MicroRNA-219a-5p和AFAP1L2对胰腺癌细胞的作用研究

MicroRNA-219a-5p targeted-regulates AFAP1L2 expression to modulate pancreatic cells proliferation

摘要:

目的 探讨microRNA-219a-5p（miR-219a-5p）和肌动蛋白丝相关蛋白1相似蛋白2（AFAP1L2）对胰腺癌细胞的作用。方法 分别采用实时荧光定量聚合酶链反应（qRT-PCR）和Western blotting法检测胰腺癌细胞株（PANC-1、BXPC-3、SW1990、Capan-1）及正常胰腺导管上皮细胞（HPDE）miR-219a-5p、AFAP1L2的表达，采用siRNA及过表达质粒下调或上调AFAP1L2表达，CCK-8法观察胰腺癌细胞株增殖变化。采用mimics或inhibitor上调或下调miR-219a-5p表达，CCK-8法观察胰腺癌细胞株增殖变化。流式细胞术检测不同干预条件下细胞凋亡和细胞周期。生物信息学预测两者可能具有靶向调控关系。qRT-PCR和Western blotting法检测AFAP1L2 mRNA及蛋白表达，荧光素酶实验观察二者碱基配对关系，进一步验证miR-219a-5p对AFAP1L2具有靶向调控作用。结果 与HPDE细胞比较，胰腺癌细胞株中miR-219a-5p表达较低（P <0.05）。AFAP1L2 mRNA及蛋白在胰腺癌细胞株中的表达高于在HPDE细胞中的表达（P <0.05）。Capan-1或SW1990细胞培养48 h后，与空白对照组（NC组）比较，pCMV-EGFP-AFAP1L2组光密度（OD）值升高（P <0.05），siAFAP1L2组OD值下降（P <0.05），AFAP1L2对胰腺癌细胞增殖具有正向调控作用。Capan-1及SW1990细胞培养48 h后，与miR-NC组比较，miR-219a-5p mimics组OD值下降（P <0.05），miR-219a-5p inhibitor组OD值升高（P <0.05），miR-219a-5p表达对胰腺癌细胞增殖具有负向调控作用；上调miR-219a-5p表达，可诱导细胞S期阻滞，导致细胞凋亡增加。miR-219a-5p负向调控AFAP1L2表达；荧光素酶实验显示，miR-219a-5p与AFAP1L2的3'-URT互补结合，miR-219a-5p与AFAP1L2存在靶向调控关系。结论 miR-219a-5p通过靶向调控AFAP1L2表达负向介导胰腺癌细胞增殖及凋亡。

Abstract:

Objective To observe microRNA-219a-5p(miR-219a-5p) target-modulates AFAP-1L2 expression to influence pancreatic cancer cell proliferation, and to discuss the mechanism.Methods Expression of miR-219a-5p and AFAP1L2 in pancreatic cancer cell lines (PANC-1, BXPC-3, SW1990, Capan-1) and normal pancreatic ductal epithelial cells (HPDE) were detected by qRT-PCR and Western blot assay. AFAP1L2 was down-regulated or up-regulated by siRNA or over-expressed plasmid, and pancreatic cancer cell proliferation was observed by CCK assay. MiR-219a-5p was down-regulated or up-regulated by inhibitor or mimics, and pancreatic cancer cell proliferation was observed by CCK assay. Flow cytometry assay was used to detect cell cycle and apoptosis. Bioinformatic analysis predicted they were target-related, AFAP1L2 mRNA and protein were detected by qRT-PCR and Western blotting assay. Luciferase assay was used to observe the base pairing relation and to prove the target effect.Results Compared with HPDE, miR-219a-5p in pancreatic cancer cells expression was lower (P < 0.05). AFAP1L2 mRNA and protein in pancreatic cancer cells were higher than in HPDE cells (P < 0.05). Capan-1 and SW1990 were cultured for 48 h. Compared with NC group, OD value was higher in pCMV-EGFP-AFAP1L2 group (P < 0.05), and was lower in siAFAP1L2 group (P < 0.05), which means AFAP1L2 positively controlled pancreatic cancer cell proliferation. In addition, compared with miR-NC group, OD value was lower (P < 0.05) in miR-219a-5p mimics group, and was higher in miR-219a-5p inhibitor group (P < 0.05), which showed miR-219a-5p negatively controlled pancreatic cancer cell proliferation; Up-regulating miR-219a-5p expression induce S phase stagnated and increased cell apoptosis. MiR-219a-5p negatively controlled AFAP1L2 expression; Luciferase assay showed that miR-219a-5p and AFAP1L2 have targeted relation as the result of that miR-219a-5p was combined with AFAP1L2 3'-URT complementarily.Conclusions MiR-219a-5p targeted-regulates AFAP1L2 expression to negatively modulate pancreatic cancer cell proliferation and apoptosis.