[目的]探究miR-206是否能通过靶向核因子κBp65-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。22vs0。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。59vs69。51±14。06;P＜0。05)、SOD、p65、p-p65、GLS1 蛋白表达降低，a-KG、Glu、MDA 表达升高;与 NC inhibi-tor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。78vs268。15±16。53;P＜0。05)、SOD、p65、p-p65、GLS1 蛋白表达升高，a-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-GLSl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。
Effect of miR-206 on proliferation,migration and invasion of hepatocellular carcinoma cells via targeting p65-GLS1 signal pathway
[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 tar-geting 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 of α-KGand 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 of α-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.
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