Circulating Biomarkers for the Detection of Human Liver Diseases

肝细胞癌（HCC）是全球癌症相关死亡的第三大常见原因。风险 丙型肝炎病毒（HCV）携带者伴肥胖和糖尿病的肝纤维化和HCC的发生率显著增加， 这在美国是一个上升的趋势。因此，HCC是一个广泛的公共卫生问题。我们最近 研究已经揭示了双皮质素样激酶（DCLK 1）的过表达和 肝癌、结肠癌、肠癌和胰腺癌。最近，我们已经证明， DCLK 1在肝硬化和HCC患者中的表达。此外，我们证明了DCLK 1表达倾向于 在肝硬化和HCC等肝脏疾病进展期间增加。DCLK 1的下调导致 免疫缺陷小鼠中HCC细胞增殖和肿瘤样生长的显著减少。我们的研究表明 DCLK 1影响胰腺癌和结肠癌的生物学过程，包括上皮细胞到 间充质转化（EMT）和cMYC途径。例如，DCLK 1的敲低导致显著的 肿瘤抑制miRNAs（let-7a、miR-200、miR-144和miR-145/143）的上调， cMYC、ZEB 1/ZEB 2、KRAS、NOTCH 1、VEGFR 1和2以及多能性因子OCT 4、SOX 2的减少， NANOG和KLF 4。因此，DCLK 1似乎是抗肿瘤治疗的合理靶点，并且是一种治疗肿瘤的有效靶点。 用于检测/鉴定/区分肝硬化的各个阶段的潜在预后生物标志物（基于 Child-Pugh评分A-C）和HCC。此外，几种miRNAs（miR-21、miR-199 a和miR-301）是 在肝硬化和HCC中上调，与疾病结局相关。此外，已知这些miRNAs 调节/诱导EMT和肿瘤发生。基于这些观察，我们假设DCLK 1和 miRNA在HCC患者血浆中表达上调，可作为HCC的生物标志物 用于检测肝硬化和HCC。DCLK 1和miRNA的表达将与 纤维化、肝硬化和HCC患者的AFP-L3水平在这里，我们将收集血液从4组， 患者-健康对照，纤维化患者（但非纤维化和非HCC;基于 FIB 4评分）、肝硬化（非HCC; Child-Pugh A-C）和HCC（Child A、B或C）。样本将 在OKC VAMC（测试队列）和VA St. Louis Health Care System（验证）采集 群组）。我们将以下列具体目标来检验这一假设。目标1。鉴定候选miRNAs- 调节肝纤维化或HCC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、肝硬化和HCC收集）中提取并进行微阵列分析 为了鉴定与EMT调节相关的各种上调的miRNAs， 肝脏疾病我们还将确定由DCLK 1调控的特定miRNA的表达。目标2.确定 DCLK 1的血浆水平是否与HCC的发展相关。我们会用酶- 连接的免疫吸附测定（ELISA）（市售）以检测患者血浆中的DCLK 1（纤维化， 肝硬化和HCC的不同阶段）。我们还将确定DCLK 1是否在患有以下疾病的患者中增加： 肝硬化和/或HCC之间的差异。我们还将估计所有受试者的血浆AFP-L3水平。 研究对象并将其与DCLK 1和miRNA水平进行比较。目标3：为了确定信号通路 其调节从HCC患者分离的循环肿瘤细胞的EMT和转移以及干性。 在这里，我们将分离循环癌细胞（来自HCC患者）并确定其EMT特征，DCLK 1 和miRNA状态，以及它们的致凝性、迁移和入侵能力。成功完成这些 这些研究将为在早期阶段防治肝癌提供新的方向。由于78%的VA患者有肝脏 疾病诊断为HCC（中位生存期为4.5-9.2个月），通过我们的方法早期检测HCC 将帮助退伍军人管理局的临床医生决定早期治疗策略，以增加退伍军人的生存。

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.