miR-200b-3p下调VEGFA调控胰腺癌细胞增殖、侵袭、迁移和凋亡的分子机制研究

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

目的探讨miR-200b-3p通过血管内皮生长因子A(VEGFA)调控胰腺癌细胞增殖、侵袭、迁移和凋亡的分子机制。方法采用实时荧光定量PCR(qRT-PCR)检测胰腺癌组织和细胞中miR-200b-3p表达水平;胰腺癌细胞PANC-1分为NC组、miR-200b-3p mimic组、si-VEGFA组及si-VEGFA+miR-200b-3p inhibitor组。CCK-8和Transwell检测胰腺癌PANC-1细胞增殖活力以及细胞迁移和侵袭能力;Annexin V-FITC/PI双染细胞凋亡实验检测PANC-1细胞凋亡;Western blotting和双荧光素酶报告基因验证miR-200b-3p与VEGFA的靶向关系。结果miR-200b-3p在胰腺癌组织和细胞中低表达。PANC-1细胞过表达miR-200b-3p 48 h后,NC组和miR-200b-3p mimic组细胞增殖活性分别为1.250±0.028、0.983±0.044;迁移细胞数分别为402.700±21.530、158.000±17.620,侵袭细胞数分别为478.300±31.050、170.000±32.470,细胞凋亡百分比为(5.280±0.352)%、(7.430±0.393)%;miR-200b-3p mimic组细胞增殖活性、迁移和侵袭能力均显著低于NC组(t=5.060,P=0.007;t=8.796,P=0.001;t=6.863,P=0.002),细胞凋亡百分比显著高于NC组(t=4.076,P=0.015)。VEGFA-WT单独转染细胞荧光强度为1.000±0.027,显著高于VEGFA-WT+miR-200b-3p mimic共转染细胞(0.632±0.048;t=6.637,P=0.003)。VEGFA-MUT单独转染细胞荧光强度为1.000±0.049,与VEGFA-MUT+miR-200b-3p mimic共转染细胞差异无统计学意义(0.868±0.047;t=1.944,P=0.124)。PANC-1细胞过表达miR-200b-3p 48 h后,NC组和miR-200b-3p mimic组中VEGFA相对表达量分别为1.000±0.058、0.762±0.020,miR-200b-3p mimic组显著低于NC组(t=3.908,P=0.017)。PANC-1细胞转染si-VEGFA或同时转染si-VEGFA+miR-200b-3p inhibitor 48 h后,NC组、si-VEGFA组及si-VEGFA+miR-200b-3p inhibitor组PANC-1细胞增殖活性分别为1.300±0.058、0.943±0.047、1.143±0.023,迁移细胞数分别为446.000±17.350、206.300±19.360、428.300±30.330,侵袭细胞数分别为510.300±24.550、175.700±24.290、473.700±35.530,组间差异具有统计学意义(F=15.830,P=0.004;F=33.530,P=0.001;F=38.860,P<0.001)。si-VEGFA组细胞增殖活性、迁移和侵袭能力均显著低于NC组(P=0.003,P<0.001,P<0.001),而si-VEGFA+miR-200b-3p inhibitor组细胞增殖活性、迁移和侵袭能力与NC组差异无统计学意义(P=0.107,P=0.854,P=0.671)。NC组、si-VEGFA组及si-VEGFA+miR-200b-3p inhibitor组细胞凋亡百分比分别为(3.810±0.577)%、(7.373±0.482)%、(3.650±0.514)%,组间差异具有统计学意义(F=16.020,P=0.004),si-VEGFA组细胞凋亡百分比显著高于NC组(P=0.007),而si-VEGFA+miR-200b-3p inhibitor组与NC组差异无统计学意义(P=0.975)。结论miR-200b-3p通过靶向下调VEGFA表达,进而抑制胰腺癌细胞增殖、侵袭和迁移,并促进细胞凋亡。

ObjectiveTo 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).MethodsThe 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.ResultsThe 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).ConclusionmiR-200b-3p suppresses the proliferation,invasion and migration and promotes the apoptosis of pancreatic cells by down-regulating VEGFA.