新型分子标志物snoRNA在非小细胞肺癌中的研究与应用

Emerging evidence indicates that small nucleolar RNAs (snoRNAs), a class of small non-coding RNAs, may play important function in tumorigenesis. A large proportion of genes hosting snoRNAs in their introns, and the expression were in the same transcriptional process with snoRNAs. Non-small cell lung cancer (NSCLC) is the most common cancer killer for people. Systematically characterizing snoRNAs in NSCLC will develop biomarkers for its detection and prognostication. Since snoRNAs have a very special co-expression pattern with the host genes, we hypothesize that snoRNAs processed from oncogenes will be highly transcribed and show high levels of production, and could be an effective biomarker for rapid diagnosis. We hope the amount of snoRNAs in serum could reflect the expression levels of oncogenes in tumor. In our previous study, we found dozens of snoRNAs had higher expression levels in NSCLC by next generation sequencing and tissue sample verifications. SNORA30 and SNORD10 were selected for further study, whose host genes are SRCAP and EIF4A1, repectively. The results showed there is a correlation between the snoRNAs and host gene expression levels in lung cancer cell lines. We will further verify the correlation between snoRNAs and host genes expression levels in NSCLC tissue samples, also expression correlation between snoRNAs (SNORA30 and SNORD10) in serum and their host genes in tumor; in addition, the availablity of regulation mechanism between them will also be explored. Our research will provide potential biomarkers for the detection and prognostication of NSCLC，also a new screening method of biomarkers for tumor and other diseases.

核仁小RNA（snoRNAs）是一类非编码RNA，在脊椎动物中由基因的内含子加工生成，与宿主基因的表达在同一转录过程中完成。基于二者共表达这一特殊模式，我们提出以癌基因内含子加工生成的snoRNAs作为分子标志物，通过检测血清中snoRNAs的含量反映肿瘤中的宿主癌基因的表达水平，进而得到分子标志物的新的筛选方法。前期通过NGS平台及大样本量检测筛选到数十个在非小细胞肺癌（NSCLC）中特异性高表达的snoRNAs。我们将以SNORA30 和 SNORD10为例（宿主基因为癌基因SRCAP和EIF4A1），确认snoRNAs与宿主基因在NSCLC组织样本中的表达相关性，探究二者表达间相互调控的可能性。其次，分析血清样品中snoRNAs与肿瘤中宿主癌基因表达的相关性。从而为NSCLC的诊断提供新的分子标志物，并为肿瘤及其他疾病分子标志物的筛选提供新的模式。

本研究基于临床推进无创诊断的大背景，期望利用与宿主基因存在共表达可能性的核仁小RNA（snoRNA） 作为NSCLC早期诊断的分子标志物。研究选取由癌基因内含子加工而成且在肿瘤患者体内存在差异性表达的SNORA30和SNORD10进行验证。二者与其宿主基因的共表达模式在体外水平、模式动物水平均得到了有效的验证。更重要的是，在89组非小细胞肺癌患者的成对组织和术前血清样本中发现血清SNORA30与组织SRCAP水平存在显著关联（r=0.2870，p=0.0064）；血清SNORD10与组织EIF4A1水平也存在显著关联（r=0.2029，p=0.0430），呈正相关趋势。这一结果有力的说明NSCLC患者体内snoRNA与其宿主癌基因存在捆绑表达模式，通过对血清样本中snoRNA含量的检测可以反映肿瘤中宿主癌基因的异常表达情况，这实现了我们期望利用血清snoRNA进行无创诊断的初衷。而后的临床有效性验证结果显示，在255例受试者的血清样本（正常人53例，NSCLC患者193例）中，SNORA30和SNORD10针对非小细胞肺癌检测的灵敏度与特异性均较高，ROC曲线下面积分别为0.7589和0.8725；而与其相比较的CEA为0.642，CA125为0.511。同时我们发现在宿主基因的启动子受到影响后，snoRNA的表达也随之发生改变，SNORD10与宿主基因EIF4A1具有表达下调的一致性。但由于仅在体外水平进行了检测，我们期望在后续的体内水平实验中对他们的调控方式作进一步的明确。综上所述，本研究证明癌基因内含子加工生成的snoRNAs是临床诊断的有效分子指标，这一验证为肿瘤分子标志物的筛选提供一个全新的模式和途径。