Abstract
Emerging evidence suggests that long non-coding RNAs (lncRNAs) are critical regulators of diverse biological processes, including adipogenesis. Despite being considered an ideal animal model for studying adipogenesis, little is known about the roles of lncRNAs in the regulation of rabbit preadipocyte differentiation. In the present study, visceral preadipocytes isolated from newborn rabbits were cultured in vitro and induced for differentiation, and global lncRNA expression profiles of adipocytes collected at days 0, 3, and 9 of differentiation were analyzed by RNA-seq. A total of 2066 lncRNAs were identified from nine RNA-seq libraries. Compared to protein-coding transcripts, lncRNA transcripts exhibited characteristics of a longer length and lower expression level. Furthermore, 486 and 357 differentially expressed (DE) lncRNAs were identified when comparing day 3 vs. day 0 and day 9 vs. day 3, respectively. Target genes of DE lncRNAs were predicted by the cis-regulating approach. Prediction of functions revealed that DE lncRNAs when comparing day 3 vs. day 0 were involved in gene ontology (GO) terms of developmental growth, growth, developmental cell growth, and stem cell proliferation, and involved in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of PI3K-Akt signaling pathway, fatty acid biosynthesis, and the insulin signaling pathway. The DE lncRNAs when comparing day 9 vs. day 3 were involved in GO terms that associated with epigenetic modification and were involved in the KEGG pathway of cAMP signaling pathway. This study provides further insight into the regulatory function of lncRNAs in rabbit visceral adipose and facilitates a better understanding of different stages of preadipocyte differentiation.
Similar content being viewed by others
References
Bakhtiarizadeh MR, Salami SA (2019) Identification and expression analysis of long noncoding RNAs in fat-tail of sheep breeds. G3 (Bethesda) 9:1263–1276
Batista PJ, Chang HY (2013) Long noncoding RNAs: cellular address codes in development and disease. Cell 152:1298–1307
Billert M, Wojciechowicz T, Jasaszwili M, Szczepankiewicz D, Wasko J, Kazmierczak S, Strowski MZ, Nowak KW, Skrzypski M (2018) Phoenixin-14 stimulates differentiation of 3T3-L1 preadipocytes via cAMP/Epac-dependent mechanism. Biochim Biophys Acta Mol Cell Biol Lipids 1863:1449–1457
Cai R, Tang G, Zhang Q, Yong W, Zhang W, Xiao J, Wei C, He C, Yang G, Pang W (2019) A novel lnc-RNA, named lnc-ORA, is identified by RNA-Seq analysis, and its knockdown inhibits adipogenesis by regulating the PI3K/AKT/mTOR signaling pathway. Cells 8(5):E477
Chen S, Zhou Y, Chen Y, Gu J (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34:i884–i890
Da WH, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4(1):44–57
Deng T, Wang Y, Wang C, Yan H (2019) FABP4 silencing ameliorates hypoxia reoxygenation injury through the attenuation of endoplasmic reticulum stress-mediated apoptosis by activating PI3K/Akt pathway. Life Sci 224:149–156
Desando G, Cavallo C, Sartoni F, Martini L, Parrilli A, Veronesi F, Fini M, Giardino R, Facchini A, Grigolo B (2013) Intra-articular delivery of adipose derived stromal cells attenuates osteoarthritis progression in an experimental rabbit model. Arthritis Res Ther 15:R22
Ding F, Li QQ, Li L, Gan C, Yuan X, Gou H, He H, Han CC, Wang JW (2015) Isolation, culture and differentiation of duck (Anas platyrhynchos) preadipocytes. Cytotechnology 67:773–781
Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, Heger A, Hetherington K, Holm L, Mistry J (2014) Pfam: the protein families database. Nucleic Acids Res 42:222–230
