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Commentary Free access | 10.1172/JCI63966

miR-122 regulates hepatic lipid metabolism and tumor suppression

Jessica Wen and Joshua R. Friedman

Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

Address correspondence to: Joshua R. Friedman, Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, Perelman School of Medicine at the University of Pennsylvania, The Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., ARC 902G, Philadelphia, Pennsylvania 19104, USA. Phone: 267.426.7223; Fax: 206.984.2191; E-mail: friedmaj@mail.med.upenn.edu.

Find articles by Wen, J. in: JCI | PubMed | Google Scholar

Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

Address correspondence to: Joshua R. Friedman, Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, Perelman School of Medicine at the University of Pennsylvania, The Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., ARC 902G, Philadelphia, Pennsylvania 19104, USA. Phone: 267.426.7223; Fax: 206.984.2191; E-mail: friedmaj@mail.med.upenn.edu.

Find articles by Friedman, J. in: JCI | PubMed | Google Scholar

Published July 23, 2012 - More info

Published in Volume 122, Issue 8 on August 1, 2012
J Clin Invest. 2012;122(8):2773–2776. https://doi.org/10.1172/JCI63966.
© 2012 The American Society for Clinical Investigation
Published July 23, 2012 - Version history
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MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis
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Research Article

MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis

Abstract

MicroRNA-122 (miR-122), which accounts for 70% of the liver’s total miRNAs, plays a pivotal role in the liver. However, its intrinsic physiological roles remain largely undetermined. We demonstrated that mice lacking the gene encoding miR-122a (Mir122a) are viable but develop temporally controlled steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC). These mice exhibited a striking disparity in HCC incidence based on sex, with a male-to-female ratio of 3.9:1, which recapitulates the disease incidence in humans. Impaired expression of microsomal triglyceride transfer protein (MTTP) contributed to steatosis, which was reversed by in vivo restoration of Mttp expression. We found that hepatic fibrosis onset can be partially attributed to the action of a miR-122a target, the Klf6 transcript. In addition, Mir122a–/– livers exhibited disruptions in a range of pathways, many of which closely resemble the disruptions found in human HCC. Importantly, the reexpression of miR-122a reduced disease manifestation and tumor incidence in Mir122a–/– mice. This study demonstrates that mice with a targeted deletion of the Mir122a gene possess several key phenotypes of human liver diseases, which provides a rationale for the development of a unique therapy for the treatment of chronic liver disease and HCC.

Authors

Wei-Chih Tsai, Sheng-Da Hsu, Chu-Sui Hsu, Tsung-Ching Lai, Shu-Jen Chen, Roger Shen, Yi Huang, Hua-Chien Chen, Chien-Hsin Lee, Ting-Fen Tsai, Ming-Ta Hsu, Jaw-Ching Wu, Hsien-Da Huang, Ming-Shi Shiao, Michael Hsiao, Ann-Ping Tsou

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Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver
Shu-hao Hsu, … , Joshua T. Mendell, Kalpana Ghoshal
Shu-hao Hsu, … , Joshua T. Mendell, Kalpana Ghoshal
Research Article

Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver

Abstract

miR-122, an abundant liver-specific microRNA (miRNA), regulates cholesterol metabolism and promotes hepatitis C virus (HCV) replication. Reduced miR-122 expression in hepatocellular carcinoma (HCC) correlates with metastasis and poor prognosis. Nevertheless, the consequences of sustained loss of function of miR-122 in vivo have not been determined. Here, we demonstrate that deletion of mouse Mir122 resulted in hepatosteatosis, hepatitis, and the development of tumors resembling HCC. These pathologic manifestations were associated with hyperactivity of oncogenic pathways and hepatic infiltration of inflammatory cells that produce pro-tumorigenic cytokines, including IL-6 and TNF. Moreover, delivery of miR-122 to a MYC-driven mouse model of HCC strongly inhibited tumorigenesis, further supporting the tumor suppressor activity of this miRNA. These findings reveal critical functions for miR-122 in the maintenance of liver homeostasis and have important therapeutic implications, including the potential utility of miR-122 delivery for selected patients with HCC and the need for careful monitoring of patients receiving miR-122 inhibition therapy for HCV.

Authors

Shu-hao Hsu, Bo Wang, Janaiah Kota, Jianhua Yu, Stefan Costinean, Huban Kutay, Lianbo Yu, Shoumei Bai, Krista La Perle, Raghu R. Chivukula, Hsiaoyin Mao, Min Wei, K. Reed Clark, Jerry R. Mendell, Michael A. Caligiuri, Samson T. Jacob, Joshua T. Mendell, Kalpana Ghoshal

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Abstract

In this issue of JCI, two independent groups describe the effects of germline and liver-specific deletion of Mir122a, the predominant liver miRNA. Their findings reveal a critical role for miR-122 in fat and cholesterol metabolism but suggest that other metabolic actions of the liver are independent of miR-122. Knockout mice also displayed hepatic inflammation, fibrosis, and a high incidence of hepatocellular carcinoma, suggesting that miR-122 has a tumor suppressor role in hepatocytes.

MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate the expression of complementary messenger RNAs. Since their initial discovery in 1993 in Caenorhabditis elegans, more than 1,400 miRNAs have been detected in the human transcriptome. In addition to regulating physiologic processes, miRNAs have also been implicated in numerous disease states. The broad function of miRNA in the liver was investigated by studying mice with conditional deletion of Dicer1 in hepatocytes (1, 2). Despite the lack of mature miRNA in this model, the liver was able to perform the essential functions of blood glucose regulation, albumin production, and bilirubin metabolism. However, over time, it became clear that miRNA plays an important role in fat metabolism, inflammation, and cell cycle regulation in the liver, as these animals developed progressive hepatic steatosis, hepatitis, apoptosis, and hepatocellular carcinoma (HCC) (1, 2).

miR-122 is the predominant liver miRNA, making up 70% of the total miRNA population (3). The activity of miR-122 has previously been assessed through antisense oligonucleotide–mediated knockdown, implicating miR-122 in cholesterol and fat metabolism (4, 5). Although HCC was not observed in the time frame of these studies, several groups have reported tumor suppressor activity for miR-122, based on decreased miR-122 levels in tumor tissue and inhibitory effects of miR-122 in tumorigenicity assays (6). However, knockdown experiments are limited by their transient nature and the potential for off-target effects.

In this issue of JCI, Hsu et al. and Tsai et al. present definitive evidence of miR-122 function using genetic deletion in mice (7, 8). Mice with germline or conditional deletion of Mir122a in the liver were viable and fertile. However, as the animals aged, they developed steatohepatitis, liver fibrosis, and HCC. These groups define direct roles for miR-122 in both fat metabolism and tumor suppression, although it is less clear whether the link to fibrosis is directly or indirectly related to miR-122 loss. Thus, although miR-122 cannot be construed as a “master regulator” of liver function — as the mutant mice have generally normal liver function — it is a critical checkpoint both in hepatic fat production and hepatocellular proliferation (Figure 1).

Overview of the consequences of miR-122 loss on hepatocyte function.Figure 1

Overview of the consequences of miR-122 loss on hepatocyte function. The normal functions of the hepatocyte include carbohydrate and lipid metabolism, bilirubin excretion, and detoxification of endogenous compounds and xenobiotics. In this issue, Tsai et al. and Hsu et al. demonstrate that loss of miR-122 results in increased lipid synthesis and decreased lipid export, but other hepatocyte functions are unaltered. Loss of miR-122 also led to increased inflammation and fibrosis, and eventually the development of HCC, suggesting the miR-122 plays a tumor-suppressive role.

miR-122 regulates fat and cholesterol metabolism

Temporary miR-122 inhibition has been shown to reduce serum cholesterol via downregulation of genes involved in cholesterol biosynthesis such as HMG-CoA reductase (4). This is recapitulated in the genetic models: the serum lipid profiles of both liver-specific knockouts (LKO) and germline knockouts (KO) show a 30% reduction in total cholesterol, LDL, HDL, and serum triglyceride (TG). However, the livers of both KO and LKO mice also have progressive steatohepatitis (7, 8), a feature not seen in the previous knockdown studies (4, 5). The concordance of phenotypes in the KO and LKO mice is as expected, given the lack of miR-122 expression in non-hepatic tissues.

Hsu and colleagues identified the mechanism of steatosis in the mutants by performing gene expression profiling of Mir122a-LKO hepatocytes (7). They noted upregulation of genes known to be involved in lipid synthesis in the liver, including Agpat1, Mogat1, Agpat3, Agpat9, Ppap2a, Ppap2c, which are part of the TG biosynthesis pathway (9). Additionally, there was upregulation of Cidec, also known as FSP27, which is a protein that localizes to lipid droplets, negatively regulates lipolysis, and enhances TG accumulation. Consistent with the gene expression changes, the mutant livers synthesize more TG than controls but secrete less, resulting in TG accumulation in hepatocytes. The authors used reporter assays to confirm that Agpat1 and Cidec are direct miR-122 targets.

