The efect of hypoxic culture on miR-301a expressi on in pancreatic cancer CFPAC-1 cells
10.3760/cma.j.issn.1674-1935.2016.03.005
- VernacularTitle:低氧培养对胰腺癌 CFPAC-1细胞 miR-301 a表达的影响
- Publication Type:Journal Article
- Keywords:
Pancreatic neoplasms;
Anoxia;
Epithelial mesenchymal transformation;
MicroRNAs
- From:
Chinese Journal of Pancreatology
2016;16(3):164-169
- CountryChina
- Language:Chinese
-
Abstract:
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. 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<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 significantly downregulated (all P<0.01).Results of scratch test showed that the healing rates of scratch area in miR-301a group after 18h was(94.82 ±2.18)%, which was significantly larger than that [( 54.68 ±4.31 )%] in miR-301a-NC group ( P<0.01 ).The number of migration cells in miR-301a group and miR-301a-NC group was 95 ±11 and 25 ±6 at 100 ×magnification, respectively .Compared with control group , the cells that migrated through membrane were obviously increased in the group transfected with miR-301a (P<0.01).Conclusions EMT and upregulation of miR-301a were observed in CFPAC-1 cells after being cultured in low oxygen .miR-301 a may promote EMT by inhibiting FOXF2 expression in pancreatic cancer .
