MiD49在肝癌中的表达与促增殖作用研究

Experimental study on the expression and pro-proliferative function of MiD49 in hepatocellular carcinoma

目的探讨线粒体动力蛋白MiD49在肝癌中的表达及其促增殖作用。方法 (1)用实时定量PCR(q RT-PCR)、Western blot及免疫组化实验方法,分别检测MiD49在肝癌细胞株以及50例肝癌手术患者癌与癌旁组织中的表达;(2)siRNA下调MiD49表达后,分别用MTT与克隆形成实验检测MiD49在肝癌细胞增殖中的调控作用;(3)siRNA下调MiD49表达后,用流式细胞术检测MiD49在肝癌细胞凋亡中的调控作用。结果 (1)肝癌组织中MiD49表达的mRNA与蛋白水平均显著高于癌旁组织[mRNA水平:癌vs癌旁=(0.38±0.28)vs(0.26±0.20);蛋白水平:癌vs癌旁=(5.67±0.47)vs(3.20±0.33)],差异均有统计学意义(P<0.05);肝癌细胞株中MiD49表达高于正常肝细胞[5种肝癌细胞vs正常肝细胞=(2.42±0.23)、(4.57±0.35)、(2.53±0.22)、(3.30±0.44)、(3.11±0.32)vs(1.00±0.08)],差异有统计学意义(P<0.05)。(2)利用siRNA下调MiD49表达可显著抑制肝癌细胞增殖[siRNA vs si-MiD49#1 vs si-MiD49#2=(4.78±0.37)vs(3.07±0.28)vs(3.26±0.21)],差异有统计学意义(P<0.05);利用siRNA下调MiD49表达可显著抑制肝癌细胞克隆形成[siRNA vs si-MiD49#1 vs si-MiD49#2=(449.00±34.00)vs(239.00±20.52)vs(223.66±21.73)],差异有统计学意义(P<0.05)。(3)利用siRNA下调MiD49表达可促进肝癌细胞凋亡[siRNA vs si-MiD49#1 vs si-MiD49#2=(3.90±0.40)vs(12.67±0.96)vs(13.17±1.01)],差异有统计学意义(P<0.05)。结论线粒体动力蛋白MiD49在肝癌中表达显著上调,MiD49高表达可促进肝癌细胞增殖而抑制凋亡,提示MID49是潜在的促癌因子并可作为潜在的肿瘤治疗靶标。

Objective To evaluate the expression of mitochondrial dynamics protein 49（MiD49） and its proproliferative function in hepatocellular carcinoma（HCC）. Methods（1） q RT-PCR, western blotting and immunohistochemistry analysis were used to detect the expression levels of MiD49 in HCC cell lines and tumor and peritumor tissues of 50 patients with HCC.（2） The effect of MiD49 knockdown on the proliferation of HCC cells was analyzed by MTT and colony formation assays.（3） The effect of MiD49 knockdown on the apoptosis of HCC cells was analyzed by flow cytometry. Results（1） The expression of MiD49 mRNA and protein were higher in tumor tissues of HCC when compared with peritumor tissues：（0.38±0.28） vs（0.26±0.20） for mRNA（P〈0.05）;（5.67±0.47） vs（3.20±0.33） for protein（P〈0.05）. The expression of MiD49 was higher in five kinds of HCC cell lines when compared with normal liver cell line：（2.42±0.23）,（4.57±0.35）,（2.53±0.22）,（3.30±0.44）,（3.11±0.32） vs（1.00±0.08）, P〈0.05.（2） Knockdown of MiD49 significantly inhibited the proliferation of HCC cells： siRNA vs si-MiD49#1 vs si-MiD49#2=（4.78 ± 0.37） vs（3.07 ± 0.28） vs（3.26 ± 0.21）, P〈0.05. Knockdown of MiD49 significantly inhibited the colony formation of HCC cells： siRNA vs siMiD49#1 vs si-MiD49#2=（449.00±34.00） vs（239.00±20.52） vs（223.66±21.73）, P〈0.05.（3） Knockdown of MiD49 significantly induced the apoptosis of HCC cells： siRNA vs si-MiD49#1 vs si-MiD49#2=（3.90 ± 0.40） vs（12.67 ± 0.96） vs（13.17 ± 1.01）, P〈0.05. Conclusion MiD49 is overexpressed in HCC, which accelerates cell proliferation and colony formation, and suppresses the apoptosis of HCC cells, indicating that MiD49 acts as a potential oncogene and may serve as a drug target in HCC treatment.