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

作　　者：刘博[1] 戚诚[1] 赵爽[2] 常晓静[3] 赵晓东[1] 张立超[4] 刘学臣[5] 刘斌[6] Bo Liu;Cheng Qi;Shuang Zhao;Xiao-jing Chang;Xiao-dong Zhao;Li-chao Zhang;Xue-chen Liu;Bin Liu(The Fourth Department of General Surgery,The Second Hospital,Hebei Medical University,Shijiazhuang,Hebei 050052,China;Department of Anesthesiology,The Third Hospital,Hebei Medical University,Shijiazhuang,Hebei 050051,China;Department of Radiotherapy,The Second Hospital,Hebei Medical University,Shijiazhuang,Hebei 050052,China;The Ninth Department of General Surgery,The Second Hospital,Hebei Medical University,Shijiazhuang,Hebei 050052,China;Department of Digestive,The Second Hospital,Hebei Medical University,Shijiazhuang,Hebei 050052,China;Department of Academic Research,Affiliated Hospital of Hebei University,Baoding,Hebei 071030,China)

机构地区：[1]河北医科大学第二医院普外四科,河北石家庄050052 [2]河北医科大学第三医院麻醉科,河北石家庄050051 [3]河北医科大学第二医院放疗科,河北石家庄050052 [4]河北医科大学第二医院普外九科,河北石家庄050052 [5]河北医科大学第二医院消化内科,河北石家庄050052 [6]河北大学附属医院科研处,河北保定071030

出　　处：《中国现代医学杂志》2021年第21期38-46,共9页China Journal of Modern Medicine

基　　金：河北省医学科学研究重点课题计划(No:20160540);2018年河北省自然科学基金资助面上项目(No:H2018206180)。

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。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 AFAP1L23'-URT complementarily.Conclusions MiR-219a-5p targeted-regulates AFAP1L2 expression to negatively modulate pancreatic cancer cell proliferation and apoptosis.

关 键 词：胰腺癌 肌动蛋白丝相关蛋白1相似蛋白2 microRNA-219a-5p 增殖 

分 类 号：R735.3[医药卫生—肿瘤]