Germline Genetic Associations for Hepatobiliary Cancers.

Hepatobiliary cancers (HBCs) include hepatocellular carcinoma, cholangiocarcinoma, and gallbladder carcinoma, which originate from the liver, bile ducts, and gallbladder, respectively. They are responsible for a substantial burden of cancer-related deaths worldwide. Despite knowledge of risk factors and advancements in therapeutics and surgical interventions, the prognosis for most patients with HBC remains bleak. There is evidence from familial aggregation and case-control studies to suggest a familial risk component in HBC susceptibility. Recent progress in genomics research has led to the identification of germline variants including single nucleotide polymorphisms (SNPs) and pathogenic or likely pathogenic (P/LP) variants in cancer-associated genes associated with HBC risk. These findings emerged from genome-wide association studies and next-generation sequencing techniques such as whole-exome sequencing. Patients with other cancer types, including breast, colon, ovarian, prostate, and pancreatic cancer, are recommended by guidelines to undergo germline genetic testing, but similar recommendations are lagging in HBC. This prompts the question of whether multi-gene panel testing should be integrated into clinical guidelines for HBC management. Here, we review the hereditary genetics of HBC, explore studies investigating SNPs and P/LP variants in HBC patients, discuss the clinical implications and potential for personalized treatments and impact on patient's family members, and conclude that additional studies are needed to examine how genetic testing can be applied clinically.

Emerging and potential use of CRISPR in human liver disease.

CRISPR is a gene editing tool adapted from naturally occurring defense systems from bacteria. It is a technology that is revolutionizing the interrogation of gene functions in driving liver disease, especially through genetic screens and by facilitating animal knockout and knockin models. It is being used in models of liver disease to identify which genes are critical for liver pathology, especially in genetic liver disease, hepatitis, and in cancer initiation and progression. It holds tremendous promise in treating human diseases directly by editing DNA. It could disable gene function in the case of expression of a maladaptive protein, such as blocking transthyretin as a therapy for amyloidosis, or to correct gene defects, such as restoring the normal functions of liver enzymes fumarylacetoacetate hydrolase or alpha-1 antitrypsin. It is also being studied for treatment of hepatitis B infection. CRISPR is an exciting, evolving technology that is facilitating gene characterization and discovery in liver disease and holds the potential to treat liver diseases safely and permanently.

Association of Statin Use With Risk of Liver Disease, Hepatocellular Carcinoma, and Liver-Related Mortality.

Importance: Given the burden of chronic liver disease on the health care system, more information on the hepatoprotective association of statins in the general population is needed. Objective: To examine whether regular statin use is associated with a reduction in liver disease, particularly hepatocellular carcinoma (HCC) and liver-related deaths, in the general population. Design, Setting, and Participants: This cohort study used data from the UK Biobank (UKB) (individuals aged 37-73 years) collected from baseline (2006-2010) to the end of follow-up in May 2021, from the TriNetX cohort (individuals aged 18-90 years) enrolled from baseline (2011-2020) until end of follow-up in September 2022, and from the Penn Medicine Biobank (PMBB) (individuals aged 18-102 years) with ongoing enrollment starting in 2013 to the end of follow-up in December 2020. Individuals were matched using propensity score matching according to the following criteria: age, sex, body mass index, ethnicity, diabetes with or without insulin or biguanide use, hypertension, ischemic heart disease, dyslipidemia, aspirin use, and number of medications taken (UKB only). Data analysis was performed from April 2021 to April 2023. Exposure: Regular statin use. Main Outcomes and Measures: Primary outcomes were liver disease and HCC development as well as liver-associated death. Results: A total of 1 785 491 individuals were evaluated after matching (aged 55 to 61 years on average, up to 56% men, and up to 49% women). A total of 581 cases of liver-associated death, 472 cases of incident HCC, and 98 497 new liver diseases were registered during the follow-up period. Individuals were aged 55-61 years on average, with a slightly higher proportion of men (up to 56%). In UKB individuals (n = 205 057) without previously diagnosed liver disease, statin users (n = 56 109) had a 15% lower hazard ratio (HR) for the association of developing a new liver disease (HR, 0.85; 95% CI, 0.78-0.92; P < .001). In addition, statin users demonstrated a 28% lower HR for the association with liver-related death (HR, 0.72; 95% CI, 0.59-0.88; P = .001) and a 42% lower HR for the development of HCC (HR, 0.58; 95% CI, 0.35-0.96; P = .04). In TriNetX individuals (n = 1 568 794), the HR for the association of HCC was reduced even further for statin users (HR, 0.26; 95% CI, 0.22-0.31; P = .003). The hepatoprotective association of statins was time and dose dependent, with a significant association in PMBB individuals (n = 11 640) for incident liver diseases after 1 year of statin use (HR, 0.76; 95% CI, 0.59-0.98; P = .03). Taking statins was particularly beneficial in men, individuals with diabetes, and individuals with a high Fibrosis-4 index at baseline. Carriers of the heterozygous minor allele of PNPLA3 rs738409 benefited from statin use and had a 69% lower HR for the association with HCC (UKB HR, 0.31; 95% CI, 0.11-0.85; P = .02). Conclusions and Relevance: This cohort study indicates substantial preventive associations of statins against liver disease, with an association with duration and dose of intake.

