Circulating Biomarkers for the Detection of Human Liver Diseases

用于检测人类肝脏疾病的循环生物标志物

• 批准号: 10049186
• 负责人: Courtney Wayne Houchen
• 金额: --
• 依托单位: OKLAHOMA CITY VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-10-01 至 2022-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10049186
• 关键词: 3-Dimensional; Affect; Alcohols; Autoimmune; Biological Markers; Biological Process; Blood; Blood Circulation; Cancer Etiology; Caring; Cell Proliferation; Cessation of life; Child; Cirrhosis; Clinical; Colon Carcinoma; Colorectal Cancer; Data; Detection; Development; Diabetes Mellitus; Diagnosis; Disease Outcome; Down-Regulation; Early Diagnosis; Early treatment; Enzyme-Linked Immunosorbent Assay; Esophageal Adenocarcinoma; Exclusion; Fibrosis; Growth; Healthcare Systems; Hemochromatosis; Hepatitis B Virus; Hepatitis C virus; Hepatocyte; Hepatolenticular Degeneration; Human; Immunodeficient Mouse; Incidence; Intestinal Cancer; KRAS2 gene; Liver Fibrosis; Liver diseases; Malignant Neoplasms; Malignant neoplasm of liver; Malignant neoplasm of lung; Malignant neoplasm of pancreas; Methods; MicroRNAs; Microarray Analysis; NOTCH1 gene; Neoplasm Circulating Cells; Neoplasm Metastasis; Numerical value; Obesity; Outcome; Pancreatic Ductal Adenocarcinoma; Pathway interactions; Patients; Phosphotransferases; Physicians; Plasma; Population; Precancerous Conditions; Primary carcinoma of the liver cells; Prognostic Marker; Provider; Public Health; Publishing; Regulation; Risk; Sampling; Signal Pathway; Stream; Study Subject; Survival Rate; Testing; The Cancer Genome Atlas; Tumor Suppressor Proteins; Up-Regulation; Validation; Vascular Endothelial Growth Factor Receptor-1; Veterans; Work; Xenograft procedure; alpha 1-Antitrypsin Deficiency; alpha-Fetoproteins; base; burden of illness; c-myc Genes; circulating biomarkers; circulating cancer cell; cohort; combat; detection method; disease diagnosis; epithelial to mesenchymal transition; inclusion criteria; knock-down; migration; military veteran; minimally invasive; non-alcoholic fatty liver disease; overexpression; pluripotency factor; prevent; stemness; treatment strategy; trend; tumor; tumorigenesis

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.

肝细胞癌（HCC）是全球癌症相关死亡的第三大常见原因。风险 患有肥胖和糖尿病的丙型肝炎病毒（HCV）携带者的肝纤维化和肝癌的发生率显着增加， 这在美国呈上升趋势。因此，HCC 代表了一个广泛的公共卫生问题。我们最近的 研究表明，双皮质素样激酶 (DCLK1) 的过度表达与 肝癌、结肠癌、肠癌和胰腺癌。最近，我们证明表达量增加 肝硬化和 HCC 患者中 DCLK1 的表达。此外，我们证明 DCLK1 表达倾向于 在肝硬化和肝癌等肝脏疾病的进展过程中会增加。 DCLK1 的下调导致 免疫缺陷小鼠的肝癌细胞增殖和肿瘤样生长显着减少。我们的研究表明 DCLK1 影响胰腺癌和结肠癌的生物过程，包括上皮细胞- 间质转化 (EMT) 和 cMYC 途径。例如，DCLK1 的敲低会导致显着的 肿瘤抑制 miRNA（let-7a、miR-200、miR-144 和 miR-145/143）的上调以及伴随的 cMYC、ZEB1/ZEB2、KRAS、NOTCH1、VEGFR1 和 2 以及多能因子 OCT4、SOX2、 NANOG 和 KLF4。因此，DCLK1 似乎是抗肿瘤治疗的合理靶标，并且是 用于检测/识别/区分肝硬化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，一些 miRNA（miR-21、miR-199a 和 miR-301） 在肝硬化和肝癌中表达上调，与疾病结果相关。此外，这些 miRNA 是已知的 调节/诱导 EMT 和肿瘤发生。基于这些观察，我们假设 DCLK1 和 miRNA 表达上调，可在 HCC 患者血浆中检测到，可作为生物标志物 用于检测肝硬化和 HCC。 DCLK1 和 miRNA 的表达将被比较 纤维化、肝硬化和 HCC 患者的 AFP-L3 水平。在这里，我们将从 4 组中采集血液 患者 – 健康对照、纤维化患者（但非肝硬化和非 HCC；根据 FIB4 评分）、肝硬化（非 HCC；Child-Pugh A – C）和 HCC（Child A、B 或 C）。样品将 在 OKC VAMC（测试队列）和 VA St. Louis 医疗保健系统（验证 队列）。我们将通过以下具体目标来检验该假设。目标 1. 鉴定候选 miRNA- 调节肝纤维化或 HCC 患者血流中的 EMT。我们将分离总 miRNA 从血浆（从临床定义的纤维化、肝硬化和 HCC 中收集）并进行微阵列分析 鉴定在患有以下疾病的患者中与 EMT 调节相关的各种上调的 miRNA 肝脏疾病。我们还将确定受 DCLK1 调节的特定 miRNA 的表达。目标 2. 确定 DCLK1 的血浆水平是否与 HCC 的发展相关。我们将使用酶- 连锁免疫吸附测定 (ELISA)（市售）可检测患者血浆中的 DCLK1（纤维化、 肝硬化和 HCC 的各个阶段）。我们还将确定 DCLK1 在患有以下疾病的患者中是否增加： 与健康对照相比，肝硬化和/或肝癌。我们还将估计所有患者的血浆 AFP-L3 水平 研究受试者并将其与 DCLK1 和 miRNA 水平进行比较。目标 3. 确定信号通路 调节 EMT 和转移，以及从 HCC 患者中分离出的循环肿瘤细胞的干性。 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 DCLK1 和 miRNA 状态，及其异源性、迁移和侵袭能力。顺利完成这些 研究将为早期对抗肝癌提供新方向。 78% 的 VA 患者患有肝脏疾病 疾病被诊断为 HCC（中位生存期为 4.5-9.2 个月），通过我们的方法早期发现 HCC 将帮助退伍军人管理局临床医生决定早期治疗策略，以提高退伍军人的生存率。