miR-138-5p靶向HIF-1α对人肾癌细胞GRC-1增殖、凋亡和迁移的影响

Effects of miR-138-5p targeting HIF-1α on the proliferation, apoptosis and migration of human renal cancer GRC-1 cells

目的探讨miR-138-5p靶向HIF-1α对人肾癌细胞GRC-1增殖、凋亡和迁移的影响。方法将GRC-1细胞分为四组,分别为GRC-1组、miR-138 mimic组、pc-HIF-1α组及共转染组(mimic+pc-HIF-1α组),并将mimic-scramble组设置为miR-138 mimic转染的阴性对照组。采用荧光素酶报告实验验证miR-138-5p与HIF-1α的靶向关系,采用qRT-PCR检测miR-138 mimic对GRC-1细胞HIF-1αmRNA水平的影响,采用Western blot检测miR-138对GRC-1细胞HIF-1α蛋白水平的影响。分别采用CCK-8、流式细胞术和划痕实验检测miR-138-5p靶向HIF-1α对GRC-1细胞的增殖、凋亡和迁移的影响。结果荧光素酶报告实验结果显示miR-138-5p可靶向HIF-1α。与GRC-1组比较,miR-138 mimic组的miR-138-5p mRNA表达水平较高,HIF-1αmRNA表达水平较低(均P<0.01);与GRC-1组比较,miR-138 mimic组的HIF-1α蛋白表达水平较低,而pc-HIF-1α组的HIF-1α蛋白表达水平较高(均P<0.01);mimic+pc-HIF-1α组的HIF-1α蛋白表达水平低于pc-HIF-1α组(P<0.01);与GRC-1组比较,miR-138 mimic组的GRC-1细胞增殖倍数较低,而pc-HIF-1α组的GRC-1细胞增殖倍数较高(均P<0.01);mimic+pc-HIF-1α组的GRC-1细胞增殖倍数低于pc-HIF-1α组,差异有统计学意义(P<0.01);与GRC-1组比较,miR-138 mimic组的GRC-1细胞凋亡率较高,而pc-HIF-1α组的GRC-1细胞凋亡率较低(均P<0.01);mimic+pc-HIF-1α组的GRC-1细胞凋亡率高于pc-HIF-1α组(P<0.01);与GRC-1组比较,miR-138 mimic组的GRC-1细胞划痕愈合率较低,而pc-HIF-1α组的GRC-1细胞划痕愈合率较高(均P<0.01);mimic+pc-HIF-1α组的GRC-1细胞划痕愈合率低于pc-HIF-1α组(P<0.01)。结论miR-138-5p可通过靶向HIF-1α,负向调控HIF-1αmRNA和蛋白的表达,进而减弱人肾癌细胞GRC-1的增殖和迁移,促进其凋亡。

Objective To investigate the effects of miR-138-5p targeting HIF-1αon the proliferation,apoptosis and migration of human renal cancer GRC-1 cells.Methods GRC-1 cells were divided into a GRC-1 group,a miR-138 mimic group,a pc-HIF-1αgroup,and a mimic+pc-HIF-1αgroup.The negative control group of transfection of miR-138 mimic was also set up.Luciferase assay was used to verify the targeting relationship between miR-138-5p and HIF-1α.qRT-PCR was used to detect the effects of miR-138 mimic on HIF-1αmRNA levels of GRC-1 cells,and Western blot was used to detect the effects of miR-138 on HIF-1αprotein levels of GRC-1 cells.The effects of miR-138-5p targeting HIF-1αon the proliferation,apoptosis and migration of GRC-1 cells were determined by CCK-8,flow cytometry and scratch assay,respectively.Results Luciferase reporter assay showed that miR-138-5p could target HIF-1α.Compared with the GRC-1 group,miR-138-5p mRNA expression levels were higher and HIF-1αmRNA expression levels were lower in the miR-138 mimic group(all P<0.01).The expression levels of HIF-1αwere lower in the miR-138 mimic group and higher in the pc-HIF-1αgroup(all P<0.01).The expression level of HIF-1αprotein in the mimic+pc-HIF-1αgroup was lower than that in the pc-HIF-1αgroup(P<0.01).The proliferation rate of GRC-1 cells was lower in the miR-138 mimic group and higher in the pc-HIF-1αgroup(all P<0.01),and the proliferation rate of GRC-1 cells in the mimic+pc-HIF-1αgroup was lower than that in the pc-HIF-1αgroup(P<0.01).The apoptosis rate of GRC-1 cells was higher in the miR-138 mimic group and lower in the pc-HIF-1αgroup(all P<0.01),and the apoptosis rate of GRC-1 cells in the mimic+pc-HIF-1αgroup was higher than that in the pc-HIF-1αgroup(P<0.01).The scratch healing rate of GRC-1 cells in the miR-138 mimic group was lower,while that of pc-HIF-1αgroup was higher(all P<0.01),and the scratch healing rate of GRC-1 cells in the mimic+pc-HIF-1αgroup was lower than that in the pc-HIF-1αgroup(P<0.01).Conclusions miR-138-5p can negatively regulate the expression of HIF-1αmRNA and protein by targeting HIF-1α,thereby attenuating the proliferation and migration of human renal carcinoma cell GRC-1 and promoting its apoptosis.