【作者】 李继伟；

【导师】 闫道广；

【作者基本信息】 暨南大学 ， 遗传学， 2016， 博士

【摘要】 肿瘤细胞的特征之一是无限制的增殖,氧化固醇结合蛋白（OSBP）相关蛋白（ORPs）家族有12个成员组成,包含了一系列生物学功能,包括固醇,磷酸肌醇运输,信号转导和代谢。氧化固醇结合蛋白相关蛋白4（ORP4）是ORPs家族的一个成员,已经有研究表明ORP4敲低的小鼠会出现精子畸形,也有研究表明ORP4能够促进细胞的生存和增殖,但是这种对细胞增殖促进的详细机制解释是不清楚的。ORP4有三个剪切体分别为:ORP4L,ORP4M和ORP4S。在我们的研究中,我们报道了在人类宫颈癌细胞C33A,HeLa,CaSki中,用小干扰RNA（siRNA）特异性沉默ORP4L能够抑制细胞的增殖,相反,ORP4L过表达能够明显促进细胞的增殖。我们通过酵母双杂交发现了ORP4L能够与1,4,5-三磷酸肌醇受体1（IP3R1）相互作用,IP3R1作为细胞内Ca²⁺通道是细胞内主要的Ca²⁺维持蛋白,并且我们通过不同手段证明了ORP4L和IP3R1的相互作用。Ca²⁺是细胞内一个重要的第二信使包含一系列生理功能,包括细胞生长和增殖。我们发现ORP4L能够维持细胞内Ca²⁺平衡以及调节活化T细胞核因子（NFAT）活性,我们已知NFAT的活化是Ca²⁺依赖的,ORP4L能够通过调节NFAT活性,进而促进一系列NFAT调控的与增殖相关靶基因的表达。另外,ORP4L能够维持IP3R1蛋白和mRNA水平的表达,并且这种调节是通过调控细胞内Ca²⁺依赖的NFAT3的表达进行的,这也提供了一个ORP4L维持细胞内Ca²⁺平衡的分子机制的解释,ORP4L通过维持IP3R1稳定表达维持细胞内Ca²⁺平衡。此外,在动物水平,我们证明ORP4L沉默能够显著抑制C33A细胞异种移植瘤裸鼠模型肿瘤的生长。总的来说,我们发现ORP4L能够通过与IP3R1的蛋白相互作用维持细胞内Ca²⁺平衡以及IP3R1的稳定表达来促进细胞增殖,这些结果揭示出ORP4L可以作为癌症治疗的一个潜在的新的靶点。更多还原

【Abstract】 One of the characteristics of tumor cells is infinite proliferation. Oxysterol binding protein（OSBP）-related proteins（ORPs） comprise a mammalian gene family with 12 members implicated in a spectrum of different cellular processes including sterol and phosphoinositide sensors coordinating transport, signaling and metabolism. ORP4 is a member of the ORPs family. Studies have reported that ORP4 knockout mice exhibit a teratozoospermia, also ORP4 is essential for cell proliferation and survival, but the underlying mechanism is unclear.ORP4 is expressed as three variants, ORP4 L, ORP4 M and ORP4 S. Here, we reported that silencing of ORP4 L with specific small interfering RNA（siRNA） inhibited the proliferation of human cervical cancer cell lines C33 A, HeLa and CaSki, the reverse effect being observed in ORP4 L overexpressing cells. For molecular insight, we found that ORP4 L maintained intracellular Ca²⁺ homeostasis. We found that ORP4 L could interact with inositol-1,4,5-trisphosphate receptor 1（IP3R1） with yeast two-hybrid analysis. IP3R1 is predominant in maintenance of cellular Ca²⁺ signals as Ca²⁺ channel. We also confirmed that ORP4 L interacted with IP3R1 with different methods.Ca²⁺ is a versatile second messenger with a wide range of central physiological roles in processes such as cell growth or proliferation. We found that ORP4 L could regulate nuclear factor of activated T cells（NFAT） activity, NFAT are usually activated by increased intracellular Ca²⁺ levels. ORP4 L promoted expression of a gene cluster which supported cell proliferation through regulating NFAT activity. Of note, ORP4 L sustained IP3R1 expression at both mRNA and protein levels via Ca²⁺-dependent NFAT3 activation, This offered a mechanic explanation for the role of ORP4 L in cellular Ca²⁺ homeostasis, ORP4 L maintained cellular Ca²⁺ homeostasis through keeping IP3R1 stable expression. Furthermore, ORP4 L knockdown markedly inhibited tumor growth in a C33 A cell xenograft mouse model.To conclude, our results revealed that ORP4 L promoted cell proliferation through maintaining intracellular Ca²⁺ homeostasis and IP3R1 stable expression by ORP4 L and IP3R1 protein interaction. This indicate that ORP4 L may be a putative new candidate target for the development of cancer treatment.更多还原