Fox CS, Massaro JM, Hoffmann U, Pou KM, Maurovich-Horvat P, Liu CY, Vasan RS, Murabito JM, Meigs JB, Cupples LA, D’Agostino RB Sr, O’Donnell CJ (2007) Abdominal visceral and subcutaneous adipose tissue compartments: association with metabolic risk factors in the Framingham Heart Study. Circulation 116:39–48
Gong L, Wang C, Li Y, Sun Q, Li G, Wang D (2014) Effects of human adipose-derived stem cells on the viability of rabbit random pattern flaps. Cytotherapy 16:496–507
Hacisuleyman E, Goff LA, Trapnell C, Williams A, Henao-Mejia J, Sun L, Mcclanahan P, Hendrickson DG, Sauvageau M, Kelley DR (2014) Topological organization of multichromosomal regions by the long intergenic noncoding RNA Firre. Nat Struct Mol Biol 21:198–206
Huang Y, Jin C, Zheng Y, Li X, Zhang S, Zhang Y, Jia L, Li W (2017) Knockdown of lncRNA MIR31HG inhibits adipocyte differentiation of human adipose-derived stem cells via histone modification of FABP4. Sci Rep 7:8080
Huang W, Zhang X, Li A, Xie L, Miao X (2018) Genome-wide analysis of mRNAs and lncRNAs of intramuscular fat related to lipid metabolism in two pig breeds. Cell Physiol Biochem 50:2406–2422
Hunter JD (2007) Matplotlib: a 2D graphics environment. Comput Sci Eng 9:90–95
Johnsson P, Lipovich L, Grander D, Morris KV (2014) Evolutionary conservation of long non-coding RNAs; sequence, structure, function. Biochim Biophys Acta 1840:1063–1071
Kai S, Kusminski CM, Scherer PE (2011) Adipose tissue remodeling and obesity. J Clin Invest 121:2094
Kershaw EE, Flier JS (2004) Adipose tissue as an endocrine organ. J Clin Endocrinol Metab 89:2548–2556
Kim D, Langmead B, Salzberg SL (2015) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12:357–360
Kim HJ, Kwon H, Jeong SM, Hwang SE, Park JH (2019) Effects of abdominal visceral fat compared with those of subcutaneous fat on the association between PM10 and hypertension in Korean men: a cross-sectional study. Sci Rep 9:5951
Kong L, Zhang Y, Ye ZQ, Liu XQ, Zhao SQ, Wei L, Gao G (2007) CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine. Nucleic Acids Res 35:W345–W349
Kuang L, Lei M, Li C, Zhang X, Ren Y, Zheng J, Guo Z, Zhang C, Yang C, Mei X, Fu M, Xie X (2018) Identification of long non-coding RNAs related to skeletal muscle development in two rabbit breeds with different growth rate. Int J Mol Sci 19:E2046
Lee MJ (2017) Hormonal regulation of adipogenesis. Compr Physiol 7:1151–1195
Lee HL, Qadir AS, Park HJ, Chung E, Lee YS, Woo KM, Ryoo HM, Kim HJ, Baek JH (2018) cAMP/protein kinase A signaling inhibits Dlx5 expression via activation of CREB and subsequent C/EBPbeta induction in 3T3-L1 preadipocytes. Int J Mol Sci 19:E3161
Lin FT, Lane MD (1994) CCAAT/enhancer binding protein alpha is sufficient to initiate the 3T3-L1 adipocyte differentiation program. Proc Natl Acad Sci U S A 91:8757–8761
Liu S, Yang Y, Wu J (2011) TNFalpha-induced up-regulation of miR-155 inhibits adipogenesis by down-regulating early adipogenic transcription factors. Biochem Biophys Res Commun 414:618–624
Lo KA, Huang S, Walet ACE, Zhang ZC, Leow MK, Liu M, Sun L (2018) Adipocyte long-noncoding RNA transcriptome analysis of obese mice identified Lnc-leptin, which regulates leptin. Diabetes 67:1045–1056
Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550
Maneschi E, Vignozzi L, Morelli A, Mello T, Filippi S, Cellai I, Comeglio P, Sarchielli E, Calcagno A, Mazzanti B, Vettor R, Vannelli GB, Adorini L, Maggi M (2013) FXR activation normalizes insulin sensitivity in visceral preadipocytes of a rabbit model of MetS. J Endocrinol 218:215–231
Miao Z, Wang S, Zhang J, Wei P, Guo L, Liu D, Wang Y, Shi M (2018) Identification and comparison of long non-conding RNA in Jinhua and Landrace pigs. Biochem Biophys Res Commun 506:765–771