Comparing telemedicine and in-person gastrointestinal cancer genetic appointment outcomes during the COVID-19 pandemic.

BACKGROUND: The study purpose is to compare outcomes associated with completion of genetic testing between telemedicine and in-person gastrointestinal cancer risk assessment appointments during the COVID-19 pandemic. METHODS: Data was collected on patients with scheduled appointments between July 2020 and June 2021 in a gastrointestinal cancer risk evaluation program (GI-CREP) that utilized both telemedicine and in-person visits throughout the COVID-19 pandemic, and a survey was administered. RESULTS: A total of 293 patients had a GI-CREP appointment scheduled and completion rates of in-person versus telemedicine appointments were similar. Individuals diagnosed with cancer and those with Medicaid insurance had lower rates of appointment completion. Although telehealth was the preferred visit modality, there were no differences in recommending genetic testing nor in the consent rate for genetic testing between in-person and telemedicine visits. However, of patients who consented for genetic testing, more than three times more patients seen via telemedicine did not complete genetic testing compared to those seen in-person (18.3% versus 5.2%, p = 0.008). Furthermore, telemedicine visits had a longer turnaround time for genetic test reporting (32 days versus 13 days, p < 0.001). CONCLUSIONS: Compared to in-person GI-CREP appointments, telemedicine was associated with lower rates of genetic testing completion, and longer turnaround time for results.

Lipid Nanoparticle Delivery of Small Proteins for Potent In Vivo RAS Inhibition.

Mutated RAS proteins are potent oncogenic drivers and have long been considered "undruggable". While RAS-targeting therapies have recently shown promise, there remains a clinical need for RAS inhibitors with more diverse targets. Small proteins represent a potential new therapeutic option, including K27, a designed ankyrin repeat protein (DARPin) engineered to inhibit RAS. However, K27 functions intracellularly and is incapable of entering the cytosol on its own, currently limiting its utility. To overcome this barrier, we have engineered a lipid nanoparticle (LNP) platform for potent delivery of functional K27-D30─a charge-modified version of the protein─intracellularly in vitro and in vivo. This system efficiently encapsulates charge-modified proteins, facilitates delivery in up to 90% of cells in vitro, and maintains potency after at least 45 days of storage. In vivo, these LNPs deliver K27-D30 to the cytosol of cancerous cells in the liver, inhibiting RAS-driven growth and ultimately reducing tumor load in an HTVI-induced mouse model of hepatocellular carcinoma. This work shows that K27 holds promise as a new cancer therapeutic when delivered using this LNP platform. Furthermore, this technology has the potential to broaden the use of LNPs to include new cargo types─beyond RNA─for diverse therapeutic applications.

