中文摘要：

目的：观察低氧培养胰腺癌CFPAC-1细胞后miR-301 a表达的变化，探讨miR-301 a的可能作用机制。方法在1％O2环境中培养胰腺癌细胞CFPAC-1，相差显微镜下观察细胞形态变化。蛋白质印迹法检测细胞低氧诱导因子HIF-1α、miR-301a及其靶基因FOXF2，上皮型标志物E-cadherin、ZO-1、β-catenin，上皮间质转化（EMT）标志物Vimentin、N-cadherin、Fibronectin的表达。构建miR-301a慢病毒表达载体，建立稳定转染miR-301a的细胞株，以转染与miR-301a不匹配（ miR-301a-NC）的细胞作为对照组，观察细胞形态变化，检测FOXF2、上皮型及EMT标志物表达，划痕实验和Transwell小室实验检测细胞迁移、侵袭能力。结果 CFPAC-1细胞在低氧环境培养后细胞外形不规则、多呈棱形、伪足较多，细胞间连接松散，向间质细胞形态改变。以常氧培养细胞的表达量为1，低氧培养4 d后细胞miR-301a表达量为2．82±0．01，HIF-1α为2．17±0．36，Vimentin、N-cadherin、Fibronectin分别为5．48±0．16、1．16±0．03、1．30±0．03，均显著上调；FOXF2、E-cadherin、ZO-1、β-catenin 分别为0．45±0．05、0．61±0．05、0．45±0．02、0．37±0．02，均显著下调，差异均有统计学意义（P值均＜0．01）。以转染miR-301a-NC细胞的蛋白表达量为1，转染miR-301a细胞的miR-301a、Fibronectin、Vimentin、N-cadherin 表达量分别为7．09±0．42、15．6±0．10、1．24±0．05、1．37±0．01，表达均显著上调；FOXF2、ZO-1、E-cadherin、β-catenin表达量分别为0．33±0．03、0．71±0．02、0．67±0．06、0．47±0．03，表达均显著下调，差异均有统计学意义（P值均＜0．01）。转染miR-301a-NC组、稳转miR-301a组CFPAC-1细胞18 h的覆盖划痕区面积分别为（54．68±43．1）％、（94．82±2．18）％；24 h的穿膜细胞数分别为（25±6）、（95±11）／100倍视野，稳转miR-301a组显著高于转染miR-301a-NC组，差异均有统计学意义

英文摘要：

Objective To study the regulation on miR-301 a expression in pancreatic cancer CFPAC-1 cells by low oxygen condition and explore the potential molecular mechanism of miR -301a.Methods CFPAC-1 cells were cultured in 1%O 2 and the morphology of CFPAC-1 cells was observed by phase contrast microscopy . The expression of HIF1-a, miR-301a, its′target gene FOXF2 and EMT markers Vinentin, N-Cadherin and Fibronectin were detected by Western blot .The lentivirus vector expressing miR-301a was constructed and stably transfected CFPAC-1 cells were established using transfected with miR-301 a-NC as control .After the transfection, the cell morphology was observed, and FOXF2 and the EMT markers expression were assessed.＆amp;nbsp;The migration and invasion of CFPAC-1 cells was determined by Transwell migration assay and wound healing test.Results The CFPAC-1 cells after being cultured in hypoxic environment had an irregular shape with more fusiform and pseudopodia , loose intercellular connections , and were transformed into mesenchymal cell morphology . By setting the expression of target genes in cells cultured in normoxia as 1, the relative expression of miR-301a was 2.82 ±0.01 after being cultured in hypoxia for 4 d, and HIF-1a level was 2.17 ±0.36, and Vimentin, N-cadherin and Fibronectin level was 5.48 ±0.16, 1.16 ±0.03 and 1.30 ±0.03, respectively, which were significantly upregulated .And FOXF2, E-cadherin , ZO-1,β-catenin expression was 0.45 ± 0.049, 0.61 ±0.049, 0.45 ±0.021, 0.37 ±0.02 respectively, which were significantly downregulated （all P＆lt;0.01）.By setting the protein expression of target genes in cells transfected with miR-301a-NC as 1, the expression of miR-301a, Fibronectin, Vimentin and N-cadherin in cells transfected with miR-301a were 7.09 ±0.42、1 5.6 ±0.10、1.24 ±0.05、1.37 ±0.01, respectively,which were significantly upregulated . And the expression of FOXF2、ZO-1、E-cadherin、β-catenin was 0.33 ±0.03, 0.71 ±0.02, 0.67 ±0.06 and 0.47 ±0.03, respectively, which were