MicroRNAs (miRs) are a new class of small non-coding RNAs that may regulate up to 30% of human genes. miRs are involved in a myriad of cellular processes: proliferation, differentiation, apoptosis and responses to stress, and were shown to be involved in normal cholangiocyte physiology as well as in cholangiocarcinomas (CCA). CCAs are cancers arising from the biliary tract. They are the 2nd most common primary hepatobiliary cancers in the United States, and their incidence is increasing globally. The survival of patients with CCA is dismal, usually measured in months. Our preliminary studies in cholangiocytes identified one hundred and thirty two miR species with differential expression in cholangiocytes vs. neoplastic cholangiocytes. We hypothesize that among these miRs, there are several miR species that have an etiologic role in neoplasia formation and homeostasis. Furthermore, we hypothesize that these miRs have important role in the normal cholangiocyte homeostasis, regulating cell cycle progression, proliferation and/or apoptosis. In the current application, we propose a step- wise approach to identify miRs with phenotypic impact, followed by careful characterization of their function. Specifically, we propose to: 1. Identify 4 miRs that have a phenotypic impact on cholangiocytes in vitro (Aim 1). We will select miR species that have similar phenotypic impact on human (Aim 1a) and murine (Aim 1b) cell lines. The reason for this restriction is that we plan on using the SMAD4/PTEN murine model of cholangiocarcinoma for our in vivo studies. Next, we propose to 2. Further validate the in vitro phenotypic action of the candidate miRs through in vivo studies (Aim 2). By applying a step-wise algorithm, we plan on verifying the miR action in a xenograft model (Aim 2a) and then to further study the miRs with phenotypic impact in a liver-specific SMAD4/PTEN-deleted murine model of CCA. At the conclusion of Aims 1 and 2, we will have identified 4 miR species with phenotypic impact (cell cycle, proliferation, apoptosis) on cholangiocytes in vitro and in vivo. These miR species are very likely to be involved in normal cholangiocyte cellular processes as well as altered as cholangiocytes become neoplastic. Finally, we propose to 3. Identify mechanism of action for miR species identified in Aim 1 and confirmed in Aim 2 (Aim 3). We will identify mRNA and protein targets for these miRs as well as explore the pathways connecting these miRs to their mRNA targets. I believe that through funding this application and execution of the proposed experiments, several goals will be met: 1. my training in molecular biology will be perfected with the final goal of contributing to my independence as principal investigator;2. the current understanding of the normal physiology of cholangiocytes as well as the neoplastic transformation will be advanced. Ultimately, I am hopeful that through the findings of this project, we will make significant progress towards understanding how and why inflammatory conditions of the biliary tree, such as Primary Sclerosing Cholangitis, progress to neoplasia.

Public Health Relevance

Our preliminary studies in cholangiocytes identified 4 microRNA species with profound effects on cell growth, cell cycle progression and apoptosis in vitro. In the current study, we propose to further dissect the effects of these microRNAs on cholangiocytes in vitro, as well as testing their effects in vivo. Finally, we aim at uncovering the mechanistic links between these microRNAs and known normal or neoplastic pathways in cholangiocytes, with the final purpose of better understanding the normal physiology of cholangiocytes as well as the neoplastic transformation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Clinical Investigator Award (CIA) (K08)
Project #
1K08DK090154-01
Application #
8030017
Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Program Officer
Podskalny, Judith M,
Project Start
2011-07-01
Project End
2016-06-30
Budget Start
2011-07-01
Budget End
2012-06-30
Support Year
1
Fiscal Year
2011
Total Cost
$158,700
Indirect Cost
Name
Johns Hopkins University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Wang, Fang; Li, Ling; Piontek, Klaus et al. (2018) Exosome miR-335 as a novel therapeutic strategy in hepatocellular carcinoma. Hepatology 67:940-954
Kumbhari, Vivek; Li, Ling; Piontek, Klaus et al. (2018) Successful liver-directed gene delivery by ERCP-guided hydrodynamic injection (with videos). Gastrointest Endosc 88:755-763.e5
Li, Ling; Piontek, Klaus; Ishida, Masaharu et al. (2017) Extracellular vesicles carry microRNA-195 to intrahepatic cholangiocarcinoma and improve survival in a rat model. Hepatology 65:501-514
Li, Ling; Piontek, Klaus B; Kumbhari, Vivek et al. (2016) Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile. J Vis Exp :
Peng, Haoran; Ishida, Masaharu; Li, Ling et al. (2015) Pseudogene INTS6P1 regulates its cognate gene INTS6 through competitive binding of miR-17-5p in hepatocellular carcinoma. Oncotarget 6:5666-77
Li, Ling; Masica, David; Ishida, Masaharu et al. (2014) Human bile contains microRNA-laden extracellular vesicles that can be used for cholangiocarcinoma diagnosis. Hepatology 60:896-907
Olaru, Alexandru V; Yamanaka, Sumitaka; Vazquez, Christine et al. (2013) MicroRNA-224 negatively regulates p21 expression during late neoplastic progression in inflammatory bowel disease. Inflamm Bowel Dis 19:471-80
Gultepe, Evin; Yamanaka, Sumitaka; Laflin, Kate E et al. (2013) Biologic tissue sampling with untethered microgrippers. Gastroenterology 144:691-3
Ishida, Masaharu; Selaru, Florin M (2013) miRNA-Based Therapeutic Strategies. Curr Anesthesiol Rep 1:63-70
Jiao, Yuchen; Pawlik, Timothy M; Anders, Robert A et al. (2013) Exome sequencing identifies frequent inactivating mutations in BAP1, ARID1A and PBRM1 in intrahepatic cholangiocarcinomas. Nat Genet 45:1470-1473

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