Mota de Sa P, Richard AJ, Hang H, Stephens JM (2017) Transcriptional regulation of adipogenesis. Compr Physiol 7:635–674
Peng Y, Xiang H, Chen C, Zheng R, Chai J, Peng J, Jiang S (2013) MiR-224 impairs adipocyte early differentiation and regulates fatty acid metabolism. Int J Biochem Cell Biol 45:1585–1593
Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL (2016) Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat Protoc 11:1650–1667
Pischon T, Boeing H, Hoffmann K, Bergmann M, Schulze MB, Overvad K, van der Schouw YT, Spencer E, Moons KG, Tjonneland A, Halkjaer J, Jensen MK, Stegger J, Clavel-Chapelon F, Boutron-Ruault MC, Chajes V, Linseisen J, Kaaks R, Trichopoulou A, Trichopoulos D, Bamia C, Sieri S, Palli D, Tumino R, Vineis P, Panico S, Peeters PH, May AM, Bueno-de-Mesquita HB, van Duijnhoven FJ, Hallmans G, Weinehall L, Manjer J, Hedblad B, Lund E, Agudo A, Arriola L, Barricarte A, Navarro C, Martinez C, Quiros JR, Key T, Bingham S, Khaw KT, Boffetta P, Jenab M, Ferrari P, Riboli E (2008) General and abdominal adiposity and risk of death in Europe. N Engl J Med 359:2105–2120
Quinn JJ, Chang HY (2016) Unique features of long non-coding RNA biogenesis and function. Nat Rev Genet 17:47–62
Ren H, Wang G, Chen L, Jiang J, Liu L, Li N, Zhao J, Sun X, Zhou P (2016) Genome-wide analysis of long non-coding RNAs at early stage of skin pigmentation in goats (Capra hircus). BMC Genomics 17:67
Rinn JL, Chang HY (2012) Genome regulation by long noncoding RNAs. Annu Rev Biochem 81:145–166
Rogne M, Tasken K (2014) Compartmentalization of cAMP signaling in adipogenesis, lipogenesis, and lipolysis. Horm Metab Res 46:833–840
Song F, Jiang D, Wang T, Wang Y, Lou Y, Zhang Y, Ma H, Kang Y (2017) Mechanical stress regulates osteogenesis and adipogenesis of rat mesenchymal stem cells through PI3K/Akt/GSK-3beta/beta-catenin signaling pathway. Biomed Res Int 2017:6027402
Sun L, Goff LA, Trapnell C, Alexander R, Lo KA, Hacisuleyman E, Sauvageau M, Tazonvega B, Kelley DR, Hendrickson DG (2013a) Long noncoding RNAs regulate adipogenesis. Proc Natl Acad Sci U S A 110:3387–3392
Sun L, Luo H, Bu D, Zhao G, Yu K, Zhang C, Liu Y, Chen R, Zhao Y (2013b) Utilizing sequence intrinsic composition to classify protein-coding and long non-coding transcripts. Nucleic Acids Res 41:e166
Tong C, Chen Q, Zhao L, Ma J, Ibeagha-Awemu EM, Zhao X (2017) Identification and characterization of long intergenic noncoding RNAs in bovine mammary glands. BMC Genomics 18:468
Tontonoz P, Hu E, Spiegelman BM (1994) Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell 79:1147–1156
Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7:562–578
Ulitsky I, Bartel DP (2013) lincRNAs: genomics, evolution, and mechanisms. Cell 154:26–46
Wang L, Park HJ, Dasari S, Wang S, Kocher JP, Li W (2013) CPAT: Coding-Potential Assessment Tool using an alignment-free logistic regression model. Nucleic Acids Res 41:e74
Wang W, He N, Feng C, Liu V, Zhang L, Wang F, He J, Zhu T, Wang S, Qiao W, Li S, Zhou G, Zhang L, Dai C, Cao W (2015) Human adipose-derived mesenchymal progenitor cells engraft into rabbit articular cartilage. Int J Mol Sci 16:12076–12091
Wang Y, Xue S, Liu X, Liu H, Hu T, Qiu X, Zhang J, Lei M (2016) Analyses of long non-coding RNA and mRNA profiling using RNA sequencing during the pre-implantation phases in pig endometrium. Sci Rep 6:20238
Wang GZ, Du K, Hu SQ, Chen SY, Jia XB, Cai MC, Shi Y, Wang J, Lai SJ (2018a) Genome-wide identification and characterization of long non-coding RNAs during postnatal development of rabbit adipose tissue. Lipids Health Dis 17:271