In their analysis of the fatty liver phenotype, Tsai and colleagues focused on MTTP (microsomal TG transfer protein) and found its expression level to be reduced in the Mir122a-KO mice (8). MTTP normally functions by enhancing the rate of lipid transfer between vesicles and is necessary for the assembly of lipoproteins (10). When Mttp expression was restored, serum cholesterol and fasting TG levels normalized. This surprising result suggests that either the lipid phenotype of the KO mice is primarily due to effects on Mttp expression or that overexpression of Mttp has a dominant effect on hepatic steatosis. The authors note that Mttp is not predicted to be a direct target of miR-122; this makes sense in light of the parallel loss of Mttp in mice lacking the repressive activity of miR-122. While Hsu and colleagues do not present any data regarding Mttp, it is noteworthy that they did detect a decrease in the expression of another microsomal lipid transporter, Stard4 (7).

Based on the these reports, miR-122 appears to play an important role in cholesterol and TG metabolism within the liver by directly or indirectly regulating cholesterol and TG biosynthesis and excretion. They make it clear that a decrease in circulating TG is not beneficial if the underlying reason is excessive retention of TG within hepatocytes. Likewise, the decrease in serum lipoproteins is of interest from the cardiovascular standpoint, although both the LDL and the HDL fractions were depressed; it will be important for future studies to determine the effect miR-122 loss in the context of atherosclerosis.

miR-122 loss results in hepatic inflammation and fibrosis

Mir122a-KO animals develop steatohepatitis and liver fibrosis (7, 8), phenotypes that were not observed in prior studies using antisense oligonucleotide–mediated Mir122a knockdown. Both groups also demonstrated an increase in infiltrating inflammatory cells in miR-122–deficient liver (7, 8). The infiltrating cells were CD11bhiGr1+, a population of immature myeloid cells with monocyte and granulocyte morphology. They produce high levels of IL-6 and TNF-α, and these cells are known to promote fibrosis in the injured liver (11).

Is inflammation directly regulated by miR-122, or is the phenotype an indirect result of chronic steatosis? The experiment employing exogenous restoration of Mttp expression implies the latter, because resolution of steatosis by Mttp also led to reduced inflammation and fibrosis. On the other hand, the authors used reporter assays to show that the chemokine gene Ccl2 is a direct miR-122 target gene in Hepa cells, albeit only to a mild degree (8).

The Mttp rescue experiment also implies that miR-122 regulates hepatic fibrosis indirectly. However, Klf6 was identified as a miR-122 target gene in reporter assays, and the suppression of Klf6 by shRNA led to a reduction in TGF-β1 expression and collagen deposition in the KO mice (8). Nevertheless, the reporter assays revealed a less than 2-fold effect of miR-122 on Ccl2 and Klf6 expression. Finally, the reduction in fibrosis observed following treatment with Klf6 shRNA may be completely independent of miR-122 function, because KLF6 is also expressed in activated hepatic stellate cells (which lack miR-122), where it regulates many fibrosis genes (12). Thus, the most likely sequence of events in these models is that steatosis is caused by direct effects of miR-122 deficiency, and that inflammation and fibrosis follow, with minor effects due to de-repression of Ccl2 and Klf6 in hepatocytes.

miR-122 is a hepatic tumor suppressor

miR-122 levels are reduced in experimental models and human samples of HCC, and loss of miR-122 is associated with tumor invasiveness and cancer progression (13–15). Therefore, it has been speculated that this miR acts as a tumor suppressor. In the models described by Tsai et al. and Hsu et al., HCC develops in both the KO and LKO mice. In the germline knockout, the incidence is 50% at 1 year of age. Both groups noted a striking sex disparity in the occurrence of HCC in miR-122–deficient mice, with a male to female ratio of approximately 4:1 in the LKO mice (7) and the KO mice (8). Hsu and colleagues did not observe a sex bias in the KO mice they analyzed, but in light of both studies, this may reflect random variation from male predominance. The males also have greater tumor burden and more advanced tumor grade than the females. Both of these features are similar to those observed in human HCC (16). In the mouse model, the sex disparity may be due in part to increased IL-6 levels in the males, illustrating the complex relationship among sex hormones, inflammation, and tumor suppressors/oncogenes in HCC.

