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）是全球与癌症相关死亡的第三大最常见原因。风险 肝纤维化和HCC的肝炎病毒（HCV）携带者的肥胖症和糖尿病，肝炎病毒的增加显着增加 这是美国的一个上升趋势。这是HCC代表一个广泛的公共卫生问题。我们的最新消息 研究表明，双铁蛋白样激酶（DCLK1）的过表达与 肝脏，结肠，肠和胰腺的癌症。最近，我们证明了表达增加 肝硬化和HCC患者的DCLK1。此外，我们证明了DCLK1表达趋于 肝病和HCC等肝病进展过程中的增加。 DCLK1的下调导致 在免疫缺陷小鼠中，HCC细胞增殖和肿瘤样生长显着降低。我们的研究表明 胰腺和结肠癌中受DCLK1影响的生物学过程，包括上皮到 - 间充质转变（EMT）和CMYC途径。例如，DCLK1的敲低导致标记 肿瘤抑制剂miRNA（Let-7a，miR-200，miR-144和miR-145/143）的上调 CMYC，ZEB1/ZEB2，KRAS，NOTCH1，VEGFR1和2的减少以及多能因子OCT4，SOX2， Nanog和KLF4。那是DCLK1似乎是抗肿瘤治疗的正式靶标，是 潜在的预后生物标志物用于检测/识别/区分肝硬化的各个阶段（基于 Child-Pugh得分A-C）和HCC。此外，几个miRNA（miR-21，miR-199a和miR-301）是 在肝硬化和HCC中上调，与疾病结局相关。此外，这些miRNA是已知的 调节/诱导的EMT和肿瘤发生。基于这些观察，我们假设DCLK1和 miRNA被上调，可以在HCC患者的血浆中检测到，并且可以是生物标志物 用于检测肝硬化和HCC。 DCLK1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，我们将从4组中收集血液 患者 - 健康对照，纤维化患者（但非核酸和非HCC；根据 FIB4评分），肝硬化（非HCC； Child-Pugh A - C）和HCC（儿童A，B或C）。样品会 可以在OKC VAMC（测试队列）和VA圣路易斯医疗保健系统（验证）收集 队列）。我们将以以下特定目的检验假设。目标1。确定候选人mirnas- 调节肝纤维化或HCC患者的血液中EMT。我们将隔离总miRNA 从血浆（从临床定义的纤维化，肝硬化和HCC收集）并进行微阵列分析 确定与EMT调节有关的各种miRNA，患有EMT的调节 肝病。我们还将确定由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的发展有关。我们将采用酶 - 连接的免疫吸附测定（ELISA）（可商购）以​​检测患者血浆中的DCLK1（纤维化， 肝硬化和HCC的各个阶段）。我们还将确定患有患者的DCLK1是否增加 与健康对照相比，肝硬化和/或HCC。我们还将估计所有的等离子体AFP-L3水平 研究对象并将其与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，我们将分离循环的癌细胞（来自HCC患者），并确定其EMT签名DCLK1 和mirna地位及其神职人员，迁移和入侵能力。这些成功完成 研究将为早期阶段与HCC战斗提供新的方向。作为肝脏的VA患者中有78％ 疾病被诊断为HCC（中位生存期4.5-9.2个月），通过我们的方法早期检测HCC 将帮助VA临床医生决定提高退伍军人生存的早期治疗策略。