miR-137靶向AURKA在肝癌多药耐药中的作用机制研究

The observation that the expected incidence and mortality for hepatocellular carcinoma (HCC) runs closely parallel highlights the paucity of effective treatment modalities for this fatal disease. HCC is a tumor with rapid growth and early vascular invasion. Most HCC patients present with advanced symptomatic tumors with underlying cirrhotic changes that are not amenable to surgical resection or liver transplantation. Even after surgical resection, the recurrence rate is very high. Transarterial chemoembolization (TACE) and systemic therapy with doxorubicin alone or a combination of cisplatin, IFN, doxorubicin, and 5-fluorouracil (5-FU) are being used for advanced disease, with moderate improvement in overall survival duration varying between 6.8 and 8.6 months (5–7). These dismal figures point to chemoresistance as an inherent trait of HCC. Identification of key molecules contributing to this chemoresistance and creating strategies to counteract this process will help augment the efficiency of current modalities of treatment and increase patient survival..MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression at the posttranscriptional level, are known to be involved in diverse biological processes, including embryonic development, metabolism, viral infections, and human malignancies. They suppress target gene expression by interaction with complementary sequences in the 3UTRs of target mRNA. Certain miRNAs undergo aberrant regulation during carcinogenesis and cause therapeutic resistance and metastasis by regulating multiple target genes. In previous studies, we demonstrated that miR-137 was epigenetic silenced in Hepatocellular Carcinoma (HCC). We have detected that miR-137 is dysregulated in HepG2/R cells compared with parental HepG2 cells treat with 5-FU. Using the algorithms for target gene prediction, including PicTar, TargetScan, and miRanda, AURKA were identified as the potential targets of miR-137. Luciferase assay also showed AURKA mRNA was the target of miR-137. AURKA is a serine/threonine kinase that regulates mitotic processes in mammalian cells, including centrosome maturation, spindle assembly, and chromosome segregation. The present study showed inhibiting AURKA leads to resensitizes HepG2/R cells to doxorubicin. All these results suggest that miR-137 could be involved in the regulation of MDR by targeting AURKA. .In general, this proposal will try to elucidates a regulatory mechanism that the machinery that the role of miR-137 and AURKA in HCC MDR. It can to provide new ideas and theoretical basis for HCC treatment.

前期工作发现miR-137的表达在肝癌耐药细胞株HepG2/R中显著降低，在肝癌组织中的表达也明显低于其癌旁组织。通过软件预测和荧光素酶报告实验证实miR-137靶向AURKA；HepG2/R细胞中AURKA表达较HepG2中明显降低；使用AURKA的特异性抑制剂后，流式细胞术检测发现HepG2/R细胞对阿霉素的泵出减少，说明抑制AURKA可能逆转肝癌多药耐药。既往研究报道miR-137和AURKA参与了多种肿瘤的发生发展以及耐药。基于前期工作和既往研究，我们推测肝癌细胞易发生原发性耐药与miR-137表达降低密切相关，其通过靶向AURKA后发挥重大作用。.本项目拟在前期工作基础上，通过体内外实验证实miR-137靶向AURKA对HCC 细胞耐药的影响，并阐明AURKA通过何种通路去影响HCC发生MDR。深入研究miR-137和AURKA在HCC中的作用或将为HCC的综合治疗带来新的提示。

肝癌是我国常见的恶性肿瘤之一，全世界每年约有60万例患者死于HCC，其中50％以上发生在我国，并呈逐年上升趋势。肝癌具有多血管、恶性程度高、生长速度快、转移范围广和复发率高等特点，临床治疗效果不理想[2, 3]。治疗肝癌以手术切除为首选方法，但术后复发率较高，而且大多数病人就诊时已丧失手术机会，只能进行辅助性综合治疗。药物治疗是中晚期消化道肿瘤综合治疗的重要手段，但在肝癌中疗效明显不佳，究其原因，可能与肝癌原发耐药现象严重有关。因此，研究逆转肝癌耐药的靶点及其作用机制，提高肝癌化疗效果成为亟待解决的问题之一。.本项目检测32例肝癌手术切除标本，5株肝癌细胞系（SMMC-7721, HepG2, Hep3B, QGY7701 和 MHCC97-L）。提取RNA，采用实时定量PCR检测miR-137的表达情况；将慢病毒包装的miR-137高表达载体感染肝癌细胞系HepG2和SMMC-7721，获得稳定细胞系后，使用CCK-8法增殖等方法检测药物敏感性。通过荧光素酶实验研究确定miR-137靶基因，并通过流式和免疫印迹等方法研究其在肝癌中的作用机制。. 结果表明，相比癌旁标本，miR-137在肝癌细胞系和肝癌标本中表达明显降低。通过比较已诱导建立的肝癌多药耐药细胞株HepG2/R及其亲本细胞系HepG2中miR-137的表达，发现在耐药细胞HepG2/R中miR-137的表达明显低于亲本细胞HepG2。进一步研究发现，miR-137可靶向AURKA。AURKA在肝癌中呈高表达，免疫组化结果显示，AURKA在所有106例样本中过表达的阳性率为45.3% (48/106)，其表达与肿瘤侵犯血管、肿瘤直径、肿瘤形态和包膜有无等方面相关。从生存曲线上看，患者的无瘤生存率AURKA过表达组较阴性组低（Figure 6）。COX回归方程进行多因素分析表明AURKA是影响患者无瘤生存时间的独立危险因素。通过流式分析AURKA可影响药物泵出，且免疫印迹发现降低AURKA表达后p-ATM 和p- Chk2表达降低，说明AURKA可通过DNA损伤通路影响肝癌细胞改变。.综上，miR-137在肝癌中表达明显降低，并且其靶向的AURKA表达增高，使得肝癌易发生耐药。本研究为肝癌综合治疗药物的开发提供了新的思路，奠定了基础。