A Therapeutically Targetable TAZ-TEAD2 Pathway Drives the Growth of Hepatocellular Carcinoma via ANLN and KIF23.

BACKGROUND & AIMS: Despite recent progress, long-term survival remains low for hepatocellular carcinoma (HCC). The most effective HCC therapies target the tumor immune microenvironment (TIME), and there are almost no therapies that directly target tumor cells. Here, we investigated the regulation and function of tumor cell-expressed Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in HCC. METHODS: HCC was induced in mice by Sleeping Beauty-mediated expression of MET, CTNNB1-S45Y, or TAZ-S89A, or by diethylnitrosamine plus CCl4. Hepatocellular TAZ and YAP were deleted in floxed mice via adeno-associated virus serotype 8-mediated expression of Cre. TAZ target genes were identified from RNA sequencing, confirmed by chromatin immunoprecipitation, and evaluated in a clustered regularly interspaced short palindromic repeats interference (CRISPRi) screen. TEA domain transcription factors (TEADs), anillin (ANLN), Kif23, and programmed cell death protein ligand 1 were knocked down by guide RNAs in dead clustered regularly interspaced short palindromic repeats-associated protein 9 (dCas9) knock-in mice. RESULTS: YAP and TAZ were up-regulated in murine and human HCC, but only deletion of TAZ consistently decreased HCC growth and mortality. Conversely, overexpression of activated TAZ was sufficient to trigger HCC. TAZ expression in HCC was regulated by cholesterol synthesis, as demonstrated by pharmacologic or genetic inhibition of 3-hydroxy-3-methylglutaryl- coenzyme A reductase (HMGCR), farnesyl pyrophosphate synthase, farnesyl-diphosphate farnesyltransferase 1 (FDFT1), or sterol regulatory element-binding protein 2 (SREBP2). TAZ- and MET/CTNNB1-S45Y-driven HCC required the expression of TEAD2 and, to a lesser extent, TEAD4. Accordingly, TEAD2 displayed the most profound effect on survival in patients with HCC. TAZ and TEAD2 promoted HCC via increased tumor cell proliferation, mediated by TAZ target genes ANLN and kinesin family member 23 (KIF23). Therapeutic targeting of HCC, using pan-TEAD inhibitors or the combination of a statin with sorafenib or anti-programmed cell death protein 1, decreased tumor growth. CONCLUSIONS: Our results suggest the cholesterol-TAZ-TEAD2-ANLN/KIF23 pathway as a mediator of HCC proliferation and tumor cell-intrinsic therapeutic target that could be synergistically combined with TIME-targeted therapies.

MYC and MET cooperatively drive hepatocellular carcinoma with distinct molecular traits and vulnerabilities.

Enhanced activation of the transcription factor MYC and of the receptor tyrosine kinase MET are among the events frequently occurring in hepatocellular carcinoma (HCC). Both genes individually act as drivers of liver cancer initiation and progression. However, their concomitant alteration in HCC has not been explored, nor functionally documented. Here, we analysed databases of five independent human HCC cohorts and found a subset of patients with high levels of MYC and MET (MYChigh/METhigh) characterised by poor prognosis. This clinical observation drove us to explore the functionality of MYC and MET co-occurrence in vivo, combining hydrodynamic tail vein injection for MYC expression in the R26stopMet genetic setting, in which wild-type MET levels are enhanced following the genetic deletion of a stop cassette. Results showed that increased MYC and MET expression in hepatocytes is sufficient to induce liver tumorigenesis even in the absence of pre-existing injuries associated with a chronic disease state. Intriguingly, ectopic MYC in MET tumours increases expression of the Mki67 proliferation marker, and switches them into loss of Afp, Spp1, Gpc3, Epcam accompanied by an increase in Hgma1, Vim, and Hep-Par1 levels. We additionally found a switch in the expression of specific immune checkpoints, with an increase in the Ctla-4 and Lag3 lymphocyte co-inhibitory responses, and in the Icosl co-stimulatory responses of tumour cells. We provide in vitro evidence on the vulnerability of some human HCC cell lines to combined MYC and MET targeting, which are otherwise resistant to single inhibition. Mechanistically, combined blockage of MYC and MET converts a partial cytostatic effect, triggered by individual blockage of MYC or MET, into a cytotoxic effect. Together, these findings highlight a subgroup of HCC characterised by MYChigh/METhigh, and document functional cooperativity between MYC and MET in liver tumorigenesis. Thus, the MYC-R26Met model is a relevant setting for HCC biology, patient classification and treatment.

