MBNL3及其诱导的可变剪接转录本POLDIP3-β在肝癌中的作用及机制研究

发布时间：2018-09-07 09:57

【摘要】：可变剪接(alternative splicing,AS)在许多生理病理过程中发挥重要的作用,并且剪接失调是肿瘤发生的重要特征之一。我们知道,来自同一基因的不同产物可能对肿瘤产生不同的影响。因此,深入研究可变剪接因子和可变剪接事件在肿瘤发生中的作用,可为肿瘤的靶向治疗找到一些新的思路。在研究中我们发现一个剪接因子MBNL3,它具有癌胚特性,能够促进肝癌的发生。首先,我们在胎肝和成年肝组织中检测了MBNL3的表达,发现MBNL3在胎肝组织中高表达。然后,在肝癌和癌旁肝组织中检测MBNL3的表达,并分析其与临床病理特征的相关性,发现MBNL3在肝癌组织中高表达并与肝癌病人的不良预后相关,可能驱动肿瘤的发生。进一步的研究发现干细胞转录因子OCT4、SOX2、NANOG上调MBNL3的表达。在肝癌细胞和肝细胞中过表达MBNL3,CCK-8、乙炔脱氧尿苷结合染色(EdU)和克隆形成等增殖实验,TUNEL、凋亡标志因子检测等凋亡实验,以及动物体内荷瘤实验,表明MBNL3能够促进肝癌细胞和未转化肝细胞的增殖、抑制其凋亡、促进体内肿瘤生长。而敲减MBNL3后抑制肝癌细胞增殖、促进其凋亡。最后,我们在MBNL3本底表达量不同的多种肝癌细胞系和肝细胞系中敲减MBNL3,研究其对肿瘤发生的作用。最终发现,敲减MBNL3可显著抑制肝癌细胞体内外的生长,并且对MBNL3高表达的细胞株作用更强烈。鉴于MBNL3强大的生物学功能,我们对其下游分子进行分析。转录组测序结果表明MBNL3可以调控POLDIP3的可变剪接,所以,我们锚定POLDIP3的不同转录本进行研究。POLDIP3是核糖体蛋白S6激酶1的靶标,它能够调节DNA的复制和mRNA的翻译。在本研究中,我们发现POLDIP3有POLDIP3-α和POLDIP3-β两个转录本,其中POLDIP3-α包含外显子3,POLDIP3-β缺少外显子3,进而缺失了29个氨基酸。通过检测POLDIP3-α和POLDIP3-β在临床肝癌组织和癌旁肝组织中的表达,我们发现,相较于癌旁肝组织,POLDIP3-β在肝癌组织中显著上调。通过Glo细胞活力实验,EdU实验,克隆形成实验,检测POLDIP3两个转录本对肝癌细胞增殖的影响;通过TUNEL实验,膜联蛋白V-碘化丙啶染色和流式细胞术,研究POLDIP3两个转录本在肝癌细胞凋亡方面的作用;利用transwell实验和划痕实验检测POLDIP3两个转录本对肝癌细胞迁移的作用。体外功能学实验结果证明POLDIP3-β能够显著促进肝癌细胞的增殖,抑制肝癌细胞的凋亡,加速肝癌细胞的迁移。然而,POLDIP3-α对肝癌细胞的增殖、凋亡、转移的作用相对较弱。最后,在动物体内检测POLDIP3-β对体内肿瘤的影响,发现POLDIP3-β可显著促进体内肝癌细胞的生长。以上数据表明POLDIP3-β在肝癌中上调表达,并且在肝癌中发挥着癌基因的作用。选择性的靶向抑制POLDIP3-β这个转录本有望成为肝癌的一个新的治疗策略。我们的研究揭示了癌胚性的可变剪接因子、可变剪接事件与肿瘤发生的关系,同时,确立可变剪接因子和可变剪接事件可以作为潜在的治疗靶标。
[Abstract]:Variable splicing (alternative splicing,AS) plays an important role in many physiological and pathological processes, and splicing imbalance is one of the important characteristics of tumorigenesis. We know that different products from the same gene may have different effects on tumors. Therefore, a further study on the role of variable splicing factors and splicing events in tumorigenesis can provide some new ideas for tumor targeting therapy. In our study, we found a splicing factor, MBNL3, which has carcinoembryonic properties and promotes the development of liver cancer. First, we detected the expression of MBNL3 in fetal liver and adult liver tissue, and found that MBNL3 was highly expressed in fetal liver tissue. Then, the expression of MBNL3 was detected in liver cancer and adjacent liver tissues, and its correlation with clinicopathological features was analyzed. It was found that the high expression of MBNL3 in HCC tissues and the poor prognosis of HCC patients may drive the development of tumor. Further studies revealed that stem cell transcription factor OCT4,SOX2,NANOG up-regulated the expression of MBNL3. MBNL3,CCK-8, was overexpressed in hepatoma cells and hepatocytes. The proliferative tests such as Tunel, detection of apoptotic marker factors, and tumor-bearing experiments in vivo, such as (EdU) staining and clone formation, were detected in hepatoma cells and hepatocytes. The