Objective To investigate the differential expression profile of microRNAs in BxPC-3 human pancreatic cancer cells during epithelial-mesenchymal transition(EMT).Methods BxPC-3 cells were cultured and assigned in the transforming growth factor-β1(TGF-β1) treatment group and control group.The changes in the cellular morphology were observed under inverted phase-contrast microscope.Cell invasion and migration were assessed by Transwell-Matrigel assay.The variations in the expression of EMT-related biomarkers were quantitatively measured by real-time quantitative PCR and Western blot.The changes in the miR expression before and after the EMT of BxPC-3 cells were analyzed by miR assay.Results BxPC-3 cells became elongated and intercellular junction was loose after TGF-β1 treatment.Transwell-matrigel assay revealed that the quantity of invasive BxPC-3 cells in the TGF-β1 treatment group was significantly increased compared with that in the control group (P<0.05).The expression levels of E-cadherih protein and mRNA were significantly down-regulated (both P<0.05), whereas the expression of vimentin and N-cadherin at the protein and mRNA levels was significantly up-regulated (both P<0.05).Total RNA was extracted from BxPC-3 cells.In two groups, the ratio of A260/A280 was calculated as 2.03 and 2.06.Agarose gel electrophoresis detected no signs of RNA degradation, prompting that the purity and integrity of BxPC-3 cells were qualified for experimental requirement.MiR array revealed that the expression levels of 26 miRs significantly differed before and after TGF-β1 induction (all P<0.05).Among them, the expression levels were up-regulated in 15 miRs and down-regulated in 11 miRs.Conclusions The expression profile of miR is altered during the EMT process of human pancreatic cancer cells.The expression levels of 26 miRs are changed before and after TGF-β1-induced EMT.%目的 分析人胰腺癌BxPC-3细胞上皮-间质转化(EMT)过程中微小RNA(miR)表达谱的差异.方法 培养BxPC-3细胞,设立转化生长因子-β1(TGF-β1)诱导96 h组(TGF-β1处理组)与未经诱导组(对照组),倒置相差显微镜观察细胞形态变化,Transwell-Matrigel体外侵袭试验检测细胞的侵袭迁移能力,实时定量PCR及蛋白免疫印迹法检测细胞EMT相关标记物的变化.采用miR微阵列芯片技术检测BxPC-3细胞EMT前后miR表达的变化.结果 在TGF-β1的刺激下,BxPC-3细胞拉长,细胞间连接变得疏松.Transwell试验显示,TGF-β1处理组BxPC-3细胞中侵袭细胞较对照组增多(P<0.05).上皮标记物上皮钙黏素的蛋白及mRNA表达水平均下降(P均<0.05),而其间质标记物波形蛋白、神经钙黏素蛋白及mRNA表达水平均升高(P均<0.05).提取BxPC-3细胞总RNA,2组细胞样本的A260/A280比值分别为2.03、2.06,变性琼脂糖凝胶电泳结果显示RNA无降解,提示BxPC-3细胞RNA纯度及完整性符合试验要求.RNA微阵列芯片技术筛选结果显示,BxPC-3细胞有26个miR的表达水平在诱导前后比较差异有统计学意义(P均<0.05),其中15个miR表达上调和11个miR表达下调.结论 人胰腺癌细胞EMT过程中miR表达谱发生了变化,其中26个miR的表达水平在诱导EMT前后存在差异.
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