利用高容量N19随机siRNA文库靶向全转录本筛选鉴定肝癌诊治标志物

对疾病分子机制理解的欠缺、诊断标志物灵敏度和特异性不足等原因，原发性肝癌面临“早期诊断困难、靶向治疗效果差”的难题。ncRNAs数量约占全转录本98%、与肝癌密切相关、体液中表达稳定可被检测，极有潜力用于临床肝癌诊治。但绝大部分ncRNAs功能未知且鉴定困难。课题组率先利用逆转录病毒介导的高容量N19随机siRNA文库靶向正常人源肝细胞的全转录本，通过功能表型试验模拟肝癌形成的动态过程，沿时间轴纵向筛选出一系列进行性、含缺失转录本的“肿瘤样”肝细胞（体外增殖能力强、肿瘤标志物阳性、裸鼠皮下成"瘤"性强）。为了筛选鉴定被文库沉默导致肝细胞“肿瘤样”变的缺失转录本用于临床肝癌诊治，课题组拟进一步借助NGS、生信分析、分子生物学技术等，按照“筛选鉴定缺失转录本-临床样本中验证缺失转录本的诊断价值-初步探讨缺失转录本的作用机制”的顺序，发现肝癌相关转录本功能、寻找全新诊治标志物、完善肝癌发病机制。

Early diagnosis and effective targeted therapies for primary liver cancer (PLC) have been significantly hampered due to the lack of the understanding of its molecular pathogenic mechanisms and the unavailability of clinical diagnostic biomarkers with high sensitivity and specificity. Comprehensive genome biology studies have revealed that vast majority of human genome are transcribed, while 98% of the transcripts are noncoding RNAs (ncRNAs). Emerging evidence also suggests that ncRNAs are associated with PLC development and progression. Since ncRNAs can be easily detected in blood and body fluids, they should have great potential to serve as PLC diagnostic biomarkers. However, the biological functions of most ncRNAs are either unknown or too complex. Here, we proposed to use our recently-developed, high capacity randomized N19 siRNA library to conduct a genomewide selection to identify both coding and noncoding transcripts that are associated with the development of PLC and determine their potential as diagnostic biomarkers and/or therapeutic targets. By introducing this novel siRNA library into primary human hepatocytes and performing multiple rounds of in vivo tumorigenicity selection in athymic nude mice, we have successfully established a progressive, multi-stage liver cancer-like cell (LCLC) model. We will perform next-gen DNA-sequencing (NGS) to identify the enriched siRNA fragments, identify and validate their target coding and noncoding transcripts through a series of RNA-Seq, bioinformatics analysis, qPCR verification and/or Western blotting analysis. The top candidate transcripts (coding and noncoding) will be further validated in clinical cancer specimens. Our ultimate goals are to determine whether or not the candidate transcripts can be used sensitive and specific diagnostic biomarkers, and to set up the stage for in-depth studies on their pathogenic roles in PLC development in our next phase of investigation.