Lin28 enhances de novo fatty acid synthesis to promote cancer progression via SREBP ‐1
2019; Springer Nature; Volume: 20; Issue: 10 Linguagem: Inglês
10.15252/embr.201948115
ISSN1469-3178
AutoresYang Zhang, Chenchen Li, Chuanzhen Hu, Qian Wu, Yongping Cai, Songge Xing, Hui Lü, Lin Wang, De Huang, Linchong Sun, Tingting Li, Xiaoping He, Xiuying Zhong, Junfeng Wang, Ping Gao, Zachary J. Smith, Weidong Jia, Huafeng Zhang,
Tópico(s)Ferroptosis and cancer prognosis
ResumoArticle5 August 2019free access Source DataTransparent process Lin28 enhances de novo fatty acid synthesis to promote cancer progression via SREBP-1 Yang Zhang orcid.org/0000-0001-9923-7531 Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author Chenchen Li Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author Chuanzhen Hu Department of Precision Machinery and Precision Instrumentation, University of Science and Technology of China, Hefei, China Search for more papers by this author Qian Wu Shanghai Center for Bioinformation Technology, Shanghai, China Search for more papers by this author Yongping Cai Department of Pathology, School of Medicine, Anhui Medical University, Hefei, China Search for more papers by this author Songge Xing Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author Hui Lu Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author Lin Wang Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author De Huang Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author Linchong Sun Laboratory of Cancer and Stem Cell Metabolism, School of Medicine, Institutes for Life Sciences, South China University of Technology, Guangzhou, China Search for more papers by this author Tingting Li Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author Xiaoping He Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author Xiuying Zhong Laboratory of Cancer and Stem Cell Metabolism, School of Medicine, Institutes for Life Sciences, South China University of Technology, Guangzhou, China Search for more papers by this author Junfeng Wang High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, China Search for more papers by this author Ping Gao orcid.org/0000-0002-6930-7989 Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China Laboratory of Cancer and Stem Cell Metabolism, School of Medicine, Institutes for Life Sciences, South China University of Technology, Guangzhou, China Search for more papers by this author Zachary J Smith Department of Precision Machinery and Precision Instrumentation, University of Science and Technology of China, Hefei, China Search for more papers by this author Weidong Jia Corresponding Author [email protected] orcid.org/0000-0002-2664-2031 Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China Search for more papers by this author Huafeng Zhang Corresponding Author [email protected] Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author Yang Zhang orcid.org/0000-0001-9923-7531 Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author Chenchen Li Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author Chuanzhen Hu Department of Precision Machinery and Precision Instrumentation, University of Science and Technology of China, Hefei, China Search for more papers by this author Qian Wu Shanghai Center for Bioinformation Technology, Shanghai, China Search for more papers by this author Yongping Cai Department of Pathology, School of Medicine, Anhui Medical University, Hefei, China Search for more papers by this author Songge Xing Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author Hui Lu Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author Lin Wang Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author De Huang Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author Linchong Sun Laboratory of Cancer and Stem Cell Metabolism, School of Medicine, Institutes for Life Sciences, South China University of Technology, Guangzhou, China Search for more papers by this author Tingting Li Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author Xiaoping He Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author Xiuying Zhong Laboratory of Cancer and Stem Cell Metabolism, School of Medicine, Institutes for Life Sciences, South China University of Technology, Guangzhou, China Search for more papers by this author Junfeng Wang High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, China Search for more papers by this author Ping Gao orcid.org/0000-0002-6930-7989 Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China Laboratory of Cancer and Stem Cell Metabolism, School of Medicine, Institutes for Life Sciences, South China University of Technology, Guangzhou, China Search for