MicroRNA-105-5p/PPM1A对胰腺癌PANC-1细胞增殖、迁移及侵袭的机制研究

Mechanistic study on the role of microRNA-105-5p/PPM1A in the proliferation, migration, and invasion of pancreatic cancer PANC-1 cells

摘要:

目的 探讨microRNA-105-5p（miR-105-5p）/PPM1A对胰腺癌PANC-1细胞增殖、迁移、侵袭及上皮细胞向间质转化（EMT）进程的影响及其潜在作用机制。方法 实时荧光定量聚合酶链反应（qRT-PCR）检测miR-105-5p在人胰腺导管上皮细胞hTRET-HPNE和胰腺癌细胞PANC-1、AsPC-1、Bxpc-3中的表达。利用Kaplan-Meier Plotter在线工具探讨miR-105-5p与胰腺癌患者预后的关系。在PANC-1细胞中分别转染mimic NC、miR-105-5p mimic、inhibitor NC、miR-105-5p inhibitor。CCK-8法、划痕实验、Transwell实验分别检测各组细胞的增殖、迁移及侵袭能力；qRT-PCR检测miR-105-5p对E-cadherin、N-cadherin、Vimentin、ZEB1表达的影响。生物信息学方法预测miR-105-5p的候选靶基因，并对候选靶基因进行GO和KEGG富集分析。双萤光素酶实验检测miR-105-5p与PPM1A的靶向关系。qRT-PCR检测在PANC-1细胞中分别转染mimic NC、miR-105-5p mimic、inhibitor NC、miR-105-5p inhibitor后PPM1A的表达。免疫荧光实验检测PPM1A在人胰腺导管上皮细胞hTRET-HPNE和胰腺癌细胞PANC-1、AsPC-1、Bxpc-3中的表达。在PANC-1细胞中分别转染mimic NC+ pcDNA3.1、mimic NC+ pcDNA3.1-PPM1A、miR-105-5p mimic + pcDNA3.1-PPM1A后，通过挽救实验进一步研究miR-105-5p inhibitor与PPM1A在胰腺癌细胞中的相互作用关系。结果 胰腺癌细胞PANC-1、AsPC-1、Bxpc-3中miR-105-5p mRNA相对表达量高于hTRET-HPNE细胞中miR-105-5p mRNA相对表达量（P <0.05），以PANC-1细胞中的相对表达量最高。miR-105-5p高表达与胰腺癌患者的不良预后有关（P <0.05）。miR-105-5p mimic组细胞增殖、迁移及侵袭能力均高于mimic NC组（P <0.05）。与mimic NC比较，miR-105-5p mimic下调E-cadherin mRNA表达，上调N-cadherin、Vimentin、ZEB1 mRNA表达（P <0.05）。转染miR-105-5p inhibitor后得到相反的结果。双萤光素酶实验证实miR-105-5p与PPM1A存在靶向关系。免疫荧光实验显示在胰腺癌细胞PANC-1、AsPC-1、Bxpc-3中PPM1A的荧光强度低于人胰腺导管上皮细胞hTRET-HPNE（P <0.05）。挽救实验表明miR-105-5p可部分挽救PPM1A对PANC-1细胞增殖、迁移和侵袭的抑制作用（P <0.05）。结论 miR-105-5p靶向PPM1A促进胰腺癌PANC-1细胞的增殖、迁移及侵袭。

Abstract:

Objective To investigate the effect of microRNA-105-5p (miR-105-5p) on the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) process of pancreatic cancer PANC-1 cells, focusing on the potential mechanism involving PPM1A.Methods The expression levels of miR-105-5p in human pancreatic duct epithelial cells (hTRET-HPNE) and pancreatic cancer cells (PANC-1, AsPC-1, Bxpc-3) were detected by real-time fluorescence quantitative PCR (qRT-PCR). The relationship between miR-105-5p expression and pancreatic cancer patient prognosis was analyzed using the Kaplan-Meier Plotter online tool. PANC-1 cells were transfected with mimic NC, miR-105-5p mimic, inhibitor NC, and miR-105-5p inhibitor, respectively. Cell proliferation, migration, and invasion were assessed using CCK-8, scratch wound healing, and Transwell assays. The effects of miR-105-5p on the expression of E-cadherin, N-cadherin, Vimentin, and ZEB1 were evaluated by qRT-PCR. Bioinformatics analysis predicted candidate target genes of miR-105-5p, followed by GO and KEGG enrichment analysis. Dual-luciferase reporter assays verified the targeting relationship between miR-105-5p and PPM1A. The expression of PPM1A in PANC-1 cells was detected after transfection with mimic NC, miR-105-5p mimic, inhibitor NC, and miR-105-5p inhibitor. Immunofluorescence experiments were conducted to measure PPM1A expression in hTRET-HPNE and pancreatic cancer cells. Finally, rescue experiments were performed by transfecting PANC-1 cells with mimic NC + pcDNA3.1, mimic NC + pcDNA3.1-PPM1A, miR-105-5p mimic + pcDNA3.1-PPM1A to explore the interaction between miR-105-5p and PPM1A in pancreatic cancer cells.Results The relative expression levels of miR-105-5p mRNA were higher in PANC-1, AsPC-1, and Bxpc-3 cells compared to hTRET-HPNE cells (P < 0.05), with the highest levels in PANC-1 cells. High expression of miR-105-5p was associated with poor prognosis in pancreatic cancer patients (P < 0.05). PANC-1 cells transfected with miR-105-5p mimic showed increased proliferation, migration, and invasion compared to the mimic NC group (P < 0.05). MiR-105-5p mimic decreased E-cadherin mRNA expression and increased N-cadherin, Vimentin, and ZEB1 mRNA expression (P < 0.05), while miR-105-5p inhibitor produced the opposite effects. Dual-luciferase reporter assays confirmed the targeting relationship between miR-105-5p and PPM1A. Immunofluorescence experiments demonstrated lower PPM1A fluorescence intensity in PANC-1, AsPC-1, and Bxpc-3 cells compared to hTRET-HPNE cells (P < 0.05). Rescue experiments indicated that miR-105-5p could partially reverse the inhibitory effects of PPM1A on the proliferation, migration, and invasion of PANC-1 cells (P < 0.05).Conclusion miR-105-5p targets PPM1A to promote the proliferation, migration, and invasion of pancreatic cancer PANC-1 cells.