乳液PCR结合流式细胞术定量检测肿瘤患者APRIL基因剪接变体及其临床应用

Our previous studies have already confirmed that a proliferation-inducing ligand ( APRIL) is overexpressed in many cancer tumors, thus, APRIL holds great promise as a novel target for cancer diagnosis and treatment. However, single detection of APRIL has been showed to be lacking in specificity. In order to get the tissue specific splicing variants of APRIL gene, we plan to further analyze splicing variants mRNA of APRIL gene in various of clinical tumors.The limitations of existing clinical testing methods have restricted further application of the clinical value of transcription splicing variants. It is necessary to explore new sensitive, reliable and easy methods of detecting splicing variants for the purpose of clinical application.Based on existing technology, the applicants want to create new methods by using emulsion PCR combined with flow cytometry (FCM) to quantitive analysis of 6 splicing variants of APRIL gene. The new method is compared with the old one of multiple PCR. Meanwhile, splicing variants mRNA of serum and tissue APRIL gene would be detected in various of clinical tumors. Moreover, the follow-up is necessary to evaluate the value of these splicing variants in tumor early diagnosis, differential diagnosis, judgement of prognosis and explore the mechanism of the occurrence and development in these tumor cell strains. This study would provide novel strategies and ideas in the detection of splicing variants mRNA and gene diagnosis, prevention and treatment with cancer.

我们前期的研究表明，增殖诱导配体（APRIL）在多种肿瘤组织中表达异常增高，有望成为肿瘤诊断与治疗的新靶点。由于单纯检测APRIL缺乏特异性，我们拟进一步对临床常见肿瘤APRIL基因剪接变体mRNA进行检测，以期得到组织特异性的APRIL基因剪接变体。目前临床现有检测方法的局限，限制了转录剪接变体临床价值的进一步应用；有必要积极探索新的敏感、可靠、临床易推广的方法来检测剪接变体。此次申请者在掌握现有技术的基础上，建立乳液PCR结合流式细胞术新方法对APRIL基因6个剪接变体mRNA进行定量检测，并与多重PCR法进行比较；同时检测临床常见肿瘤患者血清以及组织中APRIL基因剪接变体mRNA；探讨其在肿瘤诊断、预后判断中的价值。并培养相关肿瘤细胞株，探讨APRIL基因剪接变体在肿瘤发生、发展中的可能作用机制。本研究可为剪接变体mRNA的检测以及肿瘤基因诊断与防治提供新的策略与思路。

我们前期的研究表明，增殖诱导配体（a proliferation- inducing ligand，APRIL）在多种肿瘤组织中表达异常增高，有望成为肿瘤诊断与治疗的新靶点。由于单纯检测APRIL缺乏特异性，我们拟进一步对临床常见肿瘤APRIL基因剪接变体mRNA进行检测，以期得到组织特异性的APRIL基因剪接变体。目前临床现有检测方法的局限，限制了转录剪接变体临床价值的进一步应用；有必要积极探索新的敏感、可靠、临床易推广的方法来检测剪接变体。我们在掌握现有技术的基础上，并在本项目的资助下，建立乳液PCR结合流式细胞术定量检测APRIL基因剪接变体mRNA的方法，我们配制的乳化液利于PCR扩增，且回收率>90%；该法无论从目的产物量、副产物干扰、特异性等方面均优于普通多重PCR；并且模板浓度、退火温度、Taq DNA酶浓度、引物浓度及dNTP浓度等几方面对乳液PCR反应条件进行了优化，使该反应更加高效、便捷。我们对结直肠癌和肝癌中APRIL基因剪接变体mRNA进行检测，结果显示，APRIL-αγ、APRIL-β和 APRIL-η在结直肠癌组织的表达明显高于癌旁组织；而APRIL-δζ的表达无明显差异。APRIL-αγ和APRIL-β在肝癌组织的表达明显高于癌旁组织；而APRIL-δζ和APRIL-η的表达无明显差异。另外，APRIL-αγ与结直肠癌患者Dukes分期、转移以及预后密切有关；而APRIL-β和 APRIL-η与结直肠癌患者临床病理参数及预后均无关。进一步对APRIL-αγ在结直肠癌中的功能研究显示，RNA干扰APRIL-αγ可抑制结直肠癌细胞的增殖、侵袭与转移、诱导细胞周期G0/G1期阻滞以及细胞凋亡、并且抑制裸鼠移植瘤的生长。机制研究发现，APRIL作为一个潜在的癌基因，可通过TGF-β1/ERK、PI3K/Akt/m-TOR以及MMPs等信号通路促进结直肠癌细胞生长增殖、侵袭与转移并且抑制凋亡，从而促进肿瘤恶性进展。另外，在肝癌中，APRIL也可通过参与miR-383-APRIL信号来调控肝癌细胞的生长。本研究有望为临床剪接变体mRNA的检测、肿瘤的基因诊断与防治以及肿瘤发病机制的研究提供新的策略和思路。