LncRNA BLACAT1调节miR-324-5p/MAP4K3轴对肝癌细胞增殖、迁移和侵袭的影响

Effect of LncRNA BLACAT1 on Proliferation,Migration and Invasion of Liver Cancer Cells via Regulation of miR-324-5p/MAP4K3 Axis

目的探讨长链非编码RNA膀胱癌相关转录本1(LncRNA BLACAT1)调节miR-324-5p/丝裂原活化蛋白激酶激酶激酶激酶3(MAP4K3)轴对人肝癌细胞增殖、迁移和侵袭的影响。方法实时荧光定量PCR(RT-qPCR)检测肝癌组织、癌旁组织以及人正常肝细胞、人肝癌细胞(Huh-7、SMMC-7721、MHCC97 L)中BLACAT1、miR-324-5p、MAP4K3 mRNA的表达水平;将肝癌细胞系Huh-7细胞分为:ctrl组、si-NC组、si-BLACAT1组、si-BLACAT1+miR-NC组、si-BLACAT1+miR-324-5p inhibitor组;实时荧光定量PCR(RT-qPCR)检测各组细胞中BLACAT1、miR-324-5p、MAP4K3 mRNA的表达;CCK-8法检测细胞增殖;划痕实验检测细胞迁移;Transwell小室检测细胞侵袭;蛋白免疫印迹检测细胞中MAP4K3、PCNA、MMP2、MMP9、c-Myc、Cyclin D1的表达;双荧光素酶报告基因实验分别验证BLACAT1和miR-324-5p、miR-324-5p和MAP4K3的关系。结果肝癌组织和人肝癌细胞系中BLACAT1、MAP4K3 mRNA表达水平显著增加,miR-324-5p表达显著降低(P<0.05);与ctrl组、si-NC组比较,si-BLACAT1组Huh-7细胞的BLACAT1、MAP4K3 mRNA表达、A450值、迁移率、侵袭率、PCNA、MMP2、MMP9、MAP4K3、c-Myc、Cyclin D1表达明显降低(P<0.05),miR-324-5p表达显著增加(P<0.05);抑制miR-324-5p表达减弱了敲低BASCAT1对Huh-7细胞的增殖、迁移和侵袭能力的抑制作用;BASCAT1靶向负调控miR-324-5p表达,miR-324-5p靶向调控MAP4K3表达。结论敲低BLACAT1可能通过靶向miR-324-5p来下调MAP4K3表达,进而抑制肝癌细胞增殖、迁移和侵袭。

Objective To investigate the effect of long non-coding RNA bladder cancer-associated transcript 1(LncRNA BLACAT1)on the proliferation,migration and invasion of human hepatoma cells by regulating miR-324-5p/mitogen-activated protein kinase kinase kinase kinase 3(MAP4K3)axis.Methods Real-time quantitative PCR(RT-qPCR)was used to detect the expression of BLACAT1,miR-324-5p and MAP4K3 mRNA in the hepatocellular carcinoma tissues,paracancerous tissues,and human normal hepatocytes and hepatoma cell lines(Huh-7,SMMC-7721,MHCC97 L).The liver cancer cells Huh-7 were divided into 5 groups:control group,si-NC group,si-BLACAT1 group,si-BLACAT1+miR-NC group,and si-BLACAT1+miR-324-5p inhibitor group.RT-qPCR was used to detect the mRNA expression levels of BLACAT1,miR-324-5p and MAP4K3 in cells of each group.CCK-8 assay was used to detect the cell proliferation.Wound-healing assay and transwell assay was used to detect the cell migration and cell invasion.Western blotting was used to detect the expression levels of MAP4K3,PCNA,MMP2,MMP9,c-Myc and Cyclin D1 in cells.The dual-luciferase reporter assays were used to verify the relationship between BLACAT1 and miR-324-5p,and the relationship between miR-324-5p and MAP4K3.Results The mRNA expression levels of BLACAT1 and MAP4K3 in hepatoma tissues and human hepatoma cell lines were greatly increased,and the expression level of miR-324-5p was greatly decreased(P<0.05).When compared with the control group and the si-NC group,the mRNA expression levels of BLACAT1,MAP4K3,the A450 value,the migration and invasion rate,and the expression levels of PCNA,MMP2,MMP9,MAP4K3,c-Myc and Cyclin D1 proteins were greatly decreased in the si-BLACAT1 group(P<0.05).The expression level of miR-324-5p was greatly increased(P<0.05).Silencing of miR-324-5p attenuated the inhibitory effect of BASCAT1 knockdown on the proliferation,migration and invasion in Huh-7 cells.BASCAT1 targeted and negatively regulated the expression of miR-324-5p,and miR-324-5p targeted and regulated the MAP4K3.Conclusion Knockdown of BLACAT1 may down-regulate the expression of MAP4K3 by targeting miR-324-5p,thereby inhibiting the proliferation,migration and invasion of liver cancer cells.