寡糖基转移酶亚基TUSC3在结直肠癌化疗耐药中的作用及分子机制研究

In previous study, we proved that the oligosaccharyltransferase complex subunit TUSC3 gene promoted colorectal cancer proliferation and invasion. We also found that the expression level of TUSC3 gene was positively associated with chemoresistance of colorectal cancer cell lines. Furthermore overexpression of TUSC3 gene upregulated the expression of both multidrug resistant associated protein ABCC1/MRP1 and Hedgehog signaling pathway activators (Smo and Gli1), as detected by microarray and Western blot. However, the actual role of TUSC gene and its underlying molecular mechanism in chemoresistance of colorectal cancer are still unclear. Based on the preliminary experimental results and literature review, we suppose that TUSC3 gene may induce drug resistance of colorectal cancer via ABCC1/MRP1 directly or indirectly. In the indirect pathway, we think that TUSC3 may activate Hedgehog signaling pathway which then transcripts the target gene ABCC1/MRP1, and finally overexpression of ABCC1/MRP1 protein leads to chemoresistance of colorectal cancer. In order to verify the above hypothesis, a large number of clinical colorectal cancer tissue and paired normal mucosa, together with the pathological characteristics and prognostic information of patients, are collected. The expression of TUSC3, ABCC1/MRP1, and factors of Hedgehog signaling pathway (Smo, Ptch, and Gli1) are detected to clarify their clinical significance. Subsequently, we construct cell line models with lentivirus-mediated silencing of TUSC3 gene and exogenous overexpression of TUSC3 gene. All these cells are treated with 5-fluorouracil and cisplatin, respectively. Then MTT assay, plate colony formation assay and invasive assay are used to detect the reaction of TUSC3 to chemotherapy in vitro. A visulizing orthotopic animal model is also built. After the node mice are treated with 5-fluorouracil and cisplatin, the effects of TUSC3 to chemotherapy are examined by observing tumorigenesis and metastasis via whole-body visualizing orthotopic animal model of colorectal cancer. We also explore the relationship between TUSC3 expression and both ABCC1/MRP1 and Hedgehog signaling pathway factors by Western blot, co-immunoprecipitation, immunofluorescence technique in the above cell line models. From this study, we aim to elucidate the function, molecular mechanism and signaling pathway of TUSC3 in chemoresistance of colorectal cancer. Furthermore, TUSC3 gene as a potential and new target for early warning of chemoresistance and therapy of colorectal cancer, together with the scientific basis, will also be supplied.

我们前期筛选并鉴定TUSC3基因与结直肠癌进展密切相关，进一步研究发现TUSC3高表达的结肠癌细胞对化疗药物耐药，且过表达TUSC3可上调多药耐药相关蛋白ABCC1/MRP1及Hedgehog通路激动因子的表达，但其在结直肠癌耐药中的确切作用及分子机制仍不清楚。结合文献，我们推测TUSC3通过直接或间接调控ABCC1/MRP1（TUSC3/ABCC1/MRP1或TUSC3/Hedgehog/ABCC1/MRP1）诱导结直肠癌化疗耐药。本研究拟以大批量临床组织标本及预后信息、整体可视化结直肠癌动物模型为基础，通过建立TUSC3稳定过表达/RNA干扰的细胞株、细胞功能实验、免疫共沉淀、糖基化检测、质谱分析等技术，从临床、细胞水平、动物模型和分子维度四个层面，揭示TUSC3在结直肠癌化疗耐药中的作用、分子机制及相关信号通路，为结直肠癌耐药机制研究提供新思路，为结直肠癌治疗和药物筛选提供新靶标。

结直肠癌是常见的消化系统恶性肿瘤，在我国其发病率和死亡率均排在各种恶性肿瘤的第三位。转移和化疗耐药是造成结直肠癌患者死亡和预后差的主要原因。我们前期研究发现TUSC3具有促进结直肠癌细胞侵袭和转移的作用，在此基础上，本项目进一步探讨TUSC3在结直肠癌化疗耐药中的生物学功能和分子机制。我们通过实验检测结合生物信息学分析发现TUSC3在结直肠癌组织中高表达，并与患者肿瘤分期呈正相关，与患者的总生存率和无病生存率呈负相关。体外细胞功能实验证实TUSC3过表达可增强结直肠癌细胞的干性特征，诱导结直肠癌细胞对5-氟尿嘧啶和顺铂的耐药性；而干扰TUSC3表达则可抑制结直肠癌细胞的干性特征，增加结直肠癌细胞对5-氟尿嘧啶和顺铂的敏感性。TUSC3表达与Hedgehog信号通路蛋白、CD133和ABCC1的表达呈正相关性。在TUSC3干扰或TUSC3过表达的细胞中加入Hedgehog信号途径激动剂或抑制剂可逆转TUSC3表达改变对结直肠癌细胞干性和耐药性的影响以及相关标记物的表达。免疫共沉淀和免疫荧光检测分析表明，TUSC3与SMO蛋白有密切关系。我们的研究结果表明，TUSC3在结直肠癌中表达增高，并通过Hedgehog信号途径增强结直肠癌细胞的干性特征及诱导结直肠癌细胞对5-氟尿嘧啶和顺铂耐药。