Ma Huan, Zhang Zheying, Wang Yang, Shang Fei, Du Baoshun, Wang Yungang, Cheng Zhenguo
Department of Neurosurgery, Xinxiang Central Hospital, Xinxiang, China.
Xinxiang Medical University, Xinxiang, China.
Gland Surg. 2020 Dec;9(6):2144-2154. doi: 10.21037/gs-20-823.
This study aims to investigate the mechanism through which Caveolin-1 (CAV-1) regulates the expression of micro ribonucleic acid (miR)-183 in invasive pituitary adenoma (IPA) tissues and GH3 cells, and explore the effects of CAV-1 and miR-183 on the invasion and migration ability of GH3 cells.
Western blotting was used to detect the expression level of CAV-1, early growth response 1 (EGR1) and Krueppel-like factor 5 (KLF5). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of miR-183. The mechanisms of interaction between CAV-1, EGR1, and KLF5 were studied by immunoprecipitation experiments. Transwell and cell scratch tests were used to determine the invasion and migration ability of GH3 cells. The dual-luciferase reporter gene experiment was used to detect the effects of EGR1 and KLF5 on miR-183 luciferase activity and verify the targeting relationship between miR-183 and ezrin.
The expression of CAV-1 was up-regulated. However, following the knockdown of CAV-1, the invasion and migration ability of GH3 cells was significantly inhibited (P<0.05). The expression of miR-183 was down-regulated, but the expression level of miR-183 was markedly increased following the knockdown of CAV-1 (P<0.05). The knockdown of CAV-1 inhibited the nuclear ectopic of the EGR1 protein in GH3 cells. At the same time, the interaction between EGR1 and KLF5 in GH3 cells was significantly inhibited (P<0.05). The luciferase activity of miR-183 increased significantly after overexpression of KLF5 while overexpression of EGR1 and KLF5 had no significant effect on intracellular luciferase activity. Overexpression of miR-183 markedly inhibited the luciferase activity of wild-type EZR and the expression of the EZR protein in GH3 cells. Furthermore, the overexpression of miR-183 or the inhibition of EZR can reduce the invasion and migration ability of GH3 cells. The simultaneous overexpression or inhibition of miR-183 and EZR expression has no obvious effect on the invasion and migration ability of GH3 cells.
CAV-1 up-regulates the expression of miR-183 by inhibiting the nuclear ectopic of EGR1 and the interaction between EGR1 and KLF5 in GH3 cells. Also, miR-183 negatively regulates the expression of EZR and inhibits the invasion and migration of GH3 cells.
本研究旨在探讨小窝蛋白-1（CAV-1）调控侵袭性垂体腺瘤（IPA）组织和GH3细胞中微小核糖核酸（miR）-183表达的机制，并探究CAV-1和miR-183对GH3细胞侵袭和迁移能力的影响。
采用蛋白质免疫印迹法检测CAV-1、早期生长反应因子1（EGR1）和克鲁ppel样因子5（KLF5）的表达水平。运用定量实时聚合酶链反应（qRT-PCR）检测miR-183的表达。通过免疫沉淀实验研究CAV-1、EGR1和KLF5之间的相互作用机制。采用Transwell和细胞划痕试验测定GH3细胞的侵袭和迁移能力。利用双荧光素酶报告基因实验检测EGR1和KLF5对miR-183荧光素酶活性的影响，并验证miR-183与埃兹蛋白（ezrin）之间的靶向关系。
CAV-1表达上调。然而，敲低CAV-1后，GH3细胞的侵袭和迁移能力显著受到抑制（P<0.05）。miR-183表达下调，但敲低CAV-1后miR-183表达水平显著升高（P<0.05）。敲低CAV-1抑制了GH3细胞中EGR1蛋白的核异位。同时，GH3细胞中EGR1与KLF5之间的相互作用也显著受到抑制（P<0.05）。KLF5过表达后miR-183的荧光素酶活性显著增加，而EGR1和KLF5过表达对细胞内荧光素酶活性无显著影响。miR-183过表达显著抑制了野生型埃兹蛋白（EZR）的荧光素酶活性以及GH3细胞中EZR蛋白的表达。此外，miR-183过表达或抑制EZR均可降低GH3细胞的侵袭和迁移能力。同时过表达或抑制miR-183和EZR表达对GH3细胞的侵袭和迁移能力无明显影响。
CAV-1通过抑制GH3细胞中EGR1的核异位以及EGR1与KLF5之间的相互作用上调miR-183的表达。此外，miR-183负向调控EZR的表达并抑制GH3细胞的侵袭和迁移。
