Li Hualei, Gai Ling, Wu Zhimei, Li Feng
Department of Urology, Affiliated Hospital of Nantong University, Nantong, 226001, China.
Department of Chemotherapy, Affiliated Hospital of Nantong University, Nantong, 226001, Jiangsu, China.
Mol Biol Rep. 2022 Apr;49(4):3015-3024. doi: 10.1007/s11033-022-07128-3. Epub 2022 Jan 10.
Chemotherapy is an important treatment strategy for advanced hepatocellular carcinoma (HCC). Sorafenib is a first-line systemic drug that has been commonly used clinically for patients with advanced HCC. However, the high resistance rate of sorafenib in HCC patients often hinders its long-term efficacy. Therefore, it is vital to reveal the molecular mechanisms of sorafenib resistance in patients with HCC.
In current study, we screened out fourteen genes that over-expressed in HCC specimens through integrative bioinformatics analysis. Here, maternal embryonic leucine zipper kinase (MELK) was highlighted as one of the most probable molecules. The Database for Annotation Visualization and Integrated Discovery (DAVID) program was utilized for functional pathway enrichment analysis. Real-time PCR (RT-PCR) and western blot were used to examine the expression levels of MELK. CCK-8, transwell, colony formation assays and flow cytometry were used to detect cell proliferation, the cell cycle. The dual luciferase assays were performed to study the targeting relationship between MELK and miR-142-5p.
MELK expressions were correlated significantly with cell proliferation by regulating cell cycle and DNA replication. High MELK expression in patients with HCC indicated a poor prognosis both the overall and diseases free survival rates. MELK knockdown suppresses cell proliferation, migration and invasion in vitro. miR-142-5p regulates MELK expression through binding to the complementary sequence in the 3'-UTR regions. MELK knockdown enhances sensitivity of sorafenib in HCC sorafenib-resistant (HCC/SR) cells.
MELK may serve as a potential prognostic marker in HCC and MELK knockdown enhanced sensitivity of HepG2/SR cells to sorafenib treatment. Our findings suggest that MELK/miR-142-5p axis could be a potentially therapeutic target for reversing the sorafenib resistance in HCC treatment.
化疗是晚期肝细胞癌（HCC）的重要治疗策略。索拉非尼是一线全身性药物，临床上常用于晚期HCC患者。然而，HCC患者中索拉非尼的高耐药率常常阻碍其长期疗效。因此，揭示HCC患者索拉非尼耐药的分子机制至关重要。
在本研究中，我们通过综合生物信息学分析筛选出在HCC标本中过表达的14个基因。在此，母源胚胎亮氨酸拉链激酶（MELK）被视为最有可能的分子之一。利用注释、可视化与综合发现数据库（DAVID）程序进行功能通路富集分析。采用实时定量聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测MELK的表达水平。使用细胞计数试剂盒-8（CCK-8）、Transwell小室实验、集落形成实验和流式细胞术检测细胞增殖、细胞周期。进行双荧光素酶实验以研究MELK与miR-142-5p之间的靶向关系。
MELK的表达通过调节细胞周期和DNA复制与细胞增殖显著相关。HCC患者中MELK高表达表明总体生存率和无病生存率均较差。敲低MELK可在体外抑制细胞增殖、迁移和侵袭。miR-142-5p通过与3'-非翻译区（UTR）区域的互补序列结合来调节MELK的表达。敲低MELK可增强HCC索拉非尼耐药（HCC/SR）细胞对索拉非尼的敏感性。
MELK可能作为HCC的潜在预后标志物，敲低MELK可增强HepG2/SR细胞对索拉非尼治疗的敏感性。我们的研究结果表明，MELK/miR-142-5p轴可能是逆转HCC治疗中索拉非尼耐药的潜在治疗靶点。