Cellular origins of regenerating liver and hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the predominant primary cancer arising from the liver and is one of the major causes of cancer-related mortality worldwide. The cellular origin of HCC has been a topic of great interest due to conflicting findings regarding whether it originates in hepatocytes, biliary cells, or facultative stem cells. These cell types all undergo changes during liver injury, and there is controversy about their contribution to regenerative responses in the liver. Most HCCs emerge in the setting of chronic liver injury from viral hepatitis, fatty liver disease, alcohol, and environmental exposures. The injuries are marked by liver parenchymal changes such as hepatocyte regenerative nodules, biliary duct cellular changes, expansion of myofibroblasts that cause fibrosis and cirrhosis, and inflammatory cell infiltration, all of which may contribute to carcinogenesis. Addressing the cellular origin of HCC is the key to identifying the earliest events that trigger it. Herein, we review data on the cells of origin in regenerating liver and HCC and the implications of these findings for prevention and treatment. We also review the origins of childhood liver cancer and other rare cancers of the liver.

Uptake and outcomes of small intestinal and urinary tract cancer surveillance in Lynch syndrome.

BACKGROUND: Lynch syndrome (LS) is a hereditary cancer predisposition syndrome associated with increased risk of multiple cancers. While colorectal cancer surveillance decreases mortality in LS and is recommended by guidelines, there is lack of evidence for the efficacy of surveillance for extra-colonic cancers associated with LS, including small intestinal cancer (SIC) and urinary tract cancer (UTC). Given the limited evidence, guidelines do not consistently recommend surveillance for SIC and UTC, and it remains unclear how often individuals will choose to undergo and follow through with extra-colonic surveillance recommendations. AIM: To study factors associated with SIC and UTC surveillance uptake and outcomes in LS. METHODS: This is an IRB-approved retrospective analysis of individuals with LS seen at a tertiary care referral center. Included individuals had a pathogenic or likely pathogenic variant in MLH1, MSH2, MSH6, PMS2, or EPCAM, or were a confirmed obligate carrier, and had at least one documented visit to our center. Information regarding SIC and UTC surveillance was captured for each individual, and detailed personal and family history was obtained for individuals who had an initial LS management visit in our center's dedicated high-risk LS clinic between January 1, 2017 and October 29, 2020. During these initial management visits, all patients had in-depth discussions of SIC and UTC surveillance with 1 of 3 providers experienced in LS management to promote informed decision-making about whether to pursue SIC and/or UTC surveillance. Statistical analysis using Pearson's chi-squared test and Wilcoxon rank-sum test was completed to understand the factors associated with pursuit and completion of SIC and UTC surveillance, and a P value below 0.05 was deemed statistically significant. RESULTS: Of 317 individuals with LS, 86 (27%) underwent a total of 105 SIC surveillance examinations, with 5 leading to additional work-up and no SICs diagnosed. Additionally, 99 (31%) patients underwent a total of 303 UTC surveillance examinations, with 19 requiring further evaluation and 1 UTC identified. Of 155 individuals who had an initial LS management visit between January 1, 2017 and October 29, 2020, 63 (41%) chose to undergo SIC surveillance and 58 (37%) chose to undergo UTC surveillance. However, only 26 (41%) and 32 (55%) of those who initially chose to undergo SIC or UTC surveillance, respectively, successfully completed their surveillance examinations. Individuals with a pathogenic variant in MSH2 or EPCAM were more likely to initially choose to undergo SIC surveillance (P = 0.034), and older individuals were more likely to complete SIC surveillance (P = 0.007). Choosing to pursue UTC surveillance was more frequent among older individuals (P = 0.018), and females more frequently completed UTC surveillance (P = 0.002). Personal history of cancer and family history of SIC or UTC were not significantly associated with electing nor completing surveillance. Lastly, the provider discussing SIC/UTC surveillance was significantly associated with subsequent surveillance choices. CONCLUSION: Pursuing and completing SIC/UTC surveillance in LS is influenced by several factors, however broad incorporation in LS management is likely unhelpful due to low yield and frequent false positive results.

