Influence of miR-221-3p on the invasion and migration of melanoma cells by regulating vasohibin-1
Objective To observe the expressions of miR-221-3p and vasohibin-1(VASH-1)in melanoma tissues and melanoma cell line A375,and to investigate the roles of miR-221-3p and VASH-1 in the invasion and migration of melanoma cells.Methods Twenty-one melanoma patients were performed surgery in the First Affiliated Hospital of Xinjiang Medical University from January 2019 to September 2021,and the surgically resected melanoma tissues and paracancerous tissues were taken.The A375 cells in logarithmic growth phase and the normal human epidermal melanoma MC cells were taken.The relative expressions of miR-221-3p and VASH-1 mRNAs were detected by real-time fluorescence quantitative PCR in melanoma tissues,paracancerous tissues,A375 cells and MC cells.The A375 cells in logarithmic growth phase were divided into negative control group(transfected with siRNA-NC)and transfected group(transfected with miR-221-3p siRNA),the proliferation rates were detected by CCK-8 assay after 12-,24-,48-and 72-h culture,the numbers of migrating cells and invasive cells were detected by Transwell assay after 24-h culture,the cell cycle was detected by flow cytometry after 48-h culture,and the relative expressions of VASH-1,GRB2,ERK1+2,VEGF-D and VEGF-α proteins were detected by Western blot after 48-h culture in two groups.A luciferase reporter gene assay was used to verify the targeting relationship between miR-221-3p and VASH-1.Results(1)The relative expressions of miR-221-3p and VASH-1 mRNAs were higher in melanoma tissues(2.469±0.161,3.909±1.124)than those in paracancerous tissues(1.399±0.169,2.950±1.117)(t=21.024,P＜0.001;t=2.776,P=0.008),and were higher in A375 cells(1.514±0.088,2.306±0.404)than those in MC cells(0.804±0.032,0.559±0.130)(t=13.188,P＜0.001;t=7.131,P=0.002).(2)There were no significant differences in the proliferation rates after 12-,24-,48-and 72-h culture between two groups(P＞0.05).(3)After 24-h culture,the numbers of migrating cells and invasive cells were less in transfected group(319.515±15.325,19.995±11.346)than those in negative control group(494.155±13.126,114.005±11.266)(t=14.991,P＜0.001;t=10.184,P＜0.001).(4)There were no significant differences in the cell ratios of G1,S and G2 phase after 48-h culture between transfected group and negative control group(P＞0.05).(5)After 48-h culture,the relative expressions of VASH-1,GRB2,ERK1+2,VEGF-D and VEGF-αproteins were lower in transfected group(0.426±0.029,0.519±0.020,0.755±0.061,0.544±0.028,0.315±0.012)than those in negative control group(0.982±0.006,1.056±0.042,0.998±0.035,1.008±0.013,0.946±0.018)(P＜0.05).(6)The luciferase activity was lower in miR-221-3p siRNA+VASH-1 WT group(0.507±0.006)than that in miR-NC+VASH-1 WT group(1.062±0.040)(t=23.990,P＜0.001),and showed no significant difference between miR-221-3p siRNA+VASH-1 MUT group(1.082±0.048)and miR-NC+VASH-1 MUT group(1.019± 0.019)(t=2.140,P=0.099).MiR-221-3p targeted VASH-1.Conclusion The miR-221-3p and VASH-1 are highly expressed in melanoma tissues,and to down-regulate miR-221-3p can silence the expressions of VASH-1 and VEGF signaling pathway GRB2,VEGF-D,VEGF-α and ERK1+2 proteins,and inhibit the invasion and migration ability of A375 cells.
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