预警和诊断早期原发性肝细胞肝癌的血清mRNA分类器的模型建立及其应用研究

The discovery of effective and reliable strategies to detect hepatocellular carcinoma (HCC) at an early stage could improve the survival of patients with this disease. We previously demonstrated that a miRNA classifier could identify small-size, early-stage, and α-fetoprotein-negative HCC in patients at risk, and be valuable to detect preclinical hepatocellular carcinoma. Recently, we found that the circulating messenger RNA(mRNA) had significantly different levels between individuals with chronic hepatitis B and those with hepatocellular carcinoma. It is unknown that whether circulating mRNA could detect preclinical early-stage disease. mRNA is a large family of RNA molecules that convey genetic information from DNA to the ribosome, where they specify the amino acid sequence of the protein products of gene expression. We plan:1、to identify a serum mRNA classifier that could differentiate individuals with HCC from healthy individuals and at-risk controls. 2、to assess the value of the classifier to identify preclinical HCC. using prospectively collected sera from patients with HCC and at-risk controls. 3、to analyze the correlation between the frequency of mRNA mutation and prognosis of patients with HCC. We will perform a large-scale validation to identify a serum mRNA classifier that could differentiateindividuals with HCC from healthy individuals and at-risk controls, and do a nested case-control study, using prospectively collected sera from patients with hepatocellular carcinoma and at-risk controls, to assess the value of the classifier to identify preclinical hepatocellular carcinoma.

缺乏有效的早期诊断和预警分子是肝细胞肝癌（简称肝癌）患者预后差的关键原因。前期我们证实：血清microRNAs（miRNAs）组成的分类器，较AFP更早更准确地预警肝癌的发生（Lancet Oncology）。最近我们新发现，肝癌患者血清中信使RNA（mRNA）的表达谱与健康对照、慢性乙型肝炎患者存在明显差异。但血清mRNA分类器在肝癌诊断和预警中的功能未见报道。mRNA传递遗传信息，直接指导蛋白质翻译，发挥独特的功能。那么血清mRNA分类器能否更早及更灵敏地预警肝癌发生？编码基因的突变mRNA，突变频率的高低能否预测患者的预后？针对以上疑问，本课题采用深度测序的方法，筛选到一批在肝癌与慢性乙型肝炎患者血清中差异表达的mRNA，在此基础上拟建立mRNA分类器，利用已建立的大样本的前瞻性队列血清验证其在肝癌诊断和预警中的作用；揭示mRNA突变频率与患者预后的关系，为肝癌早期诊断提供科学依据。

目前血清RNA在肝细胞癌（HCC）诊断中的作用研究较少。我们通过测序结果提示与慢性乙型肝炎患者组相比，HCC患者血清中存在差异表达的RNA；进一步我们筛选了在HCC患者中差异表达的RNA；我们收集了健康对照、慢性乙型肝炎、乙肝肝硬化和肝癌患者的血清标本；我们设计了相关的定量PCR引物，并拟通过实验拟选取特异性好、扩增效率高的引物，并检测了这些异常表达的RNA的表达。在此基础上，我们使用这些引物在训练组的标本中检测筛选的RNA的表达水平。我们发现除了长链非编码RNA HULC，还有Fibulin-1, NCAPG等信使RNA也在HCC患者的血清和组织中异常表达，且血清中上调的Fibulin-1, NCAPG RNA可以用于HCC的诊断。Fibulin-1 的ROC曲线下的AUC面积0.791（95% confidence interval (CI): 0.690–0.893)，比AFP高0.640 (95% CI: 0.498–0.782)。另外，我们将Fibulin-1 RNA和AFP进行联合，发现能明显提高AFP对HCC患者的诊断效能（0.868 vs 0.640）。另外，我们收集的标本中大约35.48%（11/31）的HCC患者AFP水平正常，在AFP阴性的HCC患者中，使用Fibulin-1 RNA最佳的诊断临界值（22.38），90.91%（10/11）表现出Fibulin-1水平升高，提示了Fibulin-1在HCC诊断中的重要功能。另外，我们发现Fibulin-1能通过调控Notch信号通路进而调控下游的抗凋亡分子，影响细胞凋亡。本研究为肝癌的诊断提供了新的血清学标志物，为肝癌的防治提供了新的分子靶点，如能应用于临床，将有利于肝癌的治疗。