EUS-based Pancreatic Cancer Surveillance in BRCA1/BRCA2/PALB2/ATM Carriers Without a Family History of Pancreatic Cancer.

Carriers of a pathogenic/likely pathogenic (P/LP) BRCA1/BRCA2/ATM/PALB2 variant are at increased risk of pancreatic ductal adenocarcinoma (PDAC), yet current guidelines recommend surveillance only for those with a family history of PDAC. We aimed to investigate outcomes of endoscopic ultrasound (EUS)-based PDAC surveillance in BRCA1/BRCA2/ATM/PALB2 carriers without a family history of PDAC. We performed a retrospective analysis of all P/LP BRCA1/BRCA2/ATM/PALB2 carriers who underwent EUS at a tertiary care center. Of 194 P/LP BRCA1/BRCA2/ATM/PALB2 carriers who underwent EUS, 64 (33%) had no family history of PDAC and had at least 1 EUS for PDAC surveillance. These individuals underwent 143 total EUSs, were predominantly female (72%), and BRCA2 carriers (73%), with the majority having a personal history of cancer other than PDAC (67%). The median age at time of first EUS was 62 years [interquartile range (IQR), 53-67 years] and a median of 2 EUSs (IQR 1-3) were performed per patient, with a median of 3 years (IQR 2-4.5 years) between the first and last EUS for those with more than 1 EUS. Pancreatic abnormalities were detected in 44%, including cysts in 27%, and incidental luminal abnormalities in 41%. Eight percent developed a new pancreatic mass or cyst during surveillance, 2 individuals developed PDAC, and no serious complications resulted from surveillance. After discussion of the risks, limitations, and potential benefits, PDAC surveillance can be considered in BRCA1/BRCA2/ATM/PALB2 carriers without a family history of PDAC; however, the effectiveness of PDAC surveillance in this population requires further study. PREVENTION RELEVANCE: BRCA1/BRCA2/ATM/PALB2 carriers have increased pancreatic ductal adenocarcinoma (PDAC) risk, yet are typically not eligible for PDAC surveillance in the absence of PDAC family history. Herein we describe outcomes of PDAC surveillance in BRCA1/BRCA2/ATM/PALB2 carriers without a family history of PDAC, showing that PDAC surveillance can be considered in this high-risk group.

Multigene Panel Testing in Individuals With Hepatocellular Carcinoma Identifies Pathogenic Germline Variants.