Wang X, Chen J, Rong C, Pan F, Zhao X, Hu Y (2018b) GLP-1RA promotes brown adipogenesis of C3H10T1/2 mesenchymal stem cells via the PI3K-AKT-mTOR signaling pathway. Biochem Biophys Res Commun 506:976–982
Wang N, Li Y, Li Z, Ma J, Wu X, Pan R, Wang Y, Gao L, Bao X, Xue P (2019) IRS-1 targets TAZ to inhibit adipogenesis of rat bone marrow mesenchymal stem cells through PI3K-Akt and MEK-ERK pathways. Eur J Pharmacol 849:11–21
Wei S, Du M, Jiang Z, Hausman G, Zhang L, Dodson M (2016) Long noncoding RNAs in regulating adipogenesis: new RNAs shed lights on obesity. Cell Mol Life Sci 73:1–9
Wu Y, Cheng T, Liu C, Liu D, Zhang Q, Long R, Zhao P, Xia Q (2016) Systematic identification and characterization of long non-coding RNAs in the silkworm, Bombyx mori. PLoS One 11:e0147147
Xiao T, Liu L, Li H, Yu, Luo (2015) Long noncoding RNA ADINR regulates adipogenesis by transcriptionally activating C/EBPα. Stem Cell Rep 5:856–865
Xu B, Gerin I, Miao H, Dang VP, Johnson CN, Xu R, Chen XW, Cawthorn WP, Macdougald OA, Koenig RJ (2010) Multiple roles for the non-coding RNA SRA in regulation of adipogenesis and insulin sensitivity. PLoS One 5:e14199
Yang SM, Park YK, Kim JI, Lee YH, Lee TY, Jang BC (2018) LY3009120, a pan-Raf kinase inhibitor, inhibits adipogenesis of 3T3-L1 cells by controlling the expression and phosphorylation of C/EBP-alpha, PPAR-gamma, STAT3, FAS, ACC, perilipin A, and AMPK. Int J Mol Med 42:3477–3484
Yi F, Zhang P, Wang Y, Xu Y, Zhang Z, Ma W, Xu B, Xia Q, Du Q (2019) Long non-coding RNA slincRAD functions in methylation regulation during the early stage of mouse adipogenesis. RNA Biol 16:1401–1413
Zebisch K, Voigt V, Wabitsch M, Brandsch M (2012) Protocol for effective differentiation of 3T3-L1 cells to adipocytes. Anal Biochem 425:88–90
Zhang T, Zhang X, Han K, Zhang G, Wang J, Xie K, Xue Q (2017) Genome-wide analysis of lncRNA and mRNA expression during differentiation of abdominal preadipocytes in the chicken. G3 (Bethesda) 7:953–966
Funding
This work was supported by the earmarked fund for China Agriculture Research System (CARS-44-A-2).
Author information
Authors and Affiliations
Contributions
Conceptualization: Song-Jia Lai.
Formal analysis: Kun Du, Guo-Ze Wang.
Resource: An-yong Ren, Ming-cheng Cai, Gang Luo, Xian-bo Jia, Shen-qiang Hu, Jie Wang, Shi-Yi Chen, Song-Jia Lai.
Writing: Kun Du.
Corresponding author
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Table S1.
Primers of genes used in RT-qPCR (docx format). (DOCX 15 kb)
Table S2.
Summary of RNA-seq data and reads mapping (docx format). (DOCX 16 kb)
Table S3.
Differentially expressed analysis when comparing day 3 vs. day 0 (xlsx format). (XLSX 50 kb)
Table S4.
Differentially expressed analysis when comparing day 9 vs. day 3 (xlsx format). (XLSX 39 kb)
Table S5.
FPKM of lncRNA transcripts (xlsx format). (XLSX 209 kb)
Table S6.
Result of GO enrichment between day 0 and day 3 of rabbit adipogenesis (xlsx format). (XLSX 23 kb)
Table S7.
Result of KEGG pathway analysis between day 0 and day 3 of rabbit adipogenesis (xlsx format). (XLSX 12 kb)
Table S8.
Result of GO enrichment between day 3 and day 9 of rabbit adipogenesis (xlsx format). (XLSX 22 kb)
Table S9.
Result of KEGG pathway analysis between day 3 and day 9 of rabbit adipogenesis (xlsx format). (XLSX 11 kb)
Table S10.
DE LncRNAs in key pathways that might regulate rabbit adipogenesis (xlsx format). (XLSX 10 kb)
Rights and permissions
About this article
Cite this article
Du, K., Wang, GZ., Ren, Ay. et al. Genome-wide identification and characterization of long non-coding RNAs during differentiation of visceral preadipocytes in rabbit. Funct Integr Genomics 20, 409–419 (2020). https://doi.org/10.1007/s10142-019-00729-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10142-019-00729-5