DPH1基因新可变剪接体DPH1-IR在鼻咽癌中致癌功能及机制

Ovarian cancer gene 1(DPH1) functions as a tumor suppressor gene in ovarian cancer. Our previous studies successfully found and cloned a new alternative splicing transcript(named DPH1-IR) of DPH1 gene in C666-1, a nasopharyngeal carcinoma (NPC) cell strain. One single intron between Exon 10 and Exon 11 of DPH1 pre-mRNA was retained in transcription and produced a novel transcript. But its functions and mechanisms in NPC are unknown.We detected that expression of DPH1-IR in mRNA level was higher in NPC cell lines and tissue speciems than in normal cantrols. On basis of these results，we hypothesized that DPH1-IR probably demonstrated functions of potential oncogene. To validate the hypothesis, we constructed plasmids of DPH1-IR and DPH1-WT and transfected them into HONE1, a nasopharyngeal cancer cell with low DPH1-IR-expressing level,which was proved by our preliminary foundings, respectively. Surprisingly, we found that DPH1-IR could express protein and increase the clone numbers in clone formation assay whereas DPH1-WT could dramatically decrease the clone numbers.Fist,we will develop a specific DPH1-IR antibody,and confirm the clinical significance of the new alternative splicing transcript by using the methods of polymerase chain reaction (PCR)and immunohistochemical staining.Next, we will overexpress and knockdown DPH1-IR in NPC cell lines, and study the abilities of invasive and the clone formation in vitro, and also study abilities of both tumorgenesis and tumor metastasis in nude mice mode. Finally,The oncogenic mechanism of DPH1-IR in NPC will also be explored. The target is to demenstrate the function and significance of the new alternative splicing transcript of DPH1 gene in the process of formation and development of NPC,and preliminarily explore its oncogenic mechanisms.

DPH1是卵巢癌抑癌基因。我们前期在人鼻咽癌细胞株C666-1中发现并克隆了DPH1基因一个新的可变剪接体（DPH1-IR），该可变剪接体在10号及11号外显子之间保留了一个内含子，但其功能及机制不清。我们发现：DPH1-IR mRNA在人鼻咽癌细胞、组织中表达上调。据此,我们提出DPH1-IR可能具有潜在致癌功能的假设。为证实该假设，我们在DPH1-IR低表达的鼻咽癌细胞HONE1中转染DPH1野生型及DPH1-IR基因，发现：DPH1-IR能表达蛋白，与野生型相反，DPH1-IR能增强细胞克隆及侵袭能力。为此，我们拟研制DPH1-IR特异性抗体，并通过PCR、免疫组化等方法进一步确认该剪接体表达的临床意义，继而探讨它对鼻咽癌细胞侵袭能力、裸鼠成瘤及转移能力等影响。在此基础上，进一步研究该剪接体的致癌机制。旨在明确DPH1新剪接体在鼻咽癌发生、发展中的作用及意义，并初步阐明其分子机制。

DPH1是在卵巢癌等实体瘤中表现出抑癌基因功能。作为一种“抑癌基因”，它在鼻咽癌中有无可变剪接现象？如果发生，新可变剪接体的功能及意义又是如何？这些均未见报道。.(一)首先发现DPH1具有多种可变剪接体及其理论意义.我们首先发现，DPH1存在多种可变剪接体，这些可变剪接体除了存在保留一个内含子（one-intron retained，DPH1-IR）外，还存在保留两个内含子（two-intron retained, DPH1-DIR）的现象，甚至存在基因缺失突变。具体可变剪接及缺失突变位点的示意图，详见附件图1.DPH1 在鼻咽癌中存在多种剪接模式。.在揭示了DPH1存在多种形式可变剪接体基础上，我们又成功建立了DPH1基因野生型（Wild type, DPH1-WT）、保留一个内含子、保留两个内含子这三种表达质粒，这为研究野生型基因、多种可变剪接体在细胞、分子、动物水平上的功能，进而通过siRNA沉默技术研究DPH1所保留的内含子基因序列片段敲低后基因功能变化，打下了切实的基础。最终必将为阐明DPH1及其突变体在鼻咽癌中的功能及其作用机制，揭示DPH1在鼻咽癌发生、发展中的作用规律，为鼻咽癌靶向DPH1的基因治疗提供理论依据。.(二)发现DPH1-IR在鼻咽癌中的表达、功能及其意义.对于新可变剪接体DPH1-IR基因功能，我们进行详细研究，结果如下：.（1）DPH1-IR再次验证。.通过RNA-sequencing，验证了保留一个内含子的可变剪接，EXON 10和EXON 11之间的内含子在剪接过程中被保留下来，并因其序列中恰好有终止密码子TAG而发生翻译终止。详见附件图2.DPH1 在鼻咽癌细胞株C666-1中的两种可变剪接转录本。.对于DPH1-IR功能，我们研究结果发现：DPH1-IR mRNA在人鼻咽癌细胞、组织中表达上调。据此,提出DPH1-IR可能具有致癌功能的假设。.为证实该假设，我们在DPH1-IR低表达鼻咽癌细胞HONE1中转染DPH1野生型及DPH1-IR基因，发现：DPH1-IR能表达蛋白，与野生型相反，DPH1-SIR能增强细胞克隆及侵袭能力。我们研制了DPH1-IR特异性抗体，并通过免疫组化确认该剪接体表达的临床意义，继而探讨它对鼻咽癌细胞侵袭能力、裸鼠成瘤及转移能力等影响。在此基础上，进一步研究该剪接体的的致癌机制。