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Expression Profiling of IncRNAs, miRNAs, and mRNAs and Their Differential Expression in Leiomyoma Using Next-Generation RNA Sequencing

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Abstract

The objective of this study was to identify the expression profile of long noncoding RNAs (IncRNAs) with concurrent assessment of microRNA (miRNA) and messenger RNA (mRNA) profiles in leiomyomas and paired myometrium using next-generation RNA sequencing and assembly of RNA transcripts. Total RNA was isolated from leiomyoma and paired myometrium (N = 8) and samples from 3 pairs were subjected to RNA sequencing. Normalized assembly of over 48 000 IncRNAs resulted in identification of 45 936 IncRNAs. Of these IncRNAs, 22 148 representing overlapping, intergenic, intronic, and antisense subtypes were expressed in all paired tissues, with 5941 (2813 up- and 3128 downregulated at ≥.5 fold) differentially expressed in leiomyomas. Concurrent RNA sequencing revealed the expression of 2588 miRNAs and 21814 mRNAs, of which 392 miRNAs and 16 559 mRNAs were expressed in all paired tissues. Of these transcripts, 56 and 92 miRNAs and 2030 and 1825 mRNAs were up- or downregulated at ≥ 1.5 fold, respectively, in leiomyoma as compared to myometrium. Using quantitative reverse transcription-polymerase chain reaction (QRT-PCR), we confirmed the expression of hepatocellular carcinoma upregulated (HULC), Inc-maternally expressed 3 (MEG3), long intergenic ncRNA890 (LINC00890), TSIX, long intergenic ncRNA473 (LINC00473), lnc-KLF9-1, and lnc-POTEM-3 (IncRNA-ATB) in leiomyoma and matched myometrium (N = 8). Collectively, the results presented here provide a comprehensive expression profile of IncRNAs in leiomyomas with concurrent integrated expression of miRNAs and mRNAs and implicate potential regulatory functions of IncRNAs through interactions with specific miRNAs and mRNAs which are known to be critical in the pathogenesis of leiomyoma.

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References

  1. Segars JH, Parrott EC, Nagel JD, et al. Proceedings from the third National Institutes of Health International Congress on Advances in Uterine Leiomyoma Research: comprehensive review, conference summary and future recommendations. Hum Reprod Update. 2014;20(3):309–333.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Chegini N, Verala J, Luo X, Xu J, Williams RS. Gene expression profile of leiomyoma and myometrium and the effect of gonadotropin releasing hormone analogue therapy. J Soc Gynecol Investig. 2003;10(3):161–171.

    Article  CAS  PubMed  Google Scholar 

  3. Guo H, Zhang X, Dong R, et al. Integrated analysis of long non-coding RNAs and mRNAs reveals their potential roles in the pathogenesis of uterine leiomyomas. Oncotarget. 2014;5(8):8625–8636.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Luo X, Pan Q, Liu L, Chegini N. Genomic and proteomic profiling II: comparative assessment of gene expression profiles in leiomyomas, keloids, and surgically-induced scars. Reprod Biol Endocrinol. 2007;5:35.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Pan Q, Luo X, Chegini N. Genomic and proteomic profiling I: leiomyomas in African Americans and Caucasians. Reprod Biol Endocrinol. 2007;5:34.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Tsibris JC, Segars J, Coppola D, et al. Insights from gene arrays on the development and growth regulation of uterine leiomyo-mata. Fertil Steril. 2002;78(1):114–121.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Chegini N. Proinflammatory and profibrotic mediators: principal effectors of leiomyoma development as a fibrotic disorder. Semin Reprod Med. 2010;28(3):180–203.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Yatsenko SA, Mittal P, Wood-Trageser MA, et al. Highly heterogeneous genomic landscape of uterine leiomyomas by whole exome sequencing and genome-wide arrays. Fertil Steril. 2017;107(2):457–466.e9.

    Article  CAS  PubMed  Google Scholar 

  9. Mehine M, Kaasinen E, Makinen N, et al. Characterization of uterine leiomyomas by whole-genome sequencing. N Engl J Med. 2013;369(1):43–53.

    Article  CAS  PubMed  Google Scholar 

  10. Mehine M, Makinen N, Heinonen HR, Aaltonen LA, Vahteristo P. Genomics of uterine leiomyomas: insights from high-throughput sequencing. Fertil Steril. 2014;102(3):621–629.

