长链非编码RNA SFTA1P-IFIT3-CSN5轴在宫颈癌发生中的作用及机制研究

The role of long non-coding RNA (lncRNA) in tumorigenesis emerges to be recognized. Our recent study on RNA-sequencing of cervical suquamous cell carcinomas has identified a novel lncRNA, SFTA1P (surfactant associated 1, pseudogene), which is upregulated in cervical carcinoma relevant to normal cervical tissue adjacent to the cancer. The biological functions of SFTA1P in cancers remain totally unknown. Overexpression of SFTA1P has been found to be associated with poor prognosis in cervical carcinomas. We have demonstrated that SFTA1P playes a potential oncogenic role in cervical cancer cells. SFTA1P knock-down inhibits the energy metabolism of mitochondrium, and downregulates the protein levels of cyclin D1 (CCND1) and mitofusin 2 (MFN2). These proteins have been found to be degraded by ubiquitination. RNA pull-down and RIP (RNA binding protein immunoprecipitation assay) have confirmed that IFIT3 (interferon induced protein with tetratricopeptide repeats 3) is a binding protein of SFTA1P. It has been well documented that the interaction between IFIT3 and CSN5 (COP9 signalosome 5) can block the dennedlylation of Cullins, disassemble CRL (Cullins-RING ligase) complex, and eventually suppress the process of protein ubiquitination. Hence, we hypothesize that the SFTA1P-IFIT3-CSN5 signalling axis playes a potential role in the regulation of CCND1 and MFN2 ubiquitination via dismantling the CRL complex. In this study, we aim to further consolidate the oncogenic role of SFTA1P in cervical cancers and to validate the hypothesis. We will apply a combination of various classical cellular and molecular methods, such as RNAi, gene transfection, RNA FISH, RIP, immunoprecipitation, and GST-pull down, etc. Our study will provide novel clues to the clarification on the molecular genesis of cervical carcinoma and mechanism underlying lncRNA function. SFTA1P will be a potential novel therapeutic target for cervical carcinoma.

本课题组前期从大规模宫颈癌RNA-Seq研究中发现一个新的lncRNA-SFTA1P，它在宫颈癌组织中表达高、预后差。细胞生物学研究发现它是个促癌基因。SFTA1P敲低抑制宫颈癌细胞线粒体能量代谢，下调CCND1、MFN2等蛋白表达，这些蛋白质通过泛素化途径降解。我们还发现SFTA1P的结合蛋白-IFIT3，它与CSN5结合可阻断Cullins去Nedd化、使CRL复合物解体、抑制蛋白质泛素化降解。由此，我们提出SFTA1P-IFIT3-CSN5信号轴抑制CRL复合物对CCND1、MFN2等蛋白质的降解、发挥促癌作用的科学假说。本项目将通过RNAi、基因转染、蛋白质相互作用技术等分子生物学手段来深入研究SFTA1P功能、验证相关假说，以揭示宫颈癌发生的分子新机制，为宫颈癌的诊治提供新靶点。

SFTA1P是一个我们从RNA-Seq发现的子宫颈癌组织高表达的长链非编码RNA，其功能和作用机制未明。我们发现，SFTA1P在宫颈癌中发挥了癌基因的作用，基因表达高、预后差。基因敲低则抑制子宫颈癌细胞 Caski及C-4 I 的细胞增殖、生长以及侵袭迁移与转移能力，诱导细胞G1→S期阻滞。RNA FISH发现SFTA1P主要定位于宫颈癌细胞的细胞质。SFTA1P下拉、SDSS-PAGE、白质质谱分析发现PTBP1为SFTA1P的作用靶点，并得到SFTA1P下拉-免疫印迹和RNA免疫共沉淀（RIP）证实。截断蛋白质粒转染、下拉实验表明PTBP1 RRM3或RRM4为其与SFTA1P结合结构域。PTBP1敲除明显抑制子宫颈癌细胞生长、侵袭迁移能力，提示它具有与SFTA1P相同的促癌功能。为寻找两者的下游作用共同通路，我们将分别敲除SFTA1P和PTBP1的RNA-seq结果进行交集，发现68个受两者共同调控的基因。在分别敲低SFTA1P和PTBP1的细胞中，我们发现TPM4 mRNA和蛋白表达升高，证实SFTA1P和PTBP1调控TPM4表达。敲低TPM4促进细胞生长、克隆形成能力、G1→S期细胞、侵袭迁移能力，提示TPM4在宫颈癌细胞中发挥抑癌作用。敲低TPM4 可拯救SFTA1P敲除或PTBP1敲除引起的迁移和细胞增殖抑制效应。生物信息学分析发现，PTBP1可与TPM4 3’-UTR区结合，RNA下拉和RIP实验证实PTBP1和TPM4 mRNA之间的相互作用。在放线菌素处理的子宫颈癌细胞中，稳定敲低SFTA1P或敲低PTBP1均增加TPM4 mRNA稳定性。综上所述，我们明确SFTA1P在宫颈癌中的作用机制：SFTA1P与PTBP1蛋白结合形成复合物，该复合物与肿瘤抑制基因TPM4 3’-UTR的结合，诱使TPM4 mRNA降解，从而促进细胞增殖、侵袭迁移与转移能力而参与子宫颈癌的发生和发展。