微小RNA-135a-5p对胰腺癌增殖、迁移和耐药的影响

Effect of microRNA-135a-5p on proliferation,migration and chemoresistance of pancreatic cancer cell

目的观察微小RNA(miRNA,miR)-135a-5p对胰腺癌细胞增殖、迁移和对顺铂化疗敏感性的影响。方法采用聚合酶链反应检测胰腺癌患者肿瘤组织和癌旁组织,正常胰腺导管上皮细胞HPDE和人源胰腺癌Panc1和AsPc-1细胞中miR-135a-5p的表达;采用脂质体将miR-135a-5p mimic瞬时转染至Panc1和AsPc-1细胞中,采用细胞计数试剂盒(CCK-8)增殖实验和划痕实验检测各组细胞增殖能力和迁移能力;用不同浓度梯度的顺铂处理Panc1和AsPc-1细胞,酶标仪检测各组细胞吸光度值变化,以比较各组细胞顺铂化疗敏感性;使用在线软件预测miR-135a-5p下游靶基因,并使用双荧光素酶报告基因系统进行验证;蛋白质印迹法(Western blot)检测各组细胞支链氨基酸转氨酶1(BCAT1)的表达水平。应用SPSS 18.0统计软件分析,计量数据以均值±标准差(Mean±SD)表示,组间比较使用单因素方差分析。结果胰腺癌组织中miR-135a-5p的表达显著低于癌旁组织(0.23±0.08比1.03±0.18),人源胰腺癌Panc1和AsPc-1细胞中miR-135a-5p的表达显著低于正常胰腺导管上皮细胞HPDE(0.30±0.13,0.56±0.09比0.97±0.12,P<0.05);miR-135a-5p mimic组Panc1和AsPc-1细胞增殖能力和迁移能力受到明显抑制(P<0.05),细胞中BCAT1蛋白表达水平明显降低,双荧光素酶报告基因实验证实miR-135a-5p可靶向调控BCAT1基因的表达。miR-135a-5p mimic组Panc1细胞对顺铂化疗敏感性明显提高(P<0.05)。Panc1细胞共转染miR-135a-5p mimic和BCAT1过表达载体,可部分逆转miR-135a-5p上调介导的细胞增殖、迁移能力减弱和对顺铂化疗敏感性的增加。结论miR-135a-5p在胰腺癌中低表达,上调miR-135a-5p可通过靶向BCAT1基因抑制胰腺癌细胞增殖、迁移,增加顺铂化疗敏感性。

Objective To investigate the effect of microRNA(miRNA,miR)-135a-5p on proliferation,migration and chemoresistance of pancreatic cancer.Methods Expression of miR-135a-5p in pancreatic cancer tissues and adjacent normal tissues,Panc1 and AsPc-1 cell lines as well as normal pancreatic duct epithelial cell line HPDE were detected by reverse transcription-polymerase chain reaction(RT-qPCR).The Panc1 and AsPc-1 cells were transfected with miR-135a-5p mimics,the proliferation and migration abilities of cells in each group were detected by cell counting kite-8(CCK-8)assay and scratch assay.Panc1 and AsPc-1 cells were treated with cisplatin of different concentrations,absorbance values were detected by microplate reader and the chemosensitivity to cisplatin were compared in each group.The downstream target genes of miR-135a-5p were predicted using online software and the dual-luciferase reporter gene system was used for verification.The expression levels of branched chain amino acid aminotransferase1(BCAT1)in each group were detected by Western blotting.Results The expression of miR-135a-5p in pancreatic cancer tissues was significantly lower than that in adjacent tissues(0.23±0.08 vs.1.03±0.18),and the expression of miR-135a-5p in Panc1 and AsPc-1 cells was significantly lower than that in normal pancreatic duct epithelial cell line HPDE(0.30±0.13,0.56±0.09 vs.0.97±0.12,P<0.05).The proliferation and migration ability of Panc1 and AsPc-1 cells in the miR-135a-5p mimic group were significantly inhibited(P<0.05),and the expression levels of BCAT1 protein in the cells were significantly decreased.The dual-luciferase reporter gene system confirmed that miR-135a-5p could target and regulate the expression of BCAT1 gene.The sensitivity of Panc1 cells to cisplatin chemotherapy was significantly increased in the miR-135a-5p mimic group(P<0.05).The co-transfection of Panc1 cells with miR-135a-5p mimic and BCAT1 overexpression vector partially reversed the decreased cell proliferation,decreased migration ability and increased sensitivity to cisplatin chemotherapy mediated by the upregulation of miR-135a-5p.Conclusion MiR-135a-5p is low expressed in pancreatic cancer,and upregulation of miR-135a-5p can inhibit cell proliferation and migration of pancreatic cancer cells by targeting BCAT1 gene,and increase the sensitivity of cisplatin chemotherapy.