    Article  CAS  PubMed  Google Scholar 

  11. Hodge JC, Kim TM, Dreyfuss JM, et al. Expression profiling of uterine leiomyomata cytogenetic subgroups reveals distinct signatures in matched myometrium: transcriptional profiling of the t(12;14) and evidence in support of predisposing genetic heterogeneity. Hum Mol Genet. 2012;21(10):2312–2329.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gallagher CS, Morton CC. Genetic association studies in uterine fibroids: risk alleles presage the path to personalized therapies. Semin Reprod Med. 2016;34(4):235–241.

    Article  PubMed  Google Scholar 

  13. Liegl-Atzwanger B, Heitzer E, Flicker K, et al. Exploring chromosomal abnormalities and genetic changes in uterine smooth muscle tumors. Mod Pathol. 2016;29(10):1262–1277.

    Article  CAS  PubMed  Google Scholar 

  14. Mehine M, Kaasinen E, Heinonen HR, et al. Integrated data analysis reveals uterine leiomyoma subtypes with distinct driver pathways and biomarkers. Proc Natl Acad Sci USA. 2016;113(5):1315–1320.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Mittal P, Shin YH, Yatsenko SA, Castro CA, Surti U, Rajkovic A. Med12 gain-of-function mutation causes leiomyomas and genomic instability. J Clin Investig. 2015;125(8):3280–3284.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Al-Hendy A, Laknaur A, Diamond MP, Ismail N, Boyer TG, Haider SK. Silencing Medl2 gene reduces proliferation of human leiomyoma cells mediated via Wnt/beta-catenin signaling pathway. Endocrinology. 2017;158(3):592–603.

    CAS  PubMed  Google Scholar 

  17. Georgieva B, Milev I, Minkov I, Dimitrova I, Bradford AP, Baev V. Characterization of the uterine leiomyoma microRNAome by deep sequencing. Genomics. 2012;99(5):275–281.

    Article  CAS  PubMed  Google Scholar 

  18. Luo X, Chegini N. The expression and potential regulatory function of microRNAs in the pathogenesis of leiomyoma. Semin Reprod Med. 2008;26(6):500–514.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wang T, Zhang X, Obijuru L, et al. A micro-RNA signature associated with race, tumor size, and target gene activity in human uterine leiomyomas. Genes Chromosomes Cancer. 2007;46(4):336–347.

    Article  CAS  PubMed  Google Scholar 

  20. Zavadil J, Ye H, Liu Z, et al. Profiling and functional analyses of microRNAs and their target gene products in human uterine leiomyomas. PloS One. 2010;5(8):e12362.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Chuang TD, Khorram O. miR-200c regulates IL8 expression by targeting 1KB KB: a potential mediator of inflammation in leiomyoma pathogenesis. PloS One. 2014;9(4):e95370.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Chuang TD, Khorram O. Mechanisms underlying aberrant expression of miR-29c in uterine leiomyoma. Fertil Steril. 2016;105(1):236–245.e1.

    Article  CAS  PubMed  Google Scholar 

  23. Agarwal V, Bell GW, Nam JW, Bartel DP. Predicting effective microRNA target sites in mammalian mRNAs. eLife. 2015;4.

    Google Scholar 

  24. Huntzinger E, Izaurralde E. Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat Rev Genet. 2011;12(2):99–110.

    Article  CAS  PubMed  Google Scholar 

  25. Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N. Widespread changes in protein synthesis induced by microRNAs. Nature. 2008;455(7209):58–63.

    Article  CAS  PubMed  Google Scholar 

  26. Ma X, Becker Buscaglia LE, Barker JR, Li Y. MicroRNAs in NF-kappaB signaling. Journal of Molecular Cell Biol. 2011;3(3):159–166.

    Article  CAS  Google Scholar 

  27. Schickel R, Boyerinas B, Park SM, Peter ME. MicroRNAs: key players in the immune system, differentiation, tumorigenesis and cell death. Oncogene. 2008;27(45):5959–5974.

