Study on the molecular mechanism of miR-200b-3p regulates the proliferation, invasion, migration and apoptosis of pancreatic cancer cells by down-regulating VEGFA

Abstract

Objective To explore the molecular mechanism of miR-200b-3p regulates the proliferation, invasion, migration and apoptosis of pancreatic cancer cells by targeting vascular endothelial growth factor A (VEGFA). Methods The expression of miR-200b-3p in pancreatic cancer tissues and cell lines was detected by quantitative real-time fluorescence PCR (qRT-PCR). Pancreatic cancer PANC-1 cells were divided into NC group, miR-200b-3p mimic group, si-VEGFA group and si-VEGFA+ miR-200b-3p inhibitor group. The proliferation, migration and invasion of PANC-1 cells were measured by CCK-8 and Transwell assay. The apoptosis of PANC-1 cells was detected by Annexin V-FITC/PI double staining flow cytometry assay. The targeted relationship of miR-200b-3p and VEGFA was estimated by dual luciferase reporter gene assay and Western blotting. Results The expression of miR-200b-3p in pancreatic cancer tissues and cell lines was decreased. After miR-200b-3p was overexpressed in PANC-1 cells for 48 h, the cell viabilities of PANC-1 cells in NC group and miR-200b-3p mimic group were 1.250±0.028 and 0.983±0.044, the numbers of migrated cells were 402.700±21.530 and 158.000±17.620, the numbers of invaded cells were 478.300±31.050 and 170.000±32.470, and the cell apoptosis rates were (5.280±0.352)% and (7.430±0.393)%. The cell viability, migration and invasion of PANC-1 cells in miR-200b-3p mimic group were significantly decreased than those in NC group (t=5.060, P=0.007; t=8.796, P=0.001; t=6.863, P=0.002). The cell apoptosis rate in miR-200b-3p mimic group was significantly higher than that in NC group (t=4.076, P=0.015). The fluorescence intensity in VEGFA-WT group was 1.000±0.027, which was significantly higher than that in VEGFA-WT+ miR-200b-3p mimic group (0.632±0.048; t=6.637, P=0.003). The fluorescence intensities in VEGFA-MUT group and VEGFA-MUT + miR-200b-3p mimic group were 1.000±0.049 and 0.868±0.047, with no statistically significant difference (t=1.944, P=0.124). After miR-200b-3p was overexpressed in PANC-1 cells for 48 h, the expressions of VEGFA in NC group and miR-200b-3p mimic group were 1.000±0.058 and 0.762±0.020, respectively. The expression level in miR-200b-3p mimic group was lower than that in NC group (t=3.908, P=0.017). After transfection of PANC-1 cells with si-VEGFA or si-VEGFA + miR-200b-3p inhibitor for 48 h, the cell viabilities of PANC-1 cells in NC group, si-VEGFA group and si-VEGFA + miR-200b-3p inhibitor group were 1.300±0.058, 0.943±0.047 and 1.143±0.023, the numbers of migrated cells were 446.000±17.350, 206.300±19.360 and 428.300±30.330, and the numbers of invaded cells were 510.300±24.550, 175.700±24.290 and 473.700±35.530, with statistically significant differences (F=15.830, P=0.004, F=33.530, P=0.001, F=38.860, P<0.001). The cell viability, migration and invasion of PANC-1 cells in si-VEGFA group were significantly decreased than those in NC group (P=0.003, P<0.001, P<0.001). There was no significant difference between si-VEGFA + miR-200b-3p inhibitor group and NC group (P=0.107, P=0.854, P=0.671). The cell apoptosis rates in NC group, si-VEGFA group and si-VEGFA+ miR-200b-3p inhibitor group were (3.810±0.577)%, (7.373±0.482)% and (3.650±0.514)%, with a statistically significant difference (F=16.020, P=0.004). The cell apoptosis rate in si-VEGFA group was significantly higher than that in NC group (P=0.007), but there was no significantly difference between si-VEGFA + miR-200b-3p inhibitor group and NC group (P=0.975). Conclusion miR-200b-3p suppresses the proliferation, invasion and migration and promotes the apoptosis of pancreatic cells by down-regulating VEGFA. Key words: Pancreatic neoplasms; MicroRNA-200b-3p; Vascular endothelial growth factor A