Connexin32在调控EMT影响肝细胞癌奥沙利铂化疗敏感性中的作用研究

Oxaliplatin-based combined chemotherapy has shown promise as a treatment for advanced human hepatocellular carcinoma (HCC), however, pulse treatment with oxaliplatin leads to chemoresistance of the surviving tumor cells. These cell subpopulations may contribute to the failure of chemotherapeutic treatment and thereby promote tumor recurrence or metastasis. Emerging evidence suggests that epithelial-mesenchymal transition (EMT) is involved in this process, and that targeting EMT may have new therapeutic applications for the successful treatment of HCC patients. Using two previously established in vitro cell models of oxaliplatin-resistant HCC cells (HepG2/OXA and SMMC-7721/OXA), we found that along with the acquisition of EMT, a significant decrease of connexin32, the most important core protein of gap junction channels in liver tissue, was also observed when compared to that of their parental HCC cells (HepG2 and SMMC-7721). More interestingly, connexin32 knockdown with RNA interference in parental HCC cell line HepG2 facilitated EMT-like phenotypic changes and caused a decreased chemosensitivity to oxaliplatin. Based on the above research background, we hypothesized that “connexin32 through cell-cell junctions regulating network may have the potential to modulate the EMT process associated with drug resistance in hepatocellular cells”. Thus in the present study, the specific effect of connexin32 on oxaliplatin chemosensitivity in HCC cells will be investigated, and whether these effects are related to its regulation of EMT as well as the underlying molecular mechanisms will also be explored. This study will give strong evidence for a novel link among reduced connexin32 expression, EMT process and acquired oxaliplatin resistance, and provide a potential new targeted therapeutic approach for overcoming oxaliplatin chemoresistance by inhibition of EMT.

以奥沙利铂（OXA）为基础的化疗是治疗晚期肝细胞癌的重要手段之一，然而脉冲式化疗所致残留癌细胞耐药是临床抗肝癌治疗失败及复发的重要根源。研究发现，上皮间质转化（EMT）与这一过程有关，对其进行干预是提高现有化疗疗效的重要途径。我们前期研究发现，耐OXA肝癌细胞在获得EMT表型的同时，缝隙连接蛋白（Connexin，Cx）32及其介导的细胞间通讯功能较亲本细胞显著降低，而干扰Cx32蛋白表达后的亲本细胞出现明显的间质细胞形态学及分子生物学特征，对OXA化疗敏感性下降。基于以上研究背景，我们推测Cx32可能通过复杂的细胞间连接网络调控EMT的形成进而影响肝细胞癌OXA化疗敏感性，并拟在建立的不同耐OXA肝癌细胞及其亲本细胞上，考察Cx32对OXA化疗敏感性的影响，并探讨该作用是否与调控EMT有关及可能的分子机制。本研究将为阻断化疗所致肝细胞癌EMT发生并逆转耐药提供新的治疗靶点和策略。

项目背景：无法手术切除的局部晚期和转移性肝细胞癌（Hepatocellular carcinoma，HCC）是临床治疗棘手的难题，以奥沙利铂（Oxaliplatin，OXA）为基础的系统性化疗是治疗该类疾病的重要手段，然而细胞毒药物在临床使用过程中多会遭遇耐药。至今为止，这种耐药性产生的机理仍未阐明。.主要研究内容：在不同来源HCC亲本细胞HepG2和SMMC-7721，及建立的耐药细胞亚系HepG2/OXA和SMMC-7721/OXA上，观察上皮间质转化（Epithelial-mesenchymal transition，EMT）特性及缝隙连接蛋白（Connexin，Cx）32表达的改变；通过分子生物学方法改变Cx32蛋白表达，观察OXA化疗敏感性及EMT表型的改变，并在此过程中探讨Cx32是否通过调控EMT而影响OXA化疗敏感性；最后对Cx32调控EMT的机制进行探讨。.重要结果：①较亲本细胞相比，耐药细胞HepG2/OXA和SMMC-7721/OXA获得了典型EMT表型，与此同时，Cx32表达及其组成的缝隙连接（Gap junction，GJ）通讯功能显著下调，在HCC亲本细胞上干扰Cx32蛋白表达能够模拟HCC/OXA耐药细胞出现EMT表型，相反，在HCC耐药细胞上过表达Cx32蛋白后，细胞出现MET特征并能部分逆转OXA耐药。②组织学证实包括Cx32在内的多种Cxs蛋白在HCC组织中存在表达下调及定位“内化”，其中Cx32表达与HCC组织学分级及淋巴结转移呈负相关。③Cx32调控HCC细胞EMT的机制与Wnt/β-catenin/Snail信号通路活性的改变有关，并在体内外实验中得到证实。.关键数据及其科学意义：HCC中存在包括Cx32在内的多种Cxs蛋白表达和定位的异常，两者共同致 GJ通讯功能异常进而限制了OXA细胞毒性作用的发挥，这可能是HCC低化疗反应性的的关键机制之一；其中Cx32表达的下调参与了HCC对OXA的获得性耐药，并且与其诱导EMT的形成有关；HCC中下调的Cx32通过增强Wnt/β-catenin/Snail通路活性诱导EMT形成并赋予HCC细胞高侵袭和转移特性。未来以Cx32和该信号通路相关分子为靶点设计抗肿瘤药物，可能为有效防治HCC侵袭转移并逆转OXA耐药提供新思路和新策略。