A Mouse Model of Cholestasis-Associated Cholangiocarcinoma and Transcription Factors Involved in Progression

doi:10.1053/j.gastro.2011.03.044

Gastroenterology

Volume 141, Issue 1, July 2011, Pages 378-388.e4

https://doi.org/10.1053/j.gastro.2011.03.044 Get rights and content

Background & Aims

Cholestasis contributes to hepatocellular injury and promotes liver carcinogenesis. We created a mouse model of chronic cholestasis to study its effects on progression of cholangiocarcinoma and the oncogenes involved.

Methods

To induce chronic cholestasis, Balb/c mice were given 2 weekly intraperitoneal injections of diethylnitrosamine (DEN); 2 weeks later, some mice also received left and median bile duct ligation (LMBDL) and, then 1 week later, were fed DEN, in corn oil, weekly by oral gavage (DLD). Liver samples were analyzed by immunohistochemical and biochemical assays; expression of Mnt and c-Myc was reduced by injection of small inhibitor RNAs.

Results

Chronic cholestasis was induced by DLD and accelerated progression of cholangiocarcinoma, compared with mice given only DEN. Cystic hyperplasias, cystic atypical hyperplasias, cholangiomas, and cholangiocarcinoma developed in the DLD group at weeks 8, 12, 16, and 28, respectively. LMBDL repressed expression of microRNA (miR)-34a and let-7a, up-regulating Lin-28B, hypoxia-inducible factor (HIF)-1α, HIF-2α, and miR-210. Up-regulation of Lin-28B might inhibit let-7a, which is associated with development of cystic hyperplasias, cystic atypical hyperplasias, cholangiomas, and cholangiocarcinoma. Knockdown of c-Myc reduced progression of cholangiocarcinoma, whereas knockdown of Mnt accelerated its progression. Down-regulation of miR-34a expression might up-regulate c-Myc. The up-regulation of miR-210 via HIF-2α was involved in down-regulation of Mnt. Activation of the miR-34a–c-Myc and HIF-2α–miR-210–Mnt pathways caused c-Myc to bind the E-box element of cyclin D1, instead of Mnt, resulting in cyclin D1 up-regulation.

Conclusions

DLD induction of chronic cholestasis accelerated progression of cholangiocarcinoma, which is mediated by down-regulation of miR-34a, up-regulation miR-210, and replacement of Mnt by c-Myc in binding to cyclin D1.

Section snippets

Materials

α-³²P-dCTP and γ-³²P adenosine triphosphate was purchased from PerkinElmer (Boston, MA). All other reagents were of analytical grade and obtained from commercial sources.

Animal Surgery and Experimental Conditions

The mouse procedure protocols, use, and the care of the animals were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Southern California. A midline abdominal skin and muscle incision from 7-week-old male Balb/c mice was made to expose the xiphoid process for the LMBDL procedure (

LMBDL Had a Low Mortality Rate and Accelerated Liver Injury

The current BDL models either had massive liver damage but high mortality or a high survival rate but greatly reduced liver damage.5, 16 Mice with LMBDL surgery displayed reduced activity during the first week but regained normal activity after 2 weeks. Jaundice was seen after 3 days in all animals subjected to LMBDL. The LMBDL was considered a failure if the mice did not exhibit any symptoms of jaundice. We performed a successful LMBDL surgery in 175 of 188 Balb/c mice (93%), and, of those 175

Discussion

Chemically induced CCA progression often involves initiation by a carcinogen like DEN, carbon tetrachloride, furan, 3'-methyl-4-dimethylaminoazobenezene or dimethylnitrosamine (DMN), followed by a procedure to promote the initiated cell proliferation, such as partial hepatectomy or liver flukes. Earlier reports showed cholangiocarcinogenesis promotion by LHBDL in hamsters after initiation with DMN, which supports the link between cholestasis and CCA progression.¹⁹ Our model has several

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: Conflicts of interest The authors disclose no conflicts.

: Funding Supported by a pilot/feasibility grant from the USC Research Center for Liver Diseases (P30DK48522) and NIH grants (DK45334, DK51719 and AT1576) and by the Imaging Core of the USC Research Center for Liver Diseases (P30DK48522) for pathological sections and staining.

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Original ResearchBasic and Translational—BiliaryA Mouse Model of Cholestasis-Associated Cholangiocarcinoma and Transcription Factors Involved in Progression

Background & Aims

Methods

Results

Conclusions

Section snippets

Materials

Animal Surgery and Experimental Conditions

LMBDL Had a Low Mortality Rate and Accelerated Liver Injury

Discussion

Gastroenterology

Clin Gastroenterol Hepatol

Clin Gastroenterol Hepatol

J Hepatol

Gastroenterology

Does intrahepatic cholangiocarcinoma have better prognosis compared with perihilar cholangiocarcinoma?

J Surg Oncol

Switch from Mnt-Max to Myc-Max induces p53 and cyclin D1 expression and apoptosis during cholestasis in mouse and human hepatocytes

Hepatology

p53-Independent up-regulation of miR-34a during oncogene-induced senescence represses MYC

Cell Death Differ

Tumors line up for a letdown

Nat Genet

Roles of hypoxia-inducible factor-1α (HIF-1α) versus HIF-2α in the survival of hepatocellular tumor spheroids

Hepatology

Original Research
Basic and Translational—Biliary
A Mouse Model of Cholestasis-Associated Cholangiocarcinoma and Transcription Factors Involved in Progression