Hepatocellular carcinoma (HCC) has well-defined environmental risk factors. In addition, epidemiologic studies have suggested hereditary risk factors. The goals of this study were to determine the rate of pathogenic and likely pathogenic (P/LP) germline variants in cancer predisposition genes in patients with HCC, possible enrichment of P/LP variants in particular genes, and potential impact on clinical management. MATERIALS AND METHODS: A prospective study at a tertiary medical center enrolled 217 patients with a personal history of HCC. Multigene panel testing was performed for 134 cancer predisposition genes in all patients. The rate of P/LP variants was compared with population rates. A separate retrospective cohort included 219 patients with HCC who underwent testing at a commercial laboratory. RESULTS: In the prospective cohort, P/LP germline variants were identified in 25 of 217 patients with HCC (11.5%). Four patients (1.8%) had P/LP variants in the highly penetrant cancer genes BRCA2 (n = 2), MSH6 (n = 1), and PMS2 (n = 1). In addition, multiple patients had P/LP variants in FANCA (n = 5) and BRIP1 (n = 4), which were significantly enriched in HCC compared with the general population. Detection of P/LP variants led to changes in clinical management in regard to therapy selection, screening recommendations, and cascade testing of relatives. In a separate retrospective analysis of 219 patients with HCC, 30 (13.7%) were positive for P/LP variants including 13 (5.9%) with highly penetrant genes APC (n = 2), BRCA1 (n = 1), BRCA2 (n = 6), MSH2 (n = 2), or TP53 (n = 2). CONCLUSION: P/LP germline variants in cancer predisposition genes were detected in 11%-14% of patients with HCC. Inherited genetics should not be overlooked in HCC as there are important implications for precision treatment, future risk of cancers, and familial cancer risk.

Aspirin in Hepatocellular Carcinoma.

Preclinical and clinical studies provide evidence for aspirin as a preventative agent for cancer. Compelling direct evidence supports a chemopreventive effect of aspirin in individuals at high risk of developing colorectal cancer due to Lynch syndrome, while indirect evidence indicates that aspirin may reduce the risk of and mortality from sporadic colorectal cancer. There is weaker evidence for a protective effect of aspirin against all cancers taken as a group. Nevertheless, the results of recent retrospective cohort studies consistently indicate a beneficial effect of aspirin as a chemopreventive or adjuvant chemotherapeutic agent in hepatocellular carcinoma (HCC). Epidemiologic studies conducted in the general population or in selected populations at higher risk for HCC reveal that regular aspirin use is associated with reduced HCC incidence. In addition, aspirin may act as an adjuvant to other therapies in reducing HCC recurrence. According to studies in animal models, the cancer-preventative effect of aspirin may be related to its antiplatelet and anti-inflammatory activities. Prospective studies are warranted to determine whether aspirin should be recommended to diverse populations of patients at risk for HCC.

Upper Endoscopic Surveillance in Lynch Syndrome Detects Gastric and Duodenal Adenocarcinomas.

Lynch syndrome is a prevalent hereditary cancer predisposition syndrome. While colorectal cancer is the most common gastrointestinal (GI) cancer in Lynch syndrome, there is also increased risk of gastric and small intestinal cancers. Recommendations for upper GI cancer surveillance in Lynch syndrome vary widely with limited data supporting effectiveness. Herein, we collected data on individuals with a diagnosis of Lynch syndrome seen at our tertiary care referral center. We identified individuals who underwent upper endoscopy and those with upper GI cancers, and associated demographics, genetic testing results, and endoscopic information. Standard statistical analyses were performed. Among 295 individuals with Lynch syndrome seen at our center, 217 (73.6%) underwent 660 total upper endoscopies. Of these 217, precancerous upper endoscopy findings included Barrett's esophagus (7, 3.2%), gastric intestinal metaplasia (18, 8.3%), and duodenal adenomas (4, 1.8%), and Helicobacter pylori was identified in 6 (2.8%). Upper GI cancers were diagnosed in 11 individuals (3.7%), including esophageal in 1, gastric in 6, and duodenal in 4. Five (1.7%) of these upper GI cancers were identified on surveillance. Individuals with upper GI cancers identified on surveillance were older at first surveillance endoscopy, with median age 63.3 versus 44.9 years (P < 0.001). Of the upper GI cancers detected on surveillance, 80% (4/5) occurred within 2 years of last upper endoscopy and 80% were stage I. In conclusion, upper endoscopy surveillance in Lynch syndrome identifies upper GI cancers. For individuals with Lynch syndrome who undergo upper GI surveillance, a short surveillance interval may be warranted.