To further understand the involvement of miR-122 in carcinogenesis, Hsu et al. made use of an HCC experimental model wherein transgenic mice harbor both a tetracycline-repressible MYC gene and a liver activator promoter–driven tet-transactivator protein, resulting in hepatic tumors in the absence of liver damage or inflammation. Interestingly, miR-122 levels are strongly reduced in this model. Remarkably, administration of a recombinant adeno-associated viral vector expressing MIR122 (after the establishment of small tumors) resulted in a reduction in tumor burden from 40% to 7.7% of liver mass. Thus, miR-122 appears to act as a tumor suppressor in a manner that is independent of its roles in fat metabolism or inflammation. It likely mediates this effect via a variety of target genes that are important in cell cycle regulation and hepatocyte differentiation, such as the stem cell genes Prom1, Thy1, and Epcam. To further support a direct tumor suppressor function for miR-122, it would be interesting to determine whether the restoration of Mttp also prevents HCC in the KO mice, a question not addressed by Tsai and colleagues (8). Finally, miR-122 loss may also promote epithelial-mesenchymal transition, as E-cadherin mRNA levels were reduced in the mutant livers even before the appearance of HCC.

Other functions of miR-122

A recent study has linked miR-122 to liver development through a positive feedback loop with the Onecut hepatocyte transcription factor gene family (17). Mouse embryos lacking both Onecut1 (Hnf6) and Onecut2 have reduced hepatic miR-122 levels, and Onecut1 can bind and activate the Mir122a locus. Transgenic mice overexpressing miR-122 in the developing liver have elevated Onecut1 levels and subtle defects in hepatocyte and cholangiocyte differentiation. However, loss-of-function analysis in this study was restricted to morpholino-based knockdown in zebrafish, with the analysis limited to early time points. In this respect, the Mir122a-KO mouse has the potential to confirm a developmental function for miR-122. Hsu et al. noted that liver histology was normal at birth and that Onecut1 expression was not altered in adult LKO or KO mice. Although not described in either of the articles, it will be revealing to analyze Onecut1 levels and liver cell differentiation at embryonic time points in the mutant mice; based on the Mir122a-transgenic mouse (17), there may be defects in liver cell differentiation or developmental timing.

miR-122 also has a compelling function in HCV replication. It is required for viral replication and interacts with HCV RNA, although it is not required for HCV RNA synthesis (18–21). Although miR-122 levels have not been directly correlated with viral load in liver samples from patients infected with HCV (22), in non-human primates, treatment with a miR-122 inhibitor results in a significant decrease in HCV viral load, and it is being investigated as a potential therapeutic for HCV (23). The two miR-122 studies in this issue of the JCI suggest that the use of this miR as anti-HCV therapy will need to be balanced with the potential for developing HCC, especially since patients with chronic HCV already have an increased risk for HCC. Furthermore, patients with hepatitis C have increased risk for cardiovascular disease and insulin resistance, so the effects of miR-122 inhibition on cardiovascular health will also need to be considered.

As a whole, the findings reported by Hsu et al. and Tsai et al. definitively describe the phenotype of miR-122 loss in the quiescent liver. miR-122 is a key regulator of multiple hepatic pathways, as highlighted by its direct roles in fat metabolism, tumor suppression, and perhaps inflammation and fibrosis. Despite this, liver development and hepatocyte differentiation occur normally in mice lacking miR-122. miR-122–deficient livers also continue to perform bilirubin, protein, glucose, and xenobiotic metabolism. In future studies, it will be intriguing to see whether these other functions are dependent on miR-122 in the context of stress, such as a high-fat diet or hepatotoxins. Conversely, while loss of miR-122 causes steatohepatitis, it is unknown whether exogenous miR-122 can ameliorate fatty liver disease causes by diet or toxins. Finally, the tumor-suppressive function of miR-122 illustrated by both groups suggests that the restoration of miR-122 expression may be of benefit in HCC — but as with many other tumor suppressor miRNAs, delivery of the miRNA remains a major challenge.

Acknowledgments

The authors wish to thank the Fred and Suzanne Biesecker Pediatric Liver Center for support of this work.

Address correspondence to: Joshua R. Friedman, Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, Perelman School of Medicine at the University of Pennsylvania, The Children’s Hospital of Philadelphia, 3615 Civic Center Blvd., ARC 902G, Philadelphia, Pennsylvania 19104, USA. Phone: 267.426.7223; Fax: 206.984.2191; E-mail: friedmaj@mail.med.upenn.edu.

Footnotes

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2012;122(8):2773–2776. doi:10.1172/JCI63966.

See the related articles at Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver and MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis.

