环指蛋白44基因表达对胃癌细胞增殖和凋亡的调节作用及机制

Moderating effect and mechanism of genetic expression of ring finger protein 44 on proliferation and apoptosis of gastric cancer cells

  • 摘要:
    目的 探讨环指蛋白44(RNF44)基因表达对胃癌细胞增殖和凋亡的调节作用及机制。
    方法 检测胃癌细胞株及正常胃黏膜细胞RNF44的基因和蛋白表达; 胃癌MKN45细胞株和AGS细胞株分别转染shRNF44病毒和oeRNF44病毒, 测定RNF44的基因和蛋白的表达、细胞增殖和细胞凋亡情况、蛋白激酶B(AKT)和磷酸化蛋白激酶B(p-AKT)的蛋白表达情况; 测定AKT通路抑制剂LY294002对RNF44基因过表达AGS细胞株增殖、凋亡和AKT信号蛋白的影响; 采用MKN45细胞株构建裸鼠皮下瘤模型,观察肿瘤生长情况、肿瘤组织Ki-67表达情况以及AKT和p-AKT蛋白表达情况。
    结果 正常胃黏膜GES-1细胞RNF44的基因和蛋白表达水平最低,胃癌MKN45细胞株RNF44的基因和蛋白表达水平最高。MKN45细胞株经RNF44基因干扰慢病毒转染后, RNF44的基因和蛋白表达水平降低,细胞增殖能力降低,早期和晚期凋亡率均增加, RNF44和p-AKT蛋白表达水平降低,差异均有统计学意义(P < 0.05)。AGS细胞株经RNF44基因过表达慢病毒转染后, RNF44的基因和蛋白表达水平升高,细胞增殖能力增加,细胞早期和晚期凋亡率均降低, RNF44和p-AKT蛋白表达水平增加,差异均有统计学意义(P < 0.05)。AKT抑制剂可使正常AGS细胞或RNF44基因过表达AGS细胞株增殖能力降低,细胞早期和晚期凋亡率升高, p-AKT蛋白表达水平降低。shRNF44组裸鼠肿瘤组织大小和质量均较shNC组降低, Ki-67表达较shNC组降低,肿瘤组织RNF44和p-AKT蛋白表达较shNC组降低,差异有统计学意义(P < 0.05)。
    结论 RNF44基因表达水平对胃癌细胞增殖和凋亡具有调节作用。RNF44过表达可显著抑制肿瘤细胞增殖并促进凋亡, RNF44低表达可显著促进肿瘤细胞增殖并抑制凋亡。RNF44可能通过调控p-AKT蛋白表达而发挥调节作用。

     

    Abstract:
    Objective To investigate the moderating effect and mechanism of gene expression of ring finger protein 44 (RNF44) on proliferation and apoptosis of gastric cancer cells.
    Methods The gene and protein expressions of RNF44 in gastric cancer cell lines and normal gastric mucosa cells were detected; the gastric cancer MKN45 cell lines and AGS cell lines were transfected with shRNF44 virus and oeRNF44 virus respectively, and the gene and protein expressions of RNF44, conditions of cell proliferation and cell apoptosis, the protein expressions of protein kinase B (AKT) and phosphorylated protein kinase B (p-AKT) were determined; the effect of AKT pathway inhibitor LYS294002 on the proliferation, apoptosis and AKT signaling protein of RNF44 gene overexpression cells was determined; the tumor growth, Ki-67 expression, AKT and p-AKT protein expression were determined in nude mouse subcutaneous tumor model constructed by MKN45 cell line.
    Results The gene and protein expression levels of RNF44 were the lowest in normal gastric mucosal GES-1 cells and the highest in gastric cancer MKN45 cell line. After transfected with lentivirus of RNF44 gene interference, the gene and protein expressions of RNF44 in MKN45 cell line decreased significantly, the cell proliferation ability decreased significantly, the apoptotic rate at early and late stages increased significantly, and the protein expressions of RNF44 and p-AKT decreased significantly (P < 0.05). After transfected with lentivirus of RNF44 gene overexpression, the gene and protein expression levels of RNF44 increased significantly in AGS cell line, the cell proliferation ability increased significantly, the apoptotic rate at early and late stages decreased significantly, and the protein expression levels of RNF44 and p-AKT increased significantly (P < 0.05). AKT inhibitors were able to reduce the proliferation ability of normal AGS cells or AGS cell lines with overexpression of the RNF44 gene, increase the apoptotic rate at early and late stages, and reduce the protein expression level of p-AKT. In the shRNF44 group, the size and mass of tumor tissues of nude mice were significantly lower than those in the shNC group, the expression of Ki-67 was significantly lower than that in the shNC group, and the protein expressions of RNF44 and p-AKT in tumor tissues were significantly lower than those in the shNC group (P < 0.05).
    Conclusion The expression level of RNF44 gene has a moderating effect on the proliferation and apoptosis of gastric cancer cells. Overexpression of RNF44 can significantly inhibit tumor cell proliferation and promote apoptosis, while low expression of RNF44 can significantly promote tumor cell proliferation and inhibit apoptosis. RNF44 may play a moderating role by regulating the expression of p-AKT protein.

     

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