more papers by this author Zachary J Smith Department of Precision Machinery and Precision Instrumentation, University of Science and Technology of China, Hefei, China Search for more papers by this author Weidong Jia Corresponding Author [email protected] orcid.org/0000-0002-2664-2031 Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China Search for more papers by this author Huafeng Zhang Corresponding Author [email protected] Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China Search for more papers by this author Author Information Yang Zhang1,2,‡, Chenchen Li2,‡, Chuanzhen Hu3, Qian Wu4, Yongping Cai5, Songge Xing1,2, Hui Lu2, Lin Wang2, De Huang2, Linchong Sun6, Tingting Li2, Xiaoping He2, Xiuying Zhong6, Junfeng Wang7, Ping Gao1,6, Zachary J Smith3, Weidong Jia *,1 and Huafeng Zhang *,1,2 1Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China 2Hefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Molecular Medicine, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, China 3Department of Precision Machinery and Precision Instrumentation, University of Science and Technology of China, Hefei, China 4Shanghai Center for Bioinformation Technology, Shanghai, China 5Department of Pathology, School of Medicine, Anhui Medical University, Hefei, China 6Laboratory of Cancer and Stem Cell Metabolism, School of Medicine, Institutes for Life Sciences, South China University of Technology, Guangzhou, China 7High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, China ‡These two authors contributed equally to this work *Corresponding author. Tel: +86 551 62283740; E-mail: [email protected] *Corresponding author. Tel: +86 551 63607131; E-mail: [email protected] EMBO Rep (2019)20:e48115https://doi.org/10.15252/embr.201948115 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Lin28 plays an important role in promoting tumor development, whereas its exact functions and underlying mechanisms are largely unknown. Here, we show that both human homologs of Lin28 accelerate de novo fatty acid synthesis and promote the conversion from saturated to unsaturated fatty acids via the regulation of SREBP-1. By directly binding to the mRNAs of both SREBP-1 and SCAP, Lin28A/B enhance the translation and maturation of SREBP-1, and protect cancer cells from lipotoxicity. Lin28A/B-stimulated tumor growth is abrogated by SREBP-1 inhibition and by the impairment of the RNA binding properties of Lin28A/B, respectively. Collectively, our findings uncover that post-transcriptional regulation by Lin28A/B enhances de novo fatty acid synthesis and metabolic conversion of saturated and unsaturated fatty acids via SREBP-1, which is critical for cancer progression. Synopsis Post-transcriptional regulation by Lin28A and Lin28B enhances de novo fatty acid synthesis and metabolic conversion of saturated and unsaturated fatty acids via SREBP-1, thereby promoting tumor progression. Lin28A and Lin28B promote de novo fatty acid synthesis. Lin28A/B enhance SREBP-1 translation and maturation by binding SREBP-1 and SCAP mRNAs. Lin28A/B protect cancer cells from ER stress. SREBP-1 is critical for Lin28A/B-mediated tumor proliferation. Introduction Lin28 is highly correlated with a wide range of human malignancies, including neuroblastoma, hepatocellular carcinoma, melanoma, and Wilms' tumor, and is associated with poor clinical outcome via the repression of let-7 microRNAs 1, 2. While the Lin28/let-7 axis is traditionally known to drive multiple tumor development in murine models 3-9, the major functions of Lin28A and its homologue Lin28B are not solely dependent on let-7. Lin28 (including Lin28A and Lin28B) also serves as RNA binding proteins with both a cold-shock domain (CSD) and a cys-cys-his-cys (CCHC) domain. Lin28 can directly bind mRNAs at GGAGA motifs and enhance mRNA translation, thereby regulating mRNA metabolism, cell cycle, cell growth, glycolysis, and oxidative phosphorylation (OxPhos)-related protein expression 10-15. These studies have provided extensive insights into the roles of Lin28 independent of let-7. Regarding metabolism regulation, Lin28 has been reported to regulate glucose metabolism by PI3K-AKT-mTOR signaling 14 and enhance OxPhos metabolism to stimulate tissue repair 15. Lin28 is also reported to regulate stem cell metabolism and pluripotency 16. Of note, our group recently demonstrated that the Lin28/let-7 axis regulates the Warburg effect via PDK1 under normoxic condition in cancer cells 7. Nevertheless, the illustration of the interplay between the oncogenic effects of Lin28 and the metabolic pathway is just getting started and it is yet to be explored whether Lin28 plays other critical roles in regulating metabolism during tumor progression. Metabolic reprogramming has been defined as a core hallmark of cancer 17. The cancer cell metabolism is now extended far beyond the original observation of aerobic glycolysis, the so-called Warburg effect 18, 19. Besides typical changes in glucose metabolism, cancer cells were also observed to prefer de novo fatty acid synthesis to maintain rapid cell growth and cell proliferation 20, 21. Several reactions are involved in converting carbons from nutrient to fatty acids. ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), and stearoyl-CoA desaturase (SCD) are the key enzymes involved in generating fatty acids from glucose and reductive glutamine metabolism in cancer cells 22-24. Likewise, acetate, as a carbon source, contributes to producing acetyl-CoA for de novo fatty acid synthesis in certain tumors including liver cancers 25. The crucial transcriptional regulator of lipid synthesis, sterol regulatory element-binding protein 1 (SREBP-1), which extensively targets fatty acid synthesis genes including ACLY, ACC, FASN, and SCD, is synthesized as an inactive precursor. SREBP cleavage-activating protein (SCAP) binds to the SREBP-1 precursors to form a complex, which is embedded to the endoplasmic reticulum (ER). When sterol is deficient in cells, SCAP escorts the SREBP-1 precursors to the Golgi where they are activated by a two-step cleavage 26, 27. Inhibition of SREBP-1 induces ER stress through loss of fatty acid desaturation in human glioblastoma cells 28, 29. These studies underline the importance of fatty acid synthesis in cancer cell biology, whereas the underlying regulatory mechanisms for fatty acid synthesis in cancer cells are still largely unknown. Our group has previously documented that the Lin28/let-7 axis regulates the Warburg effect via PDK1 7. It is intriguing to note that, by staining the tumor samples generated from liver cancer cells with forced expression of Lin28, we observed a significant increase in lipid accumulation, thus prompting us to hypothesize that Lin28 may regulate lipid metabolism during cancer progression. Here, we provide ample evidence to reveal that Lin28A and Lin28B promote de novo fatty acid synthesis in cancer cells. We observed that both Lin28A and Lin28B bind to mRNAs of SREBP-1 and SCAP to enhance the translation and maturation of SREBP-1, a master lipid synthesis regulator that increases multiple triglyceride species and fatty acids levels and promotes the conversion of saturated fatty acids to unsaturated ones. Furthermore, lack of Lin28 induces ER stress via de novo lipogenic disorders and, importantly, the dysfunction of Lin28 as an RBP abrogates the lipid accumulation and cancer progression. Collectively, our results establish that Lin28 enhances de novo fatty acid synthesis and cancer progression via a previously unappreciated mechanism of SREBP-1 regulation. Results Lin28A/B enhance lipid accumulation in cancer cells Our previous studies have demonstrated that the Lin28/let-7 axis facilitates the Warburg effect to promote cancer progression 7. However, little is known about Lin28 in regulation of lipid metabolism in cancer cells. It is intriguing to note that, by staining the mouse tumor samples generated from PLC or Hep3B cells overexpressing Lin28A/B with oil red O, we observed a significant increase in lipid accumulation (Fig 1A). On the other hand, suppression of Lin28A or Lin28B by shRNAs decreased lipid accumulation in mouse xenograft derived from Hep3B cells (Appendix Fig S1A), suggesting that Lin28A and Lin28B are involved in the regulation of lipid metabolism during liver cancer progression. Consistently, in cultured PLC cells, forced expression of Lin28A or Lin28B led to increased cellular lipid accumulation as well as elevated cellular triglyceride (TG) levels (Fig 1B and C). Meanwhile, knockdown of Lin28A or Lin28B suppressed cellular lipid accumulation as well as cellular TG levels in PLC cells (Fig 1D and E). Similar results were observed in HepG2 and Hep3B cells (Appendix Fig S1B), further confirming that Lin28A and Lin28B regulate lipid metabolism in liver cancer cells. Figure 1. Lin28A/B enhance lipid accumulation in cancer cells Neutral lipids were measured in the mouse tumor samples generated from PLC or Hep3B cells overexpressing Lin28A or Lin28B by oil red O staining. Scale bars, 100 μm. Bar graphs depicted the results from the analysis of image of oil red O staining by Photoshop. Data were presented as mean ± SD. Data are representative of five independent experiments. *P < 0.05 as compared to EV group; Student's t-test. Cellular neutral lipids were measured in PLC cells overexpressing Lin28A or