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目的 探讨沉默泛素载体蛋白9(Ubc9)基因表达诱导蛋白激酶B1(AKT1)去类泛素化修饰,及其对肝癌细胞增殖和转移的影响.方法 利用RNA干扰技术沉默HepG2肝癌细胞Ubc9基因表达,Western blot法检测Ubc9、小泛素相关修饰蛋白1(SUMO1)和蛋白激酶B1(AKT1)蛋白表达水平,四甲基偶氮唑蓝(MTT)法和流式细胞术检测HepG2细胞增殖活性,细胞划痕实验和Transwell方法检测细胞迁移能力.建立荷瘤鼠模型,测量肿瘤体积并绘制生长曲线,解剖肿瘤组织并称重比较.采用原位细胞凋亡实验检测肿瘤组织细胞凋亡情况,免疫组化法检测肿瘤组织中增殖细胞核抗原(PCNA)、基质金属蛋白酶2(MMP-2)和 MMP-9的表达情况.结果 siR-Ubc9基因转染可使HepG2细胞中Ubc9、共轭SUMO1、游离SUMO1和AKT1蛋白水平均明显下降(均P<0.05).对照组、siR-neg组和siR-Ubc9组的细胞增殖指数分别为53.19%、54.25%和39.17%,细胞迁移距离分别为(59.47±4.66)μm、(56.56±5.37)μm和(34.57±6.61)μm,发生迁移的细胞数量分别为(89.44±8.36)个/视野、(93.84±8.79)个/视野和(41.67±5.39)个/视野,siR-Ubc9组与对照组和siR-neg组差异均有统计学意义(均P<0.05).对照组、siR-neg组和siR-Ubc9组的皮下肿瘤重量分别为(3.78±0.69)g、(3.72± 0.72)g和(2.09±0.61)g,细胞凋亡率分别为(7.79±2.21)%、(6.45±2.48)%,和(33.59±5.44)%,siR-Ubc9组与对照组和siR-neg组差异均有统计学意义(均P<0.05).对照组和siR-neg组肿瘤组织中PCNA、MMP-2和MMP-9蛋白均高表达,而siR-Ubc9组肿瘤组织中PCNA、MMP-2和MMP-9蛋白表达均低表达,差异均有统计学意义(均P<0.05).结论 通过沉默肝癌细胞中Ubc9基因表达能够诱导AKT1去SUMO化修饰,进而抑制肝癌细胞的增殖和转移,为未来肝癌基因治疗提供了新的借鉴方法.

Objective To investigate the effect of AKT1 deSUMOylation induced by Ubc9 silencing on the proliferation and metastasis of hepatocellular carcinoma(HCC)cells. Methods The Ubc9 gene was silenced using RNA interference, and the expression levels of Ubc9, SUMO1 and AKT1 protein were detected by Western blot. Cell proliferation and cell cycle was analyzed by MTT and flow cytometry. Wound healing and transwell assays were used to detect the cell migration ability. Furthermore,the xenograft model was established, and tumor growth curves were drawn. The in situ apoptotic rates was measured using TUNEL Apoptosis Assay. The expression of proliferating cell nuclear antigen(PCNA), matrix metalloproteinase(MMP)-2 and MMP-9 were evaluated by immunohistochemical staining. Results Knockdown of Ubc9 gene significantly decreased the protein expression levels of Ubc9,conjugated SUMO1,free SUMO1 and AKT1 in HCC cells(P<0.05 for all). In control, siR-neg and siR-Ubc9 groups, the cell proliferation indexes were 53.19%,54.25% and 39.17%, respectively. Moreover, cell migration distance and migrating cells per low power field for all these three groups were(59.47±4.66)μm and 89.44±8.36,(56.56±5.37) μm and 93.84±8.79,as well as(34.57±6.61)μm and 41.67±5.39, respectively. In the xenograft model, the weights of subcutaneous tumors for these three groups were(3.78±0.69)g,(3.72±0.72)g and(2.09± 0.61)g,respectively. The corresponding apoptotic cell rates were(7.79 ± 2.21)%,(6.45 ± 2.48)% and (33.59 ± 5.44)%, respectively. The expression levels of PCNA, MMP-2 and MMP-9 protein were significantly decreased in siR-Ubc9 group(P<0.05). Conclusions Ubc9 silencing in HCC cells induces AKT1 deSUMOylation, and then inhibits the proliferation and metastasis. These results provide a new therapeutic strategy for liver cancer in the future.