RNA干扰结合慢病毒载体过表达研究HSP27基因在鼻咽癌转移中的作用及分子机制

Nasopharyngeal Carcinoma (NPC) is one of the ten most common malignant tumours in China. Metastasis might occur even in the early stage of NPC and is the chief reason for failure in NPC treatment. Whether accompanied with lymph nodes metastasis is one of the most important indicators determining the prognosis of NPC. Up to now, the molecular mechanism resulting early metastasis of NPC remain unclear. Previous studies indicated that there are close relationship between HSP27 and proliferation, invasion and metastasis of cancer cells. In our proteomics study, HSP27, Ezrin showed significantly different expression between low and high metastatic NPC cell line. MPPs (Matrix Metalloproteinases) play pivotal role in cancer metastasis.Increasing evidents show the activity and expression level of MMPs are modulated by NF-KB and MAPK signal pathway. "Whether does the HSP27 regulate MMPs directly?" "Whether does influence the level and activity of MMPs by NF-KB or MAPK signal pathway?" "What kind of internal relationship among HSP27, MMPs, NF-KB , MAPK and Ezrin?" All of these questions are waiting for being answered.In the present study, we will inhibit HSP27 in S18 (the high metastatic subclone separated from CNE2) by RNA interference technique,and will overexpress HSP27 in NP46O (the normal cell line derive from human nasopharyngeal epithelium) or CEN1 (the low migration NPC cell line) by lentivector, then we will measure the metastatic ability of the treated cells in vitro (by small chamber test) and in vivo (by injecting cells subcutaneously into the footpad of mice). The activity and expression level of MMPs family, related signal proteins (NF-KB, MAPK) and the metastasis-associated protein, Ezrin, will also be determined. By combining all of these approaches, we will try to illuminate the molecular mechanism of HSP27 in metastasis of NPC, and to provide a basis for designing new strategy which to inhibit NPC from malignant progress and to prevent cancer cells from metastasis.

鼻咽癌是我国发病率较高的十大恶性肿瘤之一，其恶性程度较高，早期即可出现淋巴结转移。迄今，对鼻咽癌早期即发生淋巴结转移的分子机制尚不清楚。初步研究显示，HSP27与多种癌的生长、侵袭和迁移密切相关；我们前期的蛋白组学研究中发现，HSP27、Ezrin等在高、低迁移鼻咽癌细胞间明显差异表达。本研究拟通过RNA干扰沉默高迁移鼻咽癌细胞S18中的HSP27，利用慢病毒载体在正常鼻咽部上皮细胞NP460或低迁移鼻咽癌细胞CNE1中过表达HSP27，于体外（small chamber实验）和体内（小鼠足掌皮下注射）检测肿瘤细胞迁移能力的改变，检测跟肿瘤侵袭和迁移密切相关的MMP家族、相关信号蛋白（NF-KB、MAPK）和肿瘤转移相关蛋白Ezrin的表达变化，以进一步探索HSP27在鼻咽癌转移中的确切角色及分子机制，寻找相关信号通路的交联位点，探求防治鼻咽癌转移及恶性演进的新策略。

鼻咽癌是我国发病率较高的十大恶性肿瘤之一，其恶性程度较高，早期即可出现淋巴结转移。迄今，对鼻咽癌早期即发生淋巴结转移的分子机制尚不清楚。初步研究显示，Hsp27与多种癌的生长、侵袭和迁移密切相关；我们前期的蛋白组学研究中发现，Hsp27、Ezrin、VCP、Keratin18在高、低迁移鼻咽癌细胞间明显差异表达。本研究利用慢病毒载体在正常鼻咽部上皮细胞NP460中过表达Hsp27，通过RNA干扰沉默高迁移鼻咽癌细胞S18中的Hsp27，于体外（Transwell 实验）和体内（尾静脉注射）检测肿瘤细胞迁移能力的改变，检测Hsp27的磷酸化水平、相关信号蛋白（NF-KB、MAPK）的表达变化以及跟肿瘤侵袭和迁移密切相关的MMP家族的表达变化和活性水平，结果发现：（1）不同转移能力及不同分化程度的鼻咽癌细胞株中总Hsp27水平及磷酸化的Hsp27（p-Hsp27）水平明显不同；（2）沉默高转移鼻咽癌细胞株S-18内的Hsp27后，S-18细胞的侵袭迁移能力明显下降，S-18细胞内的NF-κB、MMP9、MMP11转录水平相应明显下调；（3）过表达Hsp27增强了正常鼻咽上皮细胞NP460的侵袭迁移能力；（4）Hsp27可能通过调控NF-ΚB信号通路影响体内鼻咽癌细胞株成瘤及生长速度，通过MAPK-ERK1/2途径调控MMP9等因子的活性而促进鼻咽癌在体内转移；（5）天麻素可能通过抑制Hsp27磷酸化水平，进而下调MMPs的酶活性，最终抑制鼻咽癌细胞的侵袭迁移能力。这些研究结果说明，Hsp27的表达水平和磷酸化水平在鼻咽癌转移中扮演重要作用，天麻素具有抑制鼻咽癌细胞转移的潜在活性，可以设计基于Hsp27为靶点的天然药物抗鼻咽癌转移的分子治疗策略。