Yu Qin, Liu Zhao-Yu, Chen Qiong, Lin Ju-Sheng
Department of Gastroenterology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
J Huazhong Univ Sci Technolog Med Sci. 2016 Aug;36(4):494-500. doi: 10.1007/s11596-016-1614-7. Epub 2016 Jul 28.
Hepatocellular carcinoma (HCC) is a major cause of cancer-related mortality in part due to its high resistance to chemotherapeutic drugs. The anti-apoptotic Mcl-1 expression has been reported as a resistance factor in various types of tumors. Here, we investigated the expression of Mcl-1 in hepatoma cells and HCC tissues and its relationship with p53, and analyzed the possibility of the gene as a molecular target for HCC therapy. HCC specimens of 30 patients were examined by immunohistochemistry for Mcl-1 and p53 expression. Mcl-1 expression in hepatoma cell lines was measured by RT-PCR and Western blotting. The suppression of Mcl-1 by RNA interference or specific phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002, was evaluated as monotherapy, and it was combined with mitomycin C (MMC) in treating hepatoma cell line HepG2. Cell viability and apoptosis were assessed by MTT and FACS analysis. Finally, changes of Mcl-1 or p53 expression in various hepatoma cell lines were examined after transfection with Mcl-1 siRNA, the Mcl-1 expression plasmid, or the wide-type p53 expression plasmid, respectively. Mcl-1 protein was remarkably enhanced in HCC tissues as compared with adjacent non-tumor liver tissues. In addition, Mcl-1 was prominently expressed in HepG2 and Hep3B cells, weakly in SMMC7721 cells, and not in L02 cells. P53 protein was also overexpressed in HCC tissues and there was a significant correlation between the expression of p53 and Mcl-1. Silencing Mcl-1 by RNAi or LY294002 downregulated Mcl-1 expression and led to decreased cell viability and increased apoptosis. Combination of MMC and Mcl-1 RNAi or LY294002 exhibited a significant chemosensitizing effect. The expression of p53 was not influenced by Mcl-1 siRNA in HepG2 cells or transfection with the Mcl-1 expression plasmid in L02 cells. Furthermore, the expression of Mcl-1 in Hep3B cells was also not significantly changed after transfection with the wild-type p53 expression plasmid. It is concluded that Mcl-1 is overexpressed in HCC tissues. The mechanisms by which silencing Mcl-1 sensitizes hepatoma cells towards chemotherapy may be not attributed to the upregulated expression of p53 but the dysfunction of p53 through Mcl-1/p53 interaction. Mcl-1 may be a potential target of gene therapy for HCC.
肝细胞癌（HCC）是癌症相关死亡的主要原因之一，部分原因是其对化疗药物具有高度抗性。抗凋亡蛋白Mcl-1的表达已被报道为多种肿瘤中的一个抗性因素。在此，我们研究了Mcl-1在肝癌细胞和HCC组织中的表达及其与p53的关系，并分析了该基因作为HCC治疗分子靶点的可能性。通过免疫组织化学检测30例患者的HCC标本中Mcl-1和p53的表达。采用RT-PCR和蛋白质印迹法检测肝癌细胞系中Mcl-1的表达。评估RNA干扰或特异性磷脂酰肌醇-3激酶（PI3K）抑制剂LY294002对Mcl-1的抑制作用作为单一疗法，并将其与丝裂霉素C（MMC）联合用于治疗肝癌细胞系HepG2。通过MTT法和流式细胞术分析评估细胞活力和凋亡情况。最后，分别用Mcl-1 siRNA、Mcl-1表达质粒或野生型p53表达质粒转染后，检测各种肝癌细胞系中Mcl-1或p53表达的变化。与相邻的非肿瘤肝组织相比，HCC组织中Mcl-1蛋白显著增强。此外，Mcl-1在HepG2和Hep3B细胞中高表达，在SMMC7721细胞中弱表达，在L02细胞中不表达。p53蛋白在HCC组织中也过表达，且p53与Mcl-1的表达之间存在显著相关性。通过RNAi或LY294002沉默Mcl-1可下调Mcl-1表达，导致细胞活力降低和凋亡增加。MMC与Mcl-1 RNAi或LY294002联合使用表现出显著的化学增敏作用。HepG2细胞中p53的表达不受Mcl-1 siRNA的影响，L02细胞中Mcl-1表达质粒的转染也不影响p53的表达。此外，野生型p53表达质粒转染后，Hep3B细胞中Mcl-1的表达也没有明显变化。结论是Mcl-1在HCC组织中过表达。沉默Mcl-1使肝癌细胞对化疗敏感的机制可能不是由于p53表达上调，而是通过Mcl-1/p53相互作用导致p53功能障碍。Mcl-1可能是HCC基因治疗的一个潜在靶点。