References
  1. Hand NJ, Master ZR, Le Lay J, Friedman JR. Hepatic function is preserved in the absence of mature microRNAs. Hepatology. 2009;49(2):618–626.
    View this article via: PubMed CrossRef Google Scholar
  2. Sekine S, et al. Disruption of Dicer1 induces dysregulated fetal gene expression and promotes hepatocarcinogenesis. Gastroenterology. 2009;136(7):2304–2315.e4.
    View this article via: PubMed CrossRef Google Scholar
  3. Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T. Identification of tissue-specific microRNAs from mouse. Curr Biol. 2002;12(9):735–739.
    View this article via: PubMed CrossRef Google Scholar
  4. Esau C, et al. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab. 2006;3(2):87–98.
    View this article via: PubMed CrossRef Google Scholar
  5. Krutzfeldt J, et al. Silencing of microRNAs in vivo with ‘antagomirs’. Nature. 2005;438(7068):685–689.
    View this article via: PubMed CrossRef Google Scholar
  6. Saito Y, et al. MicroRNAs in hepatobiliary and pancreatic cancers. Front Genet. 2011;2:66.
    View this article via: PubMed Google Scholar
  7. Hsu S-H, et al. Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver. J Clin Invest. 2012;122(8):2871–2883.
    View this article via: JCI Google Scholar
  8. Tsai W-C, et al. MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis. J Clin Invest. 2012;122(8):2884–2897.
    View this article via: JCI Google Scholar
  9. Kim YJ, Cho SY, Yun CH, Moon YS, Lee TR, Kim SH. Transcriptional activation of Cidec by PPARgamma2 in adipocyte. Biochem Biophys Res Commun. 2008;377(1):297–302.
    View this article via: PubMed CrossRef Google Scholar
  10. Hussain MM, Shi J, Dreizen P. Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly. J Lipid Res. 2003;44(1):22–32.
    View this article via: PubMed CrossRef Google Scholar
  11. Karlmark KR, et al. Hepatic recruitment of the inflammatory Gr1+ monocyte subset upon liver injury promotes hepatic fibrosis. Hepatology. 2009;50(1):261–274.
    View this article via: PubMed CrossRef Google Scholar
  12. Miele L, et al. The Kruppel-like factor 6 genotype is associated with fibrosis in nonalcoholic fatty liver disease. Gastroenterology. 2008;135(1):282–291.e1.
    View this article via: PubMed CrossRef Google Scholar
  13. Wu X, et al. miR-122 affects the viability and apoptosis of hepatocellular carcinoma cells. Scand J Gastroenterol. 2009;44(11):1332–1339.
    View this article via: PubMed CrossRef Google Scholar
  14. Coulouarn C, Factor VM, Andersen JB, Durkin ME, Thorgeirsson SS. Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties. Oncogene. 2009;28(40):3526–3536.
    View this article via: PubMed CrossRef Google Scholar
  15. Bai S, et al. MicroRNA-122 inhibits tumorigenic properties of hepatocellular carcinoma cells and sensitizes these cells to sorafenib. J Biol Chem. 2009;284(46):32015–32027.
    View this article via: PubMed CrossRef Google Scholar
  16. McGlynn KA, London WT. Epidemiology and natural history of hepatocellular carcinoma. Best Pract Res Clin Gastroenterol. 2005;19(1):3–23.
    View this article via: PubMed CrossRef Google Scholar
  17. Laudadio I, et al. A feedback loop between the liver-enriched transcription factor network and miR-122 controls hepatocyte differentiation. Gastroenterology. 2012;142(1):119–129.
    View this article via: PubMed CrossRef Google Scholar
  18. Machlin ES, Sarnow P, Sagan SM. Masking the 5′ terminal nucleotides of the hepatitis C virus genome by an unconventional microRNA-target RNA complex. Proc Natl Acad Sci U S A. 2011;108(8):3193–3198.
    View this article via: PubMed CrossRef Google Scholar
  19. Nunnari G, Schnell MJ. MicroRNA-122: a therapeutic target for hepatitis C virus (HCV) infection. Front Biosci (Schol Ed). 2011;3:1032–1037.
    View this article via: PubMed Google Scholar
  20. Jopling CL. Regulation of hepatitis C virus by microRNA-122. Biochem Soc Trans. 2008;36(Pt 6):1220–1223.
    View this article via: PubMed CrossRef Google Scholar
  21. Shimakami T, et al. Stabilization of hepatitis C virus RNA by an Ago2-miR-122 complex. Proc Natl Acad Sci U S A. 2012;109(3):941–946.
    View this article via: PubMed CrossRef Google Scholar
  22. Sarasin-Filipowicz M, Krol J, Markiewicz I, Heim MH, Filipowicz W. Decreased levels of micro­RNA miR-122 in individuals with hepatitis C responding poorly to interferon therapy. Nat Med. 2009;15(1):31–33.
    View this article via: PubMed CrossRef Google Scholar
  23. Lanford RE, et al. Therapeutic silencing of micro­RNA-122 in primates with chronic hepatitis C virus infection. Science. 2010;327(5962):198–201.
    View this article via: PubMed CrossRef Google Scholar
Version history
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