microRNA-27b regulates hepatic lipase enzyme LIPC and reduces triglyceride degradation during hepatitis C virus infection
2022; Elsevier BV; Volume: 298; Issue: 6 Linguagem: Inglês
10.1016/j.jbc.2022.101983
ISSN1083-351X
AutoresGeneviève F. Desrochers, Roxana Filip, Micheal Bastianelli, Tiffany Stern, John Paul Pezacki,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumomiRNAs are short, noncoding RNAs that negatively and specifically regulate protein expression, the cumulative effects of which can result in broad changes to cell systems and architecture. The miRNA miR-27b is known to regulate lipid regulatory pathways in the human liver and is also induced by the hepatitis C virus (HCV). However, the functional targets of miR-27b are not well established. Herein, an activity-based protein profiling method using a serine hydrolase probe, coupled with stable isotope labeling and mass spectrometry identified direct and indirect targets of miR-27b. The hepatic lipase C (LIPC) stood out as both highly dependent on miR-27b and as a major modulator of lipid pathway misregulation. Modulation of miR-27b using both exogenous miRNA mimics and inhibitors demonstrated that transcription factors Jun, PPARα, and HNF4α, all of which also influence LIPC levels and activity, are regulated by miR-27b. LIPC was furthermore shown to affect the progress of the life cycle of HCV and to decrease levels of intracellular triglycerides, upon which HCV is known to depend. In summary, this work has demonstrated that miR-27b mediates HCV infection by downregulating LIPC, thereby reducing triglyceride degradation, which in turn increases cellular lipid levels. miRNAs are short, noncoding RNAs that negatively and specifically regulate protein expression, the cumulative effects of which can result in broad changes to cell systems and architecture. The miRNA miR-27b is known to regulate lipid regulatory pathways in the human liver and is also induced by the hepatitis C virus (HCV). However, the functional targets of miR-27b are not well established. Herein, an activity-based protein profiling method using a serine hydrolase probe, coupled with stable isotope labeling and mass spectrometry identified direct and indirect targets of miR-27b. The hepatic lipase C (LIPC) stood out as both highly dependent on miR-27b and as a major modulator of lipid pathway misregulation. Modulation of miR-27b using both exogenous miRNA mimics and inhibitors demonstrated that transcription factors Jun, PPARα, and HNF4α, all of which also influence LIPC levels and activity, are regulated by miR-27b. LIPC was furthermore shown to affect the progress of the life cycle of HCV and to decrease levels of intracellular triglycerides, upon which HCV is known to depend. In summary, this work has demonstrated that miR-27b mediates HCV infection by downregulating LIPC, thereby reducing triglyceride degradation, which in turn increases cellular lipid levels. Cellular metabolism exists as a set of interconnected systems and the functional output of the component enzymes of these systems relies upon multiple layers of regulatory mechanisms. In humans, miRNAs calibrate the expression of protein-coding genes by selectively recognizing sequences within mRNA transcripts and using the RNA interference silencing complex to sequester and degrade the targeted mRNA (1O'Brien J.J. Overview of microRNA biogenesis, mechanisms of actions, and circulation.Front. Endocrinol. 2018; 9: 402Crossref PubMed Scopus (1352) Google Scholar). As the length of base pairing is short and the complementarity imperfect, mRNAs are frequently targeted by multiple different miRNAs (1O'Brien J.J. Overview of microRNA biogenesis, mechanisms of actions, and circulation.Front. Endocrinol. 2018; 9: 402Crossref PubMed Scopus (1352) Google Scholar, 2Sun W. Julie Li Y.-S. Huang H.-D. Shyy J.Y.-J. Chien S. microRNA: a master regulator of cellular processes for bioengineering systems.Annu. Rev. Biomed. 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The response to a miRNA is not limited to its direct targets; rather, specific miRNA–mRNA interactions may alter key factors within larger pathways resulting in significant changes downstream of the protein originally targeted. In this work, functional effectors of miR-27b regulation of lipid metabolism are identified and the mechanisms by which their activity is controlled are explored. These functional effectors were identified by means of activity-based protein profiling (ABPP) techniques, which use small-molecule probes that covalently bind active enzymes, but not their inactive counterparts (36Barglow K.T. Cravatt B.F. Activity-based protein profiling for the functional annotation of enzymes.Nat. Methods. 