 Bcl-2 表达明显降低，Bax 表达升高，细胞增殖减少，G1 期细胞比例增加，S 期细胞比例减少，凋亡率增加，侵袭能力降低，DAPT 组 miR-449a 模拟组（均 P<0.05）。然而，miR-449a 抑制剂组的结果与 miR-449a 模拟组相反（均 P<0.05）。NC 组和 miR-449a 抑制剂+DAPT 组与对照组相比，细胞增殖、凋亡和侵袭均无明显差异（均 P>0.05）。miR-449a 过表达可抑制 Notch 信号通路，从而抑制甲状腺乳头状癌细胞的增殖和侵袭，促进细胞凋亡。
