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

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。

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.