2007; 4: 822-827Crossref PubMed Scopus (186) Google Scholar, 37Cravatt B.F. Wright A.T. Kozarich J.W. Activity-based protein profiling: from enzyme chemistry to proteomic chemistry.Annu. Rev. Biochem. 2008; 77: 383-414Crossref PubMed Scopus (837) Google Scholar). 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Activity-based protein profiling: the serine hydrolases.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14694-14699Crossref PubMed Scopus (805) Google Scholar) (Fig. 1C). Using this probe, differential activity profiles were established, from which the hepatic lipase C (LIPC) was confirmed as novel functional target of miR-27b. Multiple intermediary factors responsible for the miR-27b-mediated change to LIPC activity were subsequently identified. Finally, the effect of the miR-27b-induced modulation of LIPC activity was shown to be detrimental to HCV propagation. Altogether, this study has expanded our understanding of the key role played by this miRNA in liver metabolism and HCV infection and, more generally, highlighted the need for the functional profiling of miRNA activity to fully understand their roles both in normal cell function and in disease. The importance of miR-27b in the regulation of hepatic lipid metabolism, both in healthy tissue and in the context of disease, has been previously established (17Singaravelu R. Chen R. Lyn R.K. Jones D.M. O'Hara S. Rouleau Y. et al.Hepatitis C virus induced up-regulation of microRNA-27: a novel mechanism for hepatic steatosis.Hepatology. 2014; 59: 98-108Crossref PubMed Scopus (80) Google Scholar, 22Vickers K.C. Shoucri B.M. Levin M.G. Wu H. Pearson D.S. Osei-Hwedieh D. et al.MicroRNA-27b is a regulatory hub in lipid metabolism and is altered in dyslipidemia.Hepatology. 2013; 57: 533-542Crossref PubMed Scopus (170) Google Scholar); however, the mechanisms by which this modulation of the cell’s lipid profile is accomplished are not yet well understood. To investigate the functional consequences of miR-27b to cells’ lipid profile, lipidomic mass spectrometry was performed on cells transfected with miR-27b mimic versus a control (Tables S1–S9). A significant decrease in the expression of the established miR-27b target epidermal growth factor receptor (EGFR) (44Chiyomaru T. Seki N. Inoguchi S. Ishihara T. Mataki H. Matsushita R. et al.Dual regulation of receptor tyrosine kinase genes EGFR and c-Met by the tumor-suppressive microRNA-23b/27b cluster in bladder cancer.Int. J. Oncol. 2015; 46: 487-496Crossref PubMed Scopus (73) Google Scholar) was used to demonstrate efficient miR-27b transfection (Fig. 2). Consistently with previous reports (17Singaravelu R. Chen R. Lyn R.K. Jones D.M. O'Hara S. Rouleau Y. et al.Hepatitis C virus induced up-regulation of microRNA-27: a novel mechanism for hepatic steatosis.Hepatology. 2014; 59: 98-108Crossref PubMed Scopus (80) Google Scholar), a significant increase in the overall quantity of triglycerides present was observed (Fig. 2A), with a trend toward desaturation (Fig. 2B). No significant changes were observed in any other lipid groups (Fig. 2A), suggesting triglyceride metabolism is the primary target of regulation by miR-27b and warranting further investigation. Prior efforts to profile miR-27b-induced changes to cell function have relied mainly on sequencing-based techniques such as ago-HITS-CLIP (10Luna J.M. Scheel T.K.H. Danino T. Shaw K.S. Mele A. Fak J.J. et al.Hepatitis C virus RNA functionally sequesters miR-122.Cell. 2015; 160: 1099-1110Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 45Helwak A. Kudla G. Dudnakova T. Tollervey D. Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding.Cell. 2013; 153: 654-665Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar, 46Moore M.J. Scheel T.K.H. Luna J.M. Park C.Y. Fak J.J. Nishiuchi E. et al.miRNA–target chimeras reveal miRNA 3′-end pairing as a major determinant of Argonaute target specificity.Nat. 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However, this type of analysis is incapable of reporting on important potential posttranslational regulation of protein function. Use of an activity-based probe measures differential enzyme activity derived not only from direct miRNA targeting or altered gene expression but also from changes to protein localization, posttranslational modifications, and protein–protein interactions, thereby providing a more complete, not to mention relevant, picture of the functional role. A significant number of enzymes responsible for the regulation of cells’ lipid profile belong to the serine hydrolase family, whose activity can be detected by the activity-based probe FP (Fig. 1, B and C). To determine whether any serine hydrolase activities are altered by miR-27b, the fluorescent FP–tetramethylrhodamine probe was used to label the proteomes of cells transfected with miR-27b mimic or inhibitor and visualized by in-gel fluorescence. Multiple