Lin28B by Nile red staining. Scale bars, 50 μm. Cellular TG was measured in PLC cells overexpressing Lin28A or Lin28B by Biochemical Triglyceride Determination Kit. The values were normalized to cellular protein. Data were presented as mean ± SD. Data are representative of three independent experiments. *P < 0.05 as compared to EV group; Student's t-test. Cellular neutral lipids were measured in PLC cells expressing NTC, shLin28A, or shLin28B by Nile red staining. Scale bars, 50 μm. Cellular TG was measured in PLC cells expressing NTC, shLin28A, or shLin28B by Biochemical Triglyceride Determination Kit. The values were normalized to cellular protein. Data were presented as mean ± SD. Data are representative of three independent experiments. *P < 0.05 as compared to NTC group; Student's t-test. Lipids in PLC cells expressing NTC, shLin28A, or shLin28B were measured by LC-MS. The heatmap highlighted relative changes for each type of lipid metabolite (left panel) and detailed type of TG (right panel) with their individual average ion counts normalized to NTC. Data are representative of four independent experiments. Lipid species abbreviations are available in Appendix Table S3. Total cellular fatty acids were determined from PLC cells expressing NTC, shLin28A, or shLin28B by GC-MS. Average ion counts of metabolites were normalized to NTC. Data were presented as mean ± SD. Data are representative of three independent experiments. *P < 0.05 as compared to corresponding NTC group; Student's t-test. Download figure Download PowerPoint Next, liquid chromatography–mass spectrometry (LC-MS) was performed to detect the changes in global lipid species focusing mainly on glycerides and phosphatides. LC-MS data revealed that suppression of Lin28A/B markedly decreased multiple types of lipids such as triglyceride (TG), phosphatidylethanolamine (PE), sphingomyelin (SM), lysophosphatidylcholine (LPC), phosphatidylcholine (PC), and phosphatidylinositol (PI). Among them, TG species exhibited the most extensive decrease (Fig 1F, Appendix Fig S1C and D, and Appendix Tables S1 and S2), which was consistent with the cellular TG measurement data by colorimetric assay (Fig 1C and E). Further analysis by gas chromatography–mass spectrometry (GC-MS) showed that cellular long-chain fatty acids, such as palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), and oleic acid (C18:1), were markedly reduced by shLin28A or shLin28B in PLC cells (Fig 1G), indicating that Lin28A and Lin28B promote lipid accumulation probably by regulating fatty acid metabolism. Lin28A/B promote de novo fatty acid synthesis via SREBP-1 To explore how Lin28A and Lin28B regulate fatty acid metabolism, we detected the major enzymes involved in de novo fatty acid synthesis, fatty acid β-oxidation, fatty acid uptake, and glucose metabolism. Interestingly, Western blot analysis revealed that de novo fatty acid synthetic enzymes FASN, SCD1, ACC1, and ACLY were markedly increased by Lin28A/B in PLC cells (Fig 2A). Consistently, suppression of Lin28A or Lin28B by shRNAs reduced the protein levels of the fatty acid synthetic enzymes in PLC cells (Fig 2B). To explore whether de novo fatty acid synthesis contributes to the change in cellular fatty acids induced by Lin28A or Lin28B, we carried out metabolic flux analysis using U-13C2 acetate or U-13C6 glucose. As a result, 13C-incorporated palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), and oleic acid (C18:1) derived from U-13C2 acetate or U-13C6 glucose carbons were significantly decreased in PLC cells expressing shLin28A or shLin28B (Fig 2C and Appendix Fig S2A–C), demonstrating that Lin28A/B enhance de novo fatty acid synthesis. Figure 2. Lin28A/B promote de novo fatty acid synthesis via SREBP-1 A. Protein levels of metabolic enzymes were determined by Western blot in PLC cells overexpressing Lin28A or Lin28B. β-Actin served as loading control. B. Protein levels of metabolic enzymes in de novo fatty acid synthesis were determined by Western blot in PLC cells expressing NTC, shLin28A, or shLin28B. β-Actin served as loading control. C. The proportion of labeled fatty acids were determined in PLC cells expressing NTC, shLin28A, or shLin28B incubated with U-13C2 acetate by GC-MS. Average ion counts of metabolites were normalized to NTC. Data were presented as mean ± SD. Data are representative of three independent experiments. *P < 0.05 as compared to corresponding NTC group; Student's t-test. D. Protein levels of transcription factor in de novo fatty acid synthesis and SCAP were determined by Western blot in PLC cells overexpressing Lin28A or Lin28B. β-Actin served as loading control. E. Protein levels of SREBP-1 