Circulating Biomarkers for the Detection of Human Liver Diseases

肝细胞癌是全球癌症相关死亡的第三大常见原因。风险 在患有肥胖和糖尿病的丙型肝炎病毒(HCV)携带者中，肝纤维化和肝癌的风险显著增加。 这在美国是一个上升的趋势。因此，肝细胞癌是一个广泛的公共卫生问题。我们最近 研究表明，双皮质激素样激酶(DCLK1)的过度表达与 肝癌、结肠癌、肠癌和胰腺癌。最近，我们已经证明了增加的表达 DCLK1在肝硬变和肝癌患者中的表达此外，我们还证明了DCLK1的表达趋向于 在肝病进展过程中增加，如肝硬变和肝细胞癌。DCLK1的下调导致 显著降低免疫缺陷小鼠的肝癌细胞增殖和瘤样生长。我们的研究表明 DCLK1影响胰腺癌和结肠癌的生物学过程，包括上皮到结肠癌。 间充质转化(EMT)和cMYC途径。例如，DCLK1的敲除会导致标记 伴随的肿瘤抑制基因miRNAs(let-7a、miR-200、miR-144和miR-145/143)的上调 CMYC，ZEB1/ZEB2，KRAS，NOTCH1，VEGFR1和2，以及多能因子OCT4，SOX2， Nanog和KLF4。因此，DCLK1似乎是一个很好的抗肿瘤治疗的靶点，是一种 检测/识别/区分不同阶段的肝硬变的潜在预后生物标志物(基于 Child-Pugh评分A-C)和肝细胞癌。此外，几个miRNAs(miR-21、miR-199a和miR-301) 在肝硬变和肝细胞癌中表达上调，与疾病预后相关。此外，这些miRNA是已知的 调节/诱导EMT和肿瘤的发生。基于这些观察，我们假设DCLK1和 MiRNAs表达上调，可在肝细胞癌患者血浆中检测到，可作为生物标志物 用于检测肝硬变和肝细胞癌。DCLK1和miRNAs的表达将与 肝纤维化、肝硬变和肝细胞癌患者血清AFP-L3水平的变化在这里我们将从四组人中采集血液 患者--健康对照、肝纤维化患者(但非肝硬变和非肝细胞癌；根据 FIB4评分)、肝硬变(非肝细胞癌；Child-Pugh A-C)和肝细胞癌(ChildA、B或C)。样品将 在OKC VAMC(测试队列)和VA St.Louis Health Care System(验证)收集 队列)。我们将用以下具体目标来检验这一假设。目标1.确定候选miRNAs- 调节肝纤维化或肝癌患者血流中的EMT。我们将分离总的miRNAs 从血浆(从临床定义的纤维化、肝硬变和肝细胞癌中收集)并执行微阵列分析 为了确定与EMT调节相关的各种上调的miRNAs，在患有 肝脏疾病。我们还将确定DCLK1调控的特定miRNAs的表达。目标2.确定 血浆DCLK1水平与肝细胞癌发生发展的关系我们将使用酵素- 用于检测患者血浆中的DCLK1(纤维化， 不同阶段的肝硬变和肝癌)。我们还将确定DCLK1是否在以下患者中增加 肝硬变和/或肝细胞癌与健康对照组比较。我们还将估计所有患者的血浆AFP-L3水平 研究对象，并将其与DCLK1和miRNA水平进行比较。目的3.确定信号通路 调节从肝癌患者分离的循环肿瘤细胞的EMT和转移，以及干细胞的干性。 在这里，我们将分离循环癌细胞(来自肝细胞癌患者)，并确定它们的EMT信号，DCLK1 和miRNA状态，以及它们的封闭性、迁徙和入侵能力。成功完成这些任务 研究将为早期抗击肝细胞癌提供新的方向。78%的VA患者合并肝脏 疾病被诊断为肝癌(中位生存期4.5-9.2个月)，通过我们的方法早期发现肝癌 将帮助退伍军人临床医生决定早期治疗策略，以提高退伍军人的存活率。

Hepatocellular Carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. The risk of liver fibrosis and HCC increases significantly in hepatitis C virus (HCV) carriers with obesity and diabetes, which is a rising trend in the USA. Thus, HCC represents a wide-ranging public health issue. Our recent studies have revealed a positive correlation between overexpression of doublecortin-like kinase (DCLK1) and cancers of the liver, colon, intestine and pancreas. Recently, we have demonstrated that increased expression of DCLK1 in patients with cirrhosis and HCC. Furthermore, we demonstrated that DCLK1 expression tends to increase during progression of liver diseases such as cirrhosis and HCC. Downregulation of DCLK1 resulted in marked reduction of HCC cell proliferation and tumor-like growth in immunodeficient mice. Our studies indicate that DCLK1-affected biological processes in the pancreas and colon cancers including epithelial-to- mesenchymal transition (EMT) and cMYC pathways. For example, knockdown of DCLK1 results in marked upregulation of tumor-suppressor miRNAs (let-7a, miR-200, miR-144, and miR-145/143) with a concomitant decrease in cMYC, ZEB1/ZEB2, KRAS, NOTCH1, VEGFR1 and 2, and pluripotency factors OCT4, SOX2, NANOG and KLF4. Thus, DCLK1 