The Dynamic Chromatin Architecture of the Regenerating Liver.

BACKGROUND & AIMS: The adult liver is the main detoxification organ and routinely is exposed to environmental insults but retains the ability to restore its mass and function upon tissue damage. However, extensive injury can lead to liver failure, and chronic injury causes fibrosis, cirrhosis, and hepatocellular carcinoma. Currently, the transcriptional regulation of organ repair in the adult liver is incompletely understood. METHODS: We isolated nuclei from quiescent as well as repopulating hepatocytes in a mouse model of hereditary tyrosinemia, which recapitulates the injury and repopulation seen in toxic liver injury in human beings. We then performed the assay for transposase accessible chromatin with high-throughput sequencing specifically in repopulating hepatocytes to identify differentially accessible chromatin regions and nucleosome positioning. In addition, we used motif analysis to predict differential transcription factor occupancy and validated the in silico results with chromatin immunoprecipitation followed by sequencing for hepatocyte nuclear factor 4α (HNF4α) and CCCTC-binding factor (CTCF). RESULTS: Chromatin accessibility in repopulating hepatocytes was increased in the regulatory regions of genes promoting proliferation and decreased in the regulatory regions of genes involved in metabolism. The epigenetic changes at promoters and liver enhancers correspond with the regulation of gene expression, with enhancers of many liver function genes showing a less accessible state during the regenerative process. Moreover, increased CTCF occupancy at promoters and decreased HNF4α binding at enhancers implicate these factors as key drivers of the transcriptomic changes in replicating hepatocytes that enable liver repopulation. CONCLUSIONS: Our analysis of hepatocyte-specific epigenomic changes during liver repopulation identified CTCF and HNF4α as key regulators of hepatocyte proliferation and regulation of metabolic programs. Thus, liver repopulation in the setting of toxic injury makes use of both general transcription factors (CTCF) for promoter activation, and reduced binding by a hepatocyte-enriched factor (HNF4α) to temporarily limit enhancer activity. All sequencing data in this study were deposited to the Gene Expression Omnibus database and can be downloaded with accession number GSE109466.

A High-Content Screen Identifies MicroRNAs That Regulate Liver Repopulation After Injury in Mice.

BACKGROUND & AIMS: Liver regeneration is impaired in mice with hepatocyte-specific deficiencies in microRNA (miRNA) processing, but it is not clear which miRNAs regulate this process. We developed a high-throughput screen to identify miRNAs that regulate hepatocyte repopulation after toxic liver injury using fumarylacetoacetate hydrolase-deficient mice. METHODS: We constructed plasmid pools encoding more than 30,000 tough decoy miRNA inhibitors (hairpin nucleic acids designed to specifically inhibit interactions between miRNAs and their targets) to target hepatocyte miRNAs in a pairwise manner. The plasmid libraries were delivered to hepatocytes in fumarylacetoacetate hydrolase-deficient mice at the time of liver injury via hydrodynamic tail-vein injection. Integrated transgene-containing transposons were quantified after liver repopulation via high-throughput sequencing. Changes in polysome-bound transcripts after miRNA inhibition were determined using translating ribosome affinity purification followed by high-throughput sequencing. RESULTS: Analyses of tough decoy abundance in hepatocyte genomic DNA and input plasmid pools identified several thousand miRNA inhibitors that were significantly depleted or increased after repopulation. We classified a subset of miRNA binding sites as those that have strong effects on liver repopulation, implicating the targeted hepatocyte miRNAs as regulators of this process. We then generated a high-content map of pairwise interactions between 171 miRNA-binding sites and identified synergistic and redundant effects. CONCLUSIONS: We developed a screen to identify miRNAs that regulate liver repopulation after injury in live mice.