miR-29c/FBXO31信号通路在食管癌耐药机制中的调控作用及靶向治疗研究

Esophageal cancer is the 6th most common cancer in China, and the predominant histological subtype is esophageal squamous cell carcinoma (ESCC). Chemotherapy is an important component of multimodal treatment, but acquisition of resistance to standard chemotherapeutic agents is a major challenge in the management of this highly lethal disease. Emerging evidence supports that microRNAs (miRNAs) regulate chemosensitivity, and miRNA-based therapy has received increasing attention as a novel and promising approach in cancer treatment. Our previous studies confirmed that miRNAs have important functions in different types of human cancers, but identification and experimental validation of the chemoresistance-related miRNAs in ESCC are urgently needed. We have established ESCC cell line models of acquired chemoresistance to 5-FU (FR sublines). MicroRNA profiling using Taqman miRNA array and subsequent RT-PCR confirmation showed that miR-29c is the most down-regulated miRNA in FR sublines. Our functional studies showed that overexpression of miR-29c could revert acquired chemoresistance of FR cells. We also found reduced expression level of miR-29c in ESCC tumor tissues. The functional role and mechanisms of miR-29c in ESCC progression have not been reported. We found that FBXO31, a novel F Box protein with prognostic signficance in ESCC, was amongst the upregulated mRNAs identified in the FR cells using cDNA microarray and was predicted by computational algorithms to be a target of miR-29c. The functional role of FBXO31 in cancer chemoresistance and its regulatory mechanisms have not been reported. Our preliminary data showed that FBXO31 increased chemoresistance of ESCC cells in vitro and in vivo, and ectopic expression of miR-29c significantly reduced FBXO31 expression. More importantly, FBXO31 mediated the functions of miR-29c in chemoresistance of ESCC cells. We hypothesize that miR-29c can suppress chemoresistance of ESCC by directly targeting FBXO31, and that systemic delivery of miR-29c may be a useful anticancer therapy. We have designed experiments with the following aims: 1. To study the biological functions of FBXO31 in esophageal cancer chemoresistance in vitro and in vivo. 2. To establish that miR-29c directly targets FBXO31, and investigate the role of miR-29c in suppressing chemoresistance in ESCC. 3. Clinicopathological validation of the significance of miR-29c and FBXO31 dysregulation in ESCC. 4. Preclinical evaluation of the efficacy of systemically administered miR-29c in suppressing chemoresistance of ESCC. The outcome of this project will greatly enhance our understanding of the functions of miR-29c and FBXO31 in esophageal cancer, and facilitate their application as prognostic biomarkers in predicting treatment response of the patients. More importantly, systemic delivery of miR-29c as proposed in our study will provide useful preclinical data that may translate into novel systemic therapy for this locally relevant disease.

肿瘤细胞对化疗药物产生耐药性，导致药物无法杀死肿瘤细胞并引起肿瘤复发甚至转移，是目前肿瘤临床治疗失败的主要原因之一。近年研究显示，微小RNA（miRNA）在肿瘤细胞中的异常表达在肿瘤的发生和发展中起着重要作用，但是miRNA在肿瘤耐药性尤其是食管癌耐药性中的功能、机理及应用前景尚不明晰。我们最新基因芯片结果表明，耐药细胞株中表达显著降低的miR-29c可以直接负调控在耐药细胞株中出现高表达的FBXO31蛋白，很可能是食管癌耐药性发生的重要分子机制。本项目中，我们将深入探讨miR-29c/FBXO31信号通路在肿瘤耐药性中的生物功能，分子生物学确认miR-29c对FBXO31的直接靶向作用，证实其表达水平在食管癌中的临床病理学意义。同时，临床前实验评估系统给药的miR-29c对肿瘤耐药性的抑制效率。本项目将为肿瘤耐药性发生机制研究提供新线索，为临床诊断、药效评估及个性化治疗用药提供新的靶标。

癌症中失调的microRNA（miRNA）表达可能参与调控化疗耐药。本研究旨在鉴定与食管鳞状细胞癌（ESCC）中5-氟尿嘧啶（5-FU）化疗耐药相关的miRNA。我们研究了miR-29c作为ESCC新型诊断、预后和治疗预测标志物的潜力，探讨了其在克服5-FU化疗耐药中的作用机制和治疗意义。我们首先使用Taqman miRNA高密度芯片获得5-FU获得性耐药的ESCC细胞模型的miRNA表达图谱，以鉴定与5-FU耐药相关的新miRNA，使用定量实时PCR来确定患者的组织和血清样品中的miR-29c表达。通过生物信息学和一系列功能实验以及荧光素酶报告基因测定，我们发现F-box蛋白31（FBXO31）作为miR-29c的直接靶标，并鉴定控制miR-29c表达的潜在转录因子。 并且，我们在肿瘤异种移植模型中评估了基于miR-29c寡核苷酸系统给药的干预策略在克服5-FU耐药中的治疗潜力。我们的结果显示：在STAT5A的调节控制下，miR-29c在大多数ESCC患者的肿瘤和血清样品中下调，并且表达水平与总体存活相关。我们的体外和体内功能研究表明miR-29c可通过直接与FBXO31的3'UTR相互作用，导致FBXO31表达的抑制和p38MAPK的下游激活，从而增强5-FU敏感性，而系统给药的miR-29c显著改善小鼠体内移植瘤的5-FU耐药。综上所述，miR-29c通过与FBXO31相互作用调节化疗耐药性，可能作为ESCC中一种有前途的非侵袭性生物标志物和治疗靶点。