miR-153-5p通过靶向FZD3基因抑制胰腺癌细胞的增殖、迁移和侵袭

miR-153-5p inhibits proliferation,migration and invasion of pancreatic cancer cells by targeting FZD3 gene

目的:研究微RNA(microRNA,miR)-153-5p对胰腺导管腺癌细胞增殖、迁移和侵袭的影响,并探讨其相关的作用机制。方法:采用实时荧光定量PCR法检测miR-153-5p在9例胰腺导管腺癌及相应的癌旁组织中的表达水平,以及其在胰腺正常导管上皮细胞HPDE和胰腺癌各个细胞系PANC-1、MIAPaCa-2、SW1990及BxPC-3中的表达水平,从中筛出PANC-1和SW1990细胞用于后续实验。采用脂质体法将miR-153-5p-模拟物(mimics)转入PANC-1及SW1990细胞使miR-153-5p过表达,以转入阴性对照(negative control,NC)-mimics作为阴性对照组。采用CCK-8法及Transwell小室实验检测miR-153-5p过表达对PANC-1及SW1990细胞增殖、迁移和侵袭的影响。通过Targetscan等多种在线预测软件及生物信息学分析miR-153-5p可能的靶基因,并采用实时荧光定量PCR法检测腺导管腺癌及相应的癌旁组织中卷曲蛋白家族受体3(Frizzled3,FZD3)mRNA的表达水平。采用实时荧光定量PCR法和蛋白质印迹法检测miR-153-5p过表达对FZD3 mRNA和蛋白表达水平的影响,并进一步利用双荧光素酶报告实验验证FZD3基因是否为miR-153-5p的靶基因。通过脂质体法将miR-153-5p-mimics以及携带有FZD3全基因的重组质粒共转入PANC-1和SW1990细胞,再用实时荧光定量PCR法及蛋白质印迹法检测FZD3 mRNA及蛋白的表达水平,并通过CCK-8法以及Transwell小室实验检测miR-153-5p和FZD3表达水平的变化对胰腺癌细胞增殖、迁移和侵袭能力的影响。结果:相较于正常胰腺组织或细胞,miR-153-5p在胰腺癌组织及各个细胞中的表达量明显更低(P均<0.05)。CCK-8和平板克隆实验显示,过表达miR-153-5p后对PANC-1和SW1990细胞的增殖能力被明显抑制(P均<0.05);划痕愈合实验结果显示,miR-153-5p过表达能够有效地延缓划痕伤口的愈合,抑制胰腺癌细胞的横向迁移能力(P均<0.05);Transwell小室实验提示,miR-153-5p过表达能明显抑制胰腺癌细胞侵袭和纵向迁移能力(P均<0.05)。利用在线预测软件及生物信息学分析得到FZD3基因可能是miR-153-5p的下游靶点;相较于正常胰腺组织,FZD3 mRNA在胰腺癌组织中的表达量明显升高(P<0.01),FZD3 mRNA与miR-153-5p的表达呈负相关(r=-0.5515,P=0.0219)。与阴性对照组相比,过表达miR-153-5p后PANC-1和SW1990细胞中FZD3 mRNA和蛋白的表达量明显减少(P均<0.05)。双荧光素酶报告实验显示,miR-153-5p与FZD3 mRNA的3’-非翻译区(untranslated region,UTR)结合,下调FZD3蛋白的表达水平。当共转染miR-153-5p-mimics及携带有全基因的FZD3重组质粒后,过表达FZD3能够减弱miR-153-5p对胰腺癌细胞增殖、迁移和侵袭的抑制作用(P均<0.05)。结论:miR-153-5p通过靶向FZD3基因抑制胰腺癌的侵袭和迁移,提示miR-153-5p可能是胰腺癌治疗中一种潜在生物标志物。

Objective:To investigate the effects of microRNA(miR)-153-5p on the proliferation,migration and invasion of pancreatic ductal adenocarcinoma cells,and to explore its underlying mechanisms.Methods:Real-time fluorescence quantitative PCR was used to detect the expression level of miR-153-5p in 9 pancreatic ductal adenocarcinoma tissues and their corresponding paracancerous tissues,as well as its expression level in normal pancreatic ductal epithelial cells HPDE and pancreatic cancer cell lines PANC-1,MIA PaCa-2,SW1990 and BxPC-3,from which PANC-1 and SW1990 were screened out for subsequent experiments.The miR-153-5p-mimics was transfected into PANC-1 and SW1990 cells for miR-153-5p overexpression by liposome method,and the negative control(NC)-mimics were transferred as the negative control group.The effects of miR-153-5p overexpression on the proliferation,migration and invasion of PANC-1 and SW1990 cells were examined by CCK-8 method,plate clone formation experiment and Transwell assay.The possible target genes of miR-153-5p were analyzed by various online prediction softwares such as Targetscan and bioinformatics,and the expression level of frizzled 3(FZD3) mRNA in adenoductal adenocarcinoma and corresponding paracancerous tissues was detected by real-time fluorescence quantitative PCR.The effects of miR-153-5p overexpression on the expression levels of FZD3 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blotting,and whether the FZD 3 gene was the target gene of miR-153-5p was further verfied using a dual-luciferase reporter assay.The miR-153-5p-mimics and recombinant vector carrying the full length gene of FZD 3 were co-transfected into PANC-1 and SW1990 cells by liposome method,and then the expression levels of FZD3 mRNA and protein were detected by realtime fluorescence quantitative PCR and Western blotting.The effects of changes in the expression levels of miR-153-5p and FZD3 on the proliferation,migration and invasion of pancreatic cancer cells were examined by CCK-8 method and Transwell assay.Results:The expression of miR-153-5p was significantly lower in pancreatic cancer tissues and cancer cell lines compared to normal pancreatic tissues or cells(both P<0.05).CCK-8 and plate clone formation experiment assays showed that the proliferation of PANC-1and SW1990 cells was significantly inhibited after overexpression of miR-153-5p(both P<0.05).The results of wound healing assay showed that miR-153-5p overexpression could effectively delay the healing of scratch wounds and inhibit the lateral migration of pancreatic cancer cells(both P<0.05);transwell assay suggested that miR-153-5p overexpression could significantly inhibit the invasion and longitudinal migration of pancreatic cancer cells(both P<0.05).FZD 3 gene was predicted as a possible downstream target of miR-153-5p using online prediction software and bioinformatics analysis;the expression of FZD3mRNA was significantly higher in pancreatic cancer tissues compared to normal pancreatic tissues(P<0.01),and the expression of FZD3 mRNA was negatively correlated with miR-153-5p expression(r =-0.551 5,P =0.021 9).The expression of FZD3 mRNA and protein was significantly reduced in PANC-1 and SW1990 cells after overexpression of miR-153-5p compared with negative controls(both P<0.05).Dual luciferase reporter assay showed that miR-153-5p bound to the 3′-untranslated region(UTR) of FZD3 mRNA and downregulated the expression of FZD3 protein.When co-transfected with miR-153-5p-mimics and recombinant vector carrying the full length FZD 3 gene,overexpression of FZD3 was able to attenuate the inhibitory effects of miR-153-5p on proliferation,migration and invasion of pancreatic cancer cells(all P<0.05).Conclusion:MiR-153-5p inhibited pancreatic cancer cell migration and invasion by targeting the FZD 3 gene,suggesting that miR-153-5p may be a potential biomarker in pancreatic cancer therapy.