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Objective To explore the molecular mechanism of miR-200b-3p regulates the prolifera-tion,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 detec-ted 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 apop-tosis 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 apop-tosis 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 intensi-ties 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 overex-pressed 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 statisti-cally 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 apop-tosis 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.
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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.
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Objective To study the regulatory effects of microRNA-200b (miR-200b) on hypoxia-inducible factors-1α (HIF-1α） in neonatal immature rats with hypoxic-ischemic brain damage (HIBD).Method A total of 240 three-day-old neonatal Sprague-Dawley (SD) rats were randomly assigned into six groups with 40 rats in each group:the hypoxic-ischemic group (HI group),intraventricular injection of miR-200b agomir,intraventricular injection of miR-200b antagomir,intraventricular injection of agomir negative control group,intraventricular injection of antagomir negative control group and the normal control group.The HIBD models of immature neonatal rats were established except for the normal control group.The relative expressions of HIF-1 α in brain tissues of each group were detected using quantitative real-time-PCR at 12 h,1 d,3 d and 7 d after ventricular injection,and the changes of HIF-1α expression in each group were compared.Result (1) Compared with the control group,the expression of HIF-1oα of the HI group began to increase 12 h after the injection of normal saline into the lateral ventricle (P＜0.05),and reached the peak at 1d,with statistically significant difference (P＜0.05),and then gradually decreased to the normal control group level at 7 d.(2) No significant differences of HIF-1α existed among the HI group and the HI+ agomir negative control group and the HI + antagomir negative control group (P＞0.05),and the miR-200b carrier had no significant effects on the expression of HIF-1α.(3)HIF-1α continued to be highly expressed after the injection of antagomir into the lateral ventricle of HI,and was significantly higher than the HI group at 12 h (P＜0.05).No significant differences existed between the HI+antagomir group and the H1 group at 1 d,3 d and 7 d after antagomir injection (P＞0.05).The expression of HIF-1α was constantly lower than the HI group after the injection of agomir,and significantly lower than the HI group at 1d after injection (P＜0.05).Conclusion MiR-200b overexpression inhibits the expression of HIF-1α,and the low expression of miR-200b can increase the level of HIF-1oα in a limited time window.Therefore,miR-200b may participate in the regulation of brain injury in neonatal rats after HIBD by regulating the expression of HIF-1α.
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Objective:To investigate the expression and clinical significance of plasma microRNA-21 (miRNA-21) and microRNA-200b (miRNA-200b) in epithelial ovarian cancer (EOC).Methods:The levels of plasma miRNA-200b,miRNA-21 and CA125 were detected by RT-PCR in 162 patients with EOC,120 patients with benign epithelial ovarian tumors(benign group) and 108 healthy women(control group),analyze the relation between miRNA-200b and miRNA-21 expression and clinicopathological features of EOC.The sensitivity and specificity of miRNA-200b,miRNA-21 and CA125 to EOC diagnosis were evaluated by ROC curve,and the re-lationship between three indexes and EOC was analyzed by multivariate Logistic regression model.Correlation analysis of plasma miRNA-200b and miRNA-21,CA125 in patients with EOC by Pearson correlation.Results:The levels of plasma miRNA-200b,miRNA-21 and CA125 in EOC group were significantly higher than those in benign group and control group[miRNA-200b(2-ΔΔCt):3.52±1.03 vs 1.26±0.37 and 1.15±0.34;miRNA-21(2-ΔΔCt):2.32±0.45 vs 1.18±0.32 and 1.04±0.28;CA125(U/ml):78.64±30.57 vs 26.27±11.36 and 21.53±9.45,all P<0.01].Plasma miRNA-200b,miRNA-21,CA125 and three combined diagnosis EOC of AUC (95% CI) were 0.896(0.834-0.958),0.792(0.731-0.847),0.908(0.841-0.973),0.947(0.883-0.995),the optimal cut-off values were 2.08,1.46,52.84 U/ml.Logistic regression analysis showed that elevated plasma levels of miRNA-200b,miRNA-21 and CA125 were independent risk factors for EOC[OR(95% CI)= 2.518(1.563-3.547),OR(95% CI)= 1.724(1.103-2.528),OR (95% CI)=2.316(1.347-3.419)].The correlation between plasma miRNA-200b and CA125 in patients with EOC was the best(r=0.702,P<0.01).Conclusion:Plasma miRNA-200b and miRNA-21 can be used as molecular markers for the early diagnosis of EOC, and their diagnostic efficacy is comparable to that of CA125.The combined use of the three methods is expected to improve the accuracy of early diagnosis of EOC.
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Objective To investigate the effect of expression level and methylation level of miR-200b on proliferation, invasion and apoptosis of gastric cancer cell line MGC-803 in vitro. Methods Normal human gastric epithelium cell line GES-1, and gastric cancer cell line MGC-803 cells were cultured and harvested to extract total RNA. Then miR-200b ex?pression level was examined via q-PCR;Methylation in promoter of miR-200b was revealed by Bisulphite PCR. MGC-803 cells were treated with different concentrations of 5′-Aza-CdR to test its effect on miR-200b expression and methylation of its promotor. The effect of its treatment at 10μmol/L for 72 h on invasion,proliferation and apoptosis of both cell lines were detected by Transwell assay, Flow cytometry and apoptosis assay respectively. The gene related to EMT (epithelial-mesen?chymal transition ) such as E-cadherin,N-cadherin,ZEB1,Slug were checked by Western blot. Results The expression of miR-200b in GES-1 is higher than that in MGC-803(P=0.022). The methylation level in promoter region of miR-200b in GES-1 is lower than that in MGC-803(P=0.034). After treated with 5′-Aza-CdR, the expression of miR-200b in MGC-803 cell was up-regulated in a timely dependent manner. On the contrary, the methylation in promoter of mirR-200b were down-regulated when 5′-Aza-CdR were added at 10μmol/L(P=0.043). What’s more, 5′-Aza-CdR administration increas?es cell apoptosis especially in aged cells and delays cell cycle at G0/G1 which in turn decrease ratio of cells in S phages. 5′-Aza-CdR treatment also decreased invasiveness and induced expression of E-cadherin, as well as down regulated the ex?pressions of N-cadhrin, ZEB1, Slug and MMP9 in MGC-803 cells. Conclusion Expression of miR-200b could be affected by methylation of promoter of miR-200b and in turn regulate proliferation and invasion of gastric cells in vitro.