differential band intensities were observed (Fig. 3A) following miR-27b overexpression or inhibition, indicating that serine hydrolases could play a role in the miR-27b-induced changes to the lipid profile. Interestingly, when this experiment was repeated in cells infected with the JFH1T strain of HCV, a distinct banding pattern was observed, suggesting that the effects of miR-27b might vary depending on the environment of the cell. Next, we determined the identity of the functional targets of miR-27b using affinity purification and HPLC-coupled mass spectrometry (MS). Differentially active serine hydrolases were identified using a biotinylated FP probe to selectively enrich labeled enzymes. Dimethyl labeling, a type of stable isotope labeling using isotopes of formaldehyde, was used to label peptides from each samples, which could then be combined and analyzed by LC-MS/MS. Relative abundances were calculated by the relative size of the peaks in M1, and peptides positively identified, following fragmentation, in M2 (49Boersema P.J. Raijmakers R. Lemeer S. Mohammed S. Heck A.J.R. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics.Nat. Protoc. 2009; 4: 484-494Crossref PubMed Scopus (1026) Google Scholar) (Tables S10–S24). Hits were ranked by prioritizing enzymes whose activity was altered, in opposing directions, by both overexpressing and inhibiting miR-27b, and who displayed this pattern in both naïve and HCV-infected cells (Fig. 3B). Lipid metabolic serine hydrolases represented a significant proportion of enriched, differentially active proteins, followed by a smaller number of signaling proteases and peptidases (Fig. 3B). The top-ranked enzyme was LIPC, the hepatic lipase, which has not previously been associated with miR-27b. LIPC is a liver enzyme mainly localized to the cell surface of hepatocytes (50Kuusi T. Nikklä E.A. 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To confirm our MS-derived observations and to determine whether this regulation of LIPC activity was pretranslational or posttranslational, FP-biotin was again used to label proteomes of cells transfected with miR-27b mimic or its inhibitor, and LIPC in the enriched fraction as well as the unenriched cell lysates was quantified by Western blotting. A significant decrease in both LIPC activity and abundance in the presence of miR-27b was observed, paralleled by an insignificant increase in LIPC activity and abundance when miR-27b was inhibited (Fig. 4, A–C). Quantitative RT-PCR (qRT-PCR) was used to show a similar mimic-mediated decrease in mRNA, while the inhibitor left LIPC mRNA levels relatively unchanged (Fig. 4D). This indicates that the miR-27b-mediated decrease in LIPC activity is based largely on changes to protein expression regulated at the mRNA level, either via direct targeting of the LIPC mRNA by miR-27b or by changes to transcription. While a seed site of miR-27b was found in the 3′-UTR of LIPC (Fig. S1A), a reporter construct containing the 3′-UTR from LIPC and the luciferase gene was not targeted by miR-27 (Fig. S1B), indicating that LIPC expression was not directly targeted by miR-27b. The failure of miR-27b to target the luciferase construct may be explained by the lack of complementarity outside the seed sequence, which typically provides extra stability to miRNA–mRNA interactions (1O'Brien J.J. Overview of microRNA biogenesis, mechanisms of actions, and circulation.Front. Endocrinol. 2018; 9: 402Crossref PubMed Scopus (1352) Google Scholar). Peroxisome proliferator-activated receptors (PPARs) are a group of transcription factors, which regulate lipid metabolism and have been linked to the transcriptional regulation of LIPC (52Rakhshandehroo M. Knoch B. Müller M. Kersten S. 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To assess whether a reduction in PPAR activity was responsible for the decreased expression of LIPC, cells were treated with the pan-PPAR inhibitor benzamide (54Lee G. Elwood F. McNally J. Weiszmann J. Lindstrom M. Amaral K. et al.T0070907, a selective ligand for peroxisome proliferator-activated receptor γ, functions as an antagonist of biochemical and cellular activities.J. Biol. Chem. 2002; 277: 19649-19657Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar) and LIPC activity was quantified by using FP-biotin to label and enrich the active serine hydrolases. Western blotting showed a large decrease in LIPC activity and expression (Fig. 5, A and B), confirming that PPARs play a role in the transcription of LIPC in the model system used. To confirm whether the miR-27b-mediated decrease in LIPC expression was due to a decrease in PPAR signaling, cells transfected with miR-27b mimic were treated with the pan-PPAR activator bezafibrate (55Berger J. Moller D.E. 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