miR-206靶向p65-GLS1通路影响肝细胞癌增殖、迁移及侵袭

Effect of miR-206 on proliferation,migration and invasion of hepatocellular carcinoma cells via targeting p65-GLS1 signal pathway

[目的]探究miR-206是否能通过靶向核因子κB p65-谷氨酰胺酶1通路来影响肝细胞癌生物学行为。[方法]采用RT-PCR检测各肝细胞癌细胞和人正常肝细胞中miR-206表达,将HepG2细胞按转染方式不同分为NC mimic组、miR-206 mimic组、NC inhibitor组、miR-206 inhibitor组,CCK-8、划痕实验、Transwell实验检测各组细胞增殖、迁移、侵袭水平,RT-PCR检测细胞miR-206表达,Western Blot检测p-p65、GLS1蛋白表达,生物信息学和双荧光素酶实验分析miR-206与p65的靶向结合作用。[结果]各HepG2细胞中miR-206表达均低于HL-7702细胞,且HepG2细胞miR-206表达水平低于BEL-7402、Huh7细胞(P<0.05);与NC mimic组比较,miR-206 mimic组细胞增殖水平(1.36±0.22 vs 0.70±0.16;P<0.05)、迁移率(59.62±1.46 vs 30.14±1.13;P<0.05)%、侵袭数(202.16±13.59 vs 69.51±14.06;P<0.05)、SOD、p65、p-p65、GLS1蛋白表达降低,α-KG、Glu、MDA表达升高;与NC inhibitor组比较,miR-206 inhibitor组细胞增殖(1.41±0.19 vs 2.09±0.27;P<0.05)、迁移率(61.03±1.24 vs 75.62±2.84;P<0.05)、侵袭数(200.34±12.78 vs 268.15±16.53;P<0.05)、SOD、p65、p-p65、GLS1蛋白表达升高,α-KG、Glu、MDA表达降低(P<0.05);生物信息学及荧光素酶实验显示,p65是miR-206的靶基因。[结论]miR-206可通过抑制p65-GLS1通路(0.91±0.06 vs 0.15±0.04;P<0.05)来降低HepG2细胞谷氨酰胺代谢及氧化应激水平,从而抑制其增殖、迁移和侵袭能力。

[Objective]To explore the feasibility of miR-206 regulating biological behaviour of hepatocellular carcinoma(HCC)cells via targeting transcription factor nuclear factor-kappa B-glutaminase(p65-GLS1)signal pathway.[Method]Real time polymerase chain reaction(RT-PCR)was utilized to measure the relative expression of miR-206 in HCC cells and normal liver cell line HL-7702.HepG2 cells were classified into NC mimic group,miR-206 mimic group,NC inhibitor group and miR-206 inhibitor group according to the different transfection methods.CCK8 assay,scratch test and transwell chamber assay were performed to detect the proliferation,migration and invasion of cells,respectively.The expression of miR-206 was detected via RT-PCR,and the protein expression of p-p65 and GLS1 was measured by Western Blot.miR-206 targeting p65 were predicted by bioinformatics analysis and further confirmed by dual-luciferase reporter assays.[Result]The miR-206 expression in HCC cells was lower than that in HL-7702 cells,and the miR-206 expression level in HepG2 cells was lower than that in BEL-7402 and Huh7 cells(P<0.05).After transfection,assays revealed that miR-206 mimic group had decreased cell proliferation(1.36±0.22 vs 0.70±0.16;P<0.05),migration(59.62±1.46 vs 30.14±1.13;P<0.05)%,invasion(202.16±13.59 vs 69.51±14.06;P<0.05)and SOD activity,down-regulated expression of p65,p-p65 and GLS1,and up-regulated expression ofα-KG and Glu,as well as increased MDA content compared to NC mimic group.Compared with NC inhibitor group,miR-206 inhibitor group had increased cell proliferation(1.41±0.19 vs 2.09±0.27;P<0.05),migration(61.03±1.24 vs 75.62±2.84;P<0.05),invasion(200.34±12.78 vs 268.15±16.53;P<0.05)and SOD activity,up-regulated expression of p65,p-p65 and GLS1,and down-regulated expression ofα-KG and Glu,as well as decreased MDA content,with statistical difference(all P<0.05).Bioinformatics and dual-luciferase reporter assays indicated that p65 was the target gene of miR-206.[Conclusion]miR-206 attenuates the glutamine metabolism and oxidative stress by inhibiting the p65-GLS1 pathway(0.91±0.06 vs 0.15±0.04;P<0.05),thereby inhibiting HCC cell proliferation,migration,and invasive ability.