and SCAP were determined by Western blot in PLC cells expressing NTC, shLin28A, or shLin28B. β-Actin served as loading control. F. mRNA levels of SREBP-1, SCAP, and metabolic enzymes were determined by qRT–PCR in PLC cells overexpressing Lin28A or Lin28B. ACSL6 served as negative control. Data were presented as mean ± SD. Data are representative of three independent experiments. *P < 0.05 as compared to corresponding EV group; Student's t-test. G, H. PLC cells overexpressing Lin28A or Lin28B were further infected with viruses expressing NTC, shSREBP-1 (G), or shSCAP (H). Protein levels of SREBP-1, SCAP, and metabolic enzymes in de novo fatty acid synthesis were determined by Western blot. β-Actin served as loading control. I. PLC cells overexpressing Lin28A or Lin28B were further infected with viruses expressing NTC, shSREBP-1, or shSCAP. Cellular TG was measured. The values were normalized to cellular protein. Data were presented as mean ± SD. Data are representative of three independent experiments. *P < 0.05 as compared between indicated groups. NS, not significant; Student's t-test. J. PLC cells overexpressing Lin28A or Lin28B were further infected with viruses expressing NTC or shSREBP-1. Cellular neutral lipids were measured by Nile red staining. Scale bars, 50 μm. Source data are available online for this figure. Source Data for Figure 2 [embr201948115-sup-0002-SDataFig2.pdf] Download figure Download PowerPoint To address how Lin28A and Lin28B enhance the expression of fatty acid synthetic enzymes, we detected the master de novo fatty acid synthesis regulators including sterol regulatory element-binding transcription factor 1 (SREBP-1), SREBP cleavage-activating protein (SCAP, which is the direct cleavage factor for SREBP family), carbohydrate-responsive element-binding protein (ChREBP), and liver X receptor alpha (LXRα) 26, 27, 30, 31. Interestingly, overexpression of Lin28A or Lin28B resulted in a remarkable increase in SREBP-1 precursors, mature SREBP-1, and SCAP protein in PLC cells (Fig 2D). Consistently, suppression of Lin28A or Lin28B by shRNAs reduced the protein levels of SREBP-1 precursors, mature SREBP-1, and SCAP in PLC cells (Fig 2E). Similar results were observed in cultured HepG2 and Hep3B cells as well as in the xenograft tumors derived from Hep3B cells (Appendix Fig S2D–G). Furthermore, qRT–PCR experiments showed that Lin28A/B had no effect on SREBP-1 or SCAP mRNA levels, but markedly increased mRNA levels of de novo fatty acid synthetic enzymes which are the downstream targets of SREBP-1 26 (Fig 2F), indicating that SREBP-1 and SCAP are regulated by Lin28A/B at the post-transcriptional level. Consistent with a previous report that SCAP escorts the SREBP-1 precursors to the Golgi where SREBP-1 precursors are converted to mature SREBP-1 by a two-step cleavage 27, we also observed that knockdown of SCAP decreased the mature SREBP-1 levels in PLC cells (Appendix Fig S2G). Furthermore, our data confirmed that suppression of SREBP-1 or SCAP by shRNAs markedly inhibited fatty acid synthetic enzyme expression in PLC cells (Appendix Fig S2H and I). More importantly, knockdown of SREBP-1 or SCAP markedly attenuated Lin28A/B-induced increase in the protein levels of fatty acid synthetic enzymes in PLC cells (Fig 2G and H). Consistently, knockdown of SREBP-1 or SCAP significantly abolished the promoting effect of Lin28A/B on the accumulation of cellular neutral lipids as well as TG levels (Fig 2I and J, and Appendix Fig S2J). Collectively, these data demonstrate that both SREBP-1 and SCAP are critical for Lin28A/B-mediated fatty acid synthetic enzyme expression as well as the subsequent lipid metabolism. Lin28A/B bind to SREBP-1 and SCAP mRNA to enhance SREBP-1 translation and maturation To further elucidate how Lin28A and Lin28B increase SREBP-1 expression, we first established a stable PLC cell line expressing shDGCR8 (Appendix Fig S3A), which lacks the expression of let-7 mature microRNAs (Appendix Fig S3B) 32. Western blot analysis revealed that Lin28A/B still significantly enhance SREBP-1, SCAP, and fatty acid synthetic enzyme expression in these stable cells, which was not affected remarkably by restoring the expression of let-7 isoforms (Appendix Fig S3C), suggesting that Lin28A/B regulates SREBP-1 and fatty acid synthesis through let-7-independent mechanisms. Since Lin28A and Lin28B have been reported to function as RNA binding proteins 13, we next performed an RNA immunoprecipitation experiment. Interestingly, our data showed that both endogenous Lin28A and Lin28B bind to SREBP-1 as well as SCAP mRNA (Fig 3A). Bioinformatic analysis predi
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