The Effects of miR-411-3p on the Proliferation, Migration and Angiogenic Mimicry of Colorectal Cancer Cells by Regulating the HIF-1α/VEGF Pathway
LIU Yuguan1, LIU Lintao2, XU Bo2, WANG Maonan2*
This study aims to discuss the effects of miR-411-3p on the proliferation, migration and angiogen esis mimicy of CRC (colorectal cancer) cells by regulating the HIF-1α (hypoxia inducible factor 1α)/VEGF (vascular endothelial growth factor) pathway. Human CRC cells SW480 cultured in vitro were randomly separated into the Control group, miR-NC group, miR-411-3p mimics group, DMOG group (HIF-1α/VEGF pathway activator DMOG), miR-411 3p mimics+DMOG group, miR-411-3p mimics+NC group and miR-411-3p mimics+HIF-1α group. The dual-luciferase reporter assay was used to detect the relationship between miR-411-3p and HIF-1α. MTT and colony formation assay were used to detect cell proliferation. Cell apoptosis was detected by flow cytometry. The scratch test was used to de tect the migration of cells in each group. Vascular simulation formation experiments were used to detect the formation of VM (vasculogenic mimicry) of cells. Immunofluorescence experiments were used to detect the expression levels of Ki67, CD31 and VE-cadherin in cells of each group. Moreover, qRT-PCR was used to detect the mRNA expressions of miR-411-3p, HIF-1α, and VEGF in each group. Western blot was used to detect the expression of HIF-1α, apoptosis and migration-related proteins in each group. The results of dual-luciferase reporter assay showed that the luciferase activity in the miR-411-3p mimics group was significantly lower than that in the miR-NC group (P<0.05). Compared with the Control group and the miR-NC group, the miR-411-3p mimics group had serious damage to the cell lumen structure, re duced survival rate, colony formation number, scratch healing rate, the number of luminal structures, and decreased fluo rescence intensity of Ki67, CD31, and VE-cadherin. The expression levels of HIF-1α, VEGF mRNA and protein, and N cadherin were decreased, while the apoptosis rate and the expression levels of Bax, cleaved caspase-3, miR-411-3p and E cadherin were increased (P<0.05). Compared with the Control group, the DMOG group had significantly improved cell lumen structure formation, increased colony formation number, scratch healing rate, the number of lumen structures, and increased fluorescence intensity of Ki67, CD31, and VE-cadherin. The mRNA and protein levels of HIF-1α and VEGF, and the expression of N-cadherin were increased, while the apoptosis rate and the expression levels of Bax, cleaved cas pase-3, and E-cadherin were decreased (P<0.05). Compared with the DMOG group, the miR-411-3p mimics+DMOG group had aggravated damage to the cell lumen structure, reduced survival rate, colony formation number, scratch healing rate, the number of luminal structures, and decreased fluorescence intensity of Ki67, CD31 and VE-cadherin. The mRNA and protein levels of HIF-1α and VEGF, and the expression of N-cadherin were decreased, while the apoptosis rate and the expression levels of Bax, cleaved caspase-3, miR-411-3p, and E-cadherin were increased (P<0.05). Compared with the miR-411-3p mimics group, the miR-411-3p mimics+DMOG group had reduced damage to the luminal structure, increased survival rate, colony formation number, scratch healing rate, the number of luminal structures, and increased f luorescence intensity of Ki67, CD31 and VE-cadherin. The mRNA and protein levels of HIF-1α and VEGF, and the ex pression of N-cadherin were increased, while the apoptosis rate and the expression levels of Bax, cleaved caspase-3, and E-cadherin were decreased (P<0.05). Compared with miR-411-3p mimics group and miR-411-3p mimics+NC group, the cell survival rate, colony formation number, scratch healing rate and the number of luminal structures in miR-411-3p mimics+HIF-1α group were increased. The fluorescence intensity of Ki67, CD31 and VE-cadherin increased, while the mRNA and protein levels of HIF-1α and VEGF and the expression level of N-cadherin increased. The apoptosis rate and the expression levels of Bax, cleaved caspase-3 and E-cadherin were significantly decreased (P<0.05). Up-regulating the expression of miR-411-3p may inhibit the proliferation, migration and VM formation of colorectal cancer cells by sup pressing the HIF-1α/VEGF pathway.
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