appears to be a well-justified targets for anti-tumor treatment and is a potential prognostic biomarker for detecting/identifying/differentiating various stages of cirrhosis (based on Child-Pugh Score A – C) and HCC. Furthermore, several miRNAs (miR-21, miR-199a, and miR-301) are upregulated in cirrhosis and HCC, correlated with diseases outcomes. Furthermore these miRNAs are known to regulate/induce EMT and oncogenesis. Based on these observations, we hypothesize that DCLK1 and miRNAs are upregulated and can be detected in plasma of patients with HCC, and can be a biomarker for the detection of cirrhosis and HCC. The expression of DCLK1 and miRNAs will be compared to AFP-L3 levels in patients with fibrosis, cirrhosis and HCC. Here we will collect blood from 4 groups of patients – healthy controls, patients with fibrosis (but non-cirrhotic and non-HCC; classified based on FIB4 scoring), cirrhosis (non-HCC; Child-Pugh A – C) and HCC (either Child A, B or C). Samples will be collected at OKC VAMC (test cohort) and also at VA St. Louis Health Care System (validation cohort). We will test the hypothesis with the following specific aims. Aim 1. Identify candidate miRNAs- regulating EMT in the blood stream of patients with liver fibrosis or HCC. We will isolate total miRNAs from the plasma (collected from clinically defined fibrosis, cirrhosis and HCC) and perform microarray analysis to identify various miRNAs upregulated that have been associated with the regulation of EMT, in patients with liver diseases. We will also determine expression of specific miRNAs regulated by DCLK1. Aim 2. Determine whether plasma levels of DCLK1 are associated with the development of HCC. We will employ enzyme- linked immunosorbent assay (ELISA) (commercially available) to detect DCLK1 in patients' plasma (fibrosis, various stages of cirrhosis and HCC). We will also determine whether DCLK1 is increased in patients with cirrhosis and/or HCC compared to healthy controls. We will also estimate the plasma AFP-L3 levels in all the study subjects and compare it with DCLK1 and miRNA levels. Aim 3. To determine the signaling pathways that regulate EMT and metastasis, and stemness of circulating tumor cells isolated from HCC patients. Here we will isolate circulating cancer cells (from HCC patients) and determine their EMT signature, DCLK1 and miRNA status, and their clogenicity, migration and invasion capabilities. Successful completion of these studies will provide a new direction to combat HCC at an early stage. As 78% of the VA patients with liver diseases are diagnosed with HCC (median survival of 4.5–9.2 months), early detection of HCC by our methods will help the VA clinicians to decide early treatment strategies to increase the survival of the veterans.