results showed that MBNL3 could promote the proliferation of hepatoma cells and untransformed hepatocytes, inhibit their apoptosis, and promote tumor growth in vivo. Knockout MBNL3 inhibited the proliferation of hepatoma cells and promoted their apoptosis. Finally, we studied the role of knockout MBNL3, in tumorigenesis in various hepatoma cell lines and hepatocyte lines with different MBNL3 background expression. It was found that knockout MBNL3 could significantly inhibit the growth of hepatoma cells in vitro and in vivo, and had a stronger effect on cell lines with high MBNL3 expression. In view of the strong biological function of MBNL3, we analyze its downstream molecules. The results of transcriptome sequencing show that MBNL3 can regulate the variable splicing of POLDIP3. Therefore, we anchor the different transcripts of POLDIP3 to study. POLDIP3 is the target of ribosomal protein S6 kinase 1, which can regulate the replication of DNA and the translation of mRNA. In this study, we found that POLDIP3 has two transcripts of POLDIP3- 伪 and POLDIP3- 尾, in which POLDIP3- 伪 contains exon 3, POLDIP3- 尾, and then lacks 29 amino acids. By detecting the expression of POLDIP3- 伪 and POLDIP3- 尾 in clinical and para-cancerous liver tissues, we found that POLDIP3- 尾 was significantly up-regulated in HCC tissues compared with paracarcinomatous liver tissues. The effects of two transcripts of POLDIP3 on the proliferation of hepatocellular carcinoma cells were detected by Glo cell viability assay and clone formation assay. To study the role of POLDIP3 two transcripts in the apoptosis of hepatoma cells, and to detect the effect of POLDIP3 two transcripts on the migration of HCC cells by transwell assay and scratch test. The results of functional experiments in vitro showed that POLDIP3- 尾 could significantly promote the proliferation of hepatoma cells, inhibit the apoptosis of hepatoma cells, and accelerate the migration of hepatoma cells. However, POLDIP3- 伪 has a relatively weak effect on the proliferation, apoptosis and metastasis of hepatoma cells. Finally, the effect of POLDIP3- 尾 on tumor in vivo was detected in animals. It was found that POLDIP3- 尾 could significantly promote the growth of hepatoma cells in vivo. The above data suggest that POLDIP3- 尾 is up-regulated in HCC and plays an oncogene role in HCC. Selective targeting inhibition of POLDIP3- 尾 is expected to be a new therapeutic strategy for HCC. Our study revealed the relationship between the variable splicing factor, the variable splicing event and the tumorigenesis. At the same time, we established that the variable splicing factor and the variable splicing event can be used as potential therapeutic targets.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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