长寿保障同源基因2诱导Hepal-6肝癌细胞G0／G1期阻滞的作用及其机制

Mechanisms of G0/G1 phase arrest induced by longevity -assurance homologue 2 in Hepal -6 hepatoma cells

目的探讨长寿保障同源基因2（Lass2）对小鼠肝癌细胞Hepal-6细胞周期的影响及其机制。方法将前期成功构建的pEGFP-Lass2采用脂质体转染法转染Hepal-6肝癌细胞48h，流式细胞术碘化丙锭（PI）法检测各组（阴性对照、空载、实验组）细胞周期，实时荧光定量聚合酶链反应（FQ-PCR）法和Western blot法检测Lass2、细胞周期蛋白D1（Cyclin D1）、核因子-κB（NF-κB）p65mRNA及蛋白相对表达水平，并Western blot法检测核NF-κBp65磷酸化（NF-κB p-p65）表达水平；采用细胞计数试剂盒（CCK-8）法检测细胞增殖活性；为进一步验证且明确其机制，利用Hepal-6细胞悬液肝内注射法，构建C57BL／6J小鼠原位肝癌模型，采用流体力学尾静脉分次加强注射裸质粒，转染后分别提取各组小鼠肝组织总RNA、总蛋白及核蛋白，FQ-PCR、Western blot法验证Lass2、CyclinD1、NF-κBp65mRNA和蛋白相对表达水平、NF-κBp65磷酸化水平。结果Hepal-6肝癌细胞实验组Lass2 mRNA及蛋白相对表达水平较阴性对照组、空载组明显上调（mRNA：F=1486．895，均P=0．000；蛋白：F=55．964，均P=0．000），细胞周期G0／G1期百分比明显增高（F=239．867，均P=0．000）、G2／M期和S期明显减少（FG2/M=13．462，PG/M=0．003，0．005；FS=46．207，均P=0．000），同时下调实验组NF-κBp65、Cyclin D1 mRNA及蛋白表达水平（mRNA：FNF-κBP65=29．993，FcYELIN D1=113．260，均P=0．000；蛋白：FNF-κBP65=17．679，P=0．003，0．002；Fcyclin D1：176．357，均P=0．000），NF-κBp-p65蛋白磷酸化水平明显抑制（FNF-κ p-p65=33．817，P=0．000）。结论Lass2基因使肝癌细胞Hepal-6的细胞周期阻滞于G0／G1期，其作用机制可能是通过抑制NF-κB的激活，下调Cyclin D1的转录与蛋白表达有关。

Objective To investigate the effects of longevity - assurance homologue gene 2 （Lass2） on the cell cycle ofmouse hepatic carcinoma cell line Hepal -6 and the related mechanism. Methods Lass2 recombinant eukaryotic expression vector has been successfully constructed in the early stage, and were transfected in Hepal - 6 hepatoma cell line 48 h by liposome 2000. The cell cycle of each group （negative control, empty load, experimental group） was detected by flow cytometry （PI）. The relative expression levels of Lass2, Cyclin D1, nuclear factor - κB （ NF -κB） p65 mRNA and protein were detected by real - time fluorescent quantitative polymerase chain reaction （ FQ - PCR） and Western blotting. The phosphorylation of NF - κB p65 protein were detected by Western blotting; In order to further verify and clarify the mechanism, the C57BL/6J mouse hepatic carcinoma in situ model was constructed by injecting Hepal - 6 hepatic carcinoma cell suspension directly into the liver parenchyma of C57BL/6J mice. The naked plasmid was injected into the tail vein separately by hydrodynamics, and the total RNA and total protein in liver tissue of each group were extracted after transfection. The relative expression levels of Lass2, Cyclin D1, NF - κB 1965 mRNA and protein in mice liver were detected by FQ - PCR and Western blotting. The phosphorylation of NF - κB p65 protein were detected by Western blotting. Results The relative expression levels of Lass2 mRNA and protein in Hepal - 6 hepatoma cells were significantly higher than those in negative control group and blank control group （ F = 1 486. 895, all P = 0. 000 ; protein ： F = 55. 964, all P = 0. 000 ）, and the percentage of cell cycle G0/G1 phase increased significantly （ F = 239. 867, all P =0. 000） , significantly decreased in G2/M phase and S phase （FG2/M = 13. 462, PG2/M = 0. 003, 0. 005 ; Fs = 46. 207, all P = 0. 000） , at the same time, the levels of NF - κB p65, CyelinD1 mRNA and protein in the experimental group were decreased （ mRNA ： FNF-κB p65 = 29. 993, Fcyclin D1 = 113. 260, all P = 0. 000 ; protein ： FNF-κB κB p65 = 17. 679, P = 0. 003, 0. 002 ; F Cyclia, D1= 176. 357, all P = 0. 000）, the phosphorylation of NF - κB p65 were significantly downregulated （FNF-κB p-p65 = 33. 817, P = 0. 000）. Conclusion Lass2 gene blocks the cell cycle of Hepal -6 cells in G0/G1 phase, and its mechanism may be related to inhibits NF -κB p65 activation and expression and down - regulation of Cyclin D1 transcription and protein expression.