微小RNA-874-3p 通过靶向调控斑菲素蛋白3影响肺腺癌细胞生物学行为及机制

陈帆  滕召虎  房涛  任君旭  张静  李雪  王怡璇  林旭  吴靖芳

doi:10.16098/j.issn.0529-1356.2025.02.009

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解剖学报 ›› 2025, Vol. 56 ›› Issue (2) : 188-201. DOI: 10.16098/j.issn.0529-1356.2025.02.009

微小RNA-874-3p 通过靶向调控斑菲素蛋白3影响肺腺癌细胞生物学行为及机制

陈帆¹ 滕召虎¹ 房涛² 任君旭³ 张静^3* 李雪¹ 王怡璇¹ 林旭³ 吴靖芳¹

作者信息 +

Effects of microRNA-874-3p on biological behavior of lung adenocarcinoma cells through targeted regulation of plakophilin 3 and its mechanism

CHEN Fan¹ TENG Zhao-hu¹ FANG Tao² REN Jun-xu³ ZHANG Jing^3* LI Xue¹ WANG Yi-xuan¹ LIN Xu³ WU Jing-fang¹

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文章历史 +

摘要

目的探讨微小RNA-874-3p（miR-874-3p）靶向调控斑菲素蛋白3（PKP3)对肺腺癌细胞恶性生物学行为的影响及作用机制。方法免疫组织化学法和免疫细胞化学法分别检测肺腺癌组织芯片与肺腺癌细胞系A549细胞PKP3的表达水平，并分析与肺腺癌患者临床病理特征之间的关系。细胞实验分A549细胞组（空白对照组）、miR-NC组(转染miR-NC)、miR-mimics组（转染miR-874-3p mimics）、sh-NC组（转染PKP3沉默质粒的对照组）、sh-PKP3组（转染PKP3沉默质粒）、miR+pcDNA-PKP3组（转染miR-874-3p mimics+pcDNA-PKP3，挽救组）、miR+pcDNA-NC组（转染miR-874-3p mimics+pcDNA-NC），分别对各组细胞的增殖、侵袭、迁移以及凋亡能力进行检测。用ENCORI数据库预测PKP3的上游基因，双荧光素酶实验检测miR-874-3p与PKP3靶向关系。Western blotting检测MAPK/mTOR通路相关蛋白。结果 PKP3在肺腺癌组织中的表达水平明显高于癌旁组织，PKP3高表达与临床分期、肿瘤大小及淋巴结转移有关（P＜0.05），A549细胞中PKP3表达水平显著增高，miR-874-3p表达降低（P＜0.05）。过表达miR-874-3p使PKP3表达水平下降（P＜0.05）。与对照组相比，过表达miR-874-3p与沉默PKP3均能抑制A549细胞克隆及侵袭能力，引起细胞周期阻滞，降低A549细胞中细胞周期蛋白依赖性激酶4（CDK4）、细胞周期蛋白（cyclin）D1、cyclin E1的表达水平（P＜0.05），上调Bax蛋白及Caspase-3 蛋白表达（P＜0.05），增加细胞凋亡。过表达PKP3可以逆转过表达miR-874-3p产生的生物学行为。过表达miR-874-3p与沉默PKP3均显著降低P38 MAPK、mTOR磷酸化蛋白的表达水平。结论 MiR-874-3p能够靶向负调控PKP3表达并通过MAPK/mTOR通路抑制A549细胞恶性生物学行为。

Abstract

Objective The study aims to investigate the impact of microRNA-874-3p (miR-874-3p) regulation of plakophilin 3(PKP3)on the malignant biological behavior of lung adenocarcinoma cells and its underlying mechanism. Methods Immunohistochemistry and immunocytochemistry were used to detect the expression of PKP3 in lung adenocarcinoma tissue microarray and lung adenocarcinoma cell line A549 cells respectively, and the relationship between PKP3 and clinicopathological features of lung adenocarcinoma patients was analyzed. Select lung adenocarcinoma cell line A549, the experiment was divided into A549 cell group (blank control group), miR-NC group (transfected with miR-NC) and miR-mimics group (transfected with miR-874-3p mimics), sh-NC group (control group transfected with PKP3 silencing plasmid), sh-PKP3 group (transfected with PKP3 silencing plasmid), miR+pcDNA-PKP3 group (transfected with miR-874-3P mimics+pcDNA-PKP3, rescue group) and miR+pcDNA-NC group (transfected with miR-874-3p mimics+pcDNA-NC). The proliferation, invasion, migration and apoptosis of cells in each group were detected. ENCORI database was used to predict the upstream gene of PKP3, and dual luciferase assay was used to detect the targeting relationship between miR-874-3p and PKP3. MAPK/mTOR pathway-related proteins were detected by Western blotting. Results The expression of PKP3 in lung adenocarcinoma tissue was significantly higher than that in adjacent tissues. The high expression of PKP3 was related to clinical stage, tumor size, and lymph node metastasis (P<0.05).Compared with the human normal lung epithelial cells (BEAS-2B), the expression of PKP3 in A549 cells increased significantly, and the expression of miR-874-3p decreased (P<0.05). Overexpression of miR-874-3p decreased the PKP3 expression level (P<0.05). Compared with the control group, both overexpression of miR-874-3p and silenced PKP3 inhibited the cloning and invasion ability of A549 cells, caused cell cycle arrest, and decreased the expression levels of cyclin dependent kinase 4(CDK4), cyclin D1, cyclin E1 proteins in A549 cells (P<0.05). The expressions of Bax protein and Caspase-3 protein were up-regulated (P<0.05), and apoptosis increased. Overexpression of PKP3 could reverse the biological behavior of overexpression of miR-874-3p. Overexpression of miR-874-3p and silencing of PKP3 significantly decreased the expressions of P38 MAPK and mTOR phosphorylated proteins. Conclusion MiR-874-3p can negatively regulate PKP3 expression and inhibit the malignant biological behavior of A549 cells through MAPK/mTOR pathway.

导出引用

陈帆滕召虎房涛任君旭张静李雪王怡璇林旭吴靖芳. 微小RNA-874-3p 通过靶向调控斑菲素蛋白3影响肺腺癌细胞生物学行为及机制[J]. 解剖学报. 2025, 56(2): 188-201 https://doi.org/10.16098/j.issn.0529-1356.2025.02.009

CHEN Fan TENG Zhao-hu FANG Tao REN Jun-xu ZHANG Jing LI Xue WANG Yi-xuan LIN Xu WU Jing-fang. Effects of microRNA-874-3p on biological behavior of lung adenocarcinoma cells through targeted regulation of plakophilin 3 and its mechanism[J]. Acta Anatomica Sinica. 2025, 56(2): 188-201 https://doi.org/10.16098/j.issn.0529-1356.2025.02.009

中图分类号： R73

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