    Article  CAS  PubMed  Google Scholar 

  28. Chuang TD, Panda H, Luo X, Chegini N. miR-200c is aberrantly expressed in leiomyomas in an ethnic-dependent manner and targets ZEBs, VEGFA, TIMP2, and FBLN5. Endocr Relat Cancer. 2012;19(4):541–556.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Chuang TD, Luo X, Panda H, Chegini N. miR-93/106b and their host gene, MCM7, are differentially expressed in leiomyomas and functionally target F3 and IL-8. Mol Endocrinol. 2012;26(6):1028–1042.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Fitzgerald JB, Chennathukuzhi V, Koohestani F, Nowak RA, Christenson LK. Role of microRNA-21 and programmed cell death 4 in the pathogenesis of human uterine leiomyomas. Fertil Steril. 2012;98(3):726–734.e2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Klemke M, Meyer A, Hashemi Nezhad M, Beige G, Bartnitzke S, Bullerdiek J. Loss of let-7 binding sites resulting from truncations of the 3’ untranslated region of HMGA2 mRNA in uterine leiomyomas. Cancer Genet Cytogenet. 2010;196(2):119–123.

    Article  CAS  PubMed  Google Scholar 

  32. Qiang W, Liu Z, Serna VA, et al. Down-regulation of miR-29b is essential for pathogenesis of uterine leiomyoma. Endocrinology. 2014;155(3):663–669.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Beermann J, Piccoli MT, Viereck J, Thum T. Non-coding RNAs in development and disease: background, mechanisms, and therapeutic approaches. Physiol Rev. 2016;96(4):1297–1325.

    Article  CAS  PubMed  Google Scholar 

  34. Jiang C, Li X, Zhao H, Liu H. Long non-coding RNAs: potential new biomarkers for predicting tumor invasion and metastasis. Mol Cancer. 2016;15(1):62.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Li J, Tian H, Yang J, Gong Z. Long noncoding RNAs regulate cell Growth, proliferation, and apoptosis. DNA Cell Biol. 2016;35(9):459–470.

    Article  CAS  PubMed  Google Scholar 

  36. Ulitsky I, Bartel DP. lincRNAs: genomics, evolution, and mechanisms. Cell. 2013;154(1):26–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Hangauer MJ, Vaughn IW, McManus MT. Pervasive transcription of the human genome produces thousands of previously unidentified long intergenic noncoding RNAs. PLoS Genet. 2013;9(6):e1003569.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Sigova AA, Mullen AC, Molinie B, et al. Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells. Proc Natl Acad Sci USA. 2013;110(8):2876–2881.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Cao MX, Jiang YP, Tang YL, Liang XH. The crosstalk between IncRNA and microRNA in cancer metastasis: orchestrating the epithelial-mesenchymal plasticity. Oncotarget. 2017;8(7):12472–12483.

    Article  PubMed  Google Scholar 

  40. Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10(3):R25.

    Google Scholar 

  41. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357–359.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioin-formatics. 2011;12:323.

    Article  CAS  Google Scholar 

  43. Sim Z, Evans J, Bhagwate A, et al. CAP-miRSeq: a comprehensive analysis pipeline for microRNA sequencing data. BMC Genomics. 2014;15:423.

    Article  Google Scholar 

  44. Volders PJ, Helsens K, Wang X, et al. LNCipedia: a database for annotated human IncRNA transcript sequences and structures. Nucleic Acids Res. 2013;41(Database issue):D246–D251.

    Article  CAS  PubMed  Google Scholar 

  45. Volders PJ, Verheggen K, Menschaert G, et al. An update on LNCipedia: a database for annotated human IncRNA sequences. Nucleic Acids Res. 2015;43(8):4363–4364.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. McCarthy DJ, Chen Y, Smyth GK. Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res. 2012;40(10):4288–4297.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26(1):139–140.

    Article  CAS  PubMed  Google Scholar 

  48. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4(1):44–57.

    Article  CAS  Google Scholar 

  49. Huang da W, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009;37(1):1–13.

    Article  CAS  Google Scholar 

  50. Chuang TD, Khorram O. Tranilast inhibits genes functionally involved in cell proliferation, fibrosis, and epigenetic regulation and epigenetically induces miR-29c expression in leiomyoma cells [Published online January 1, 2016]. Reprod Sci. 1933719116682878.

    Google Scholar 

  51. Chuang TD, Ho M, Khorram O. The regulatory function of miR-200c on inflammatory and cell-cycle associated genes in SK-LMS-1, A leiomyosarcoma cell line. Reprod Sci. 2015;22(5):563–571.

    Article  CAS  PubMed  Google Scholar 

  52. Chuang TD, Pearce WJ, Khorram O. miR-29c induction contributes to downregulation of vascular extracellular matrix proteins by glucocorticoids. Am J Physiol Cell Physiol. 2015;309(2):C117–C125.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Khorram O, Chuang TD, Pearce WJ. Long-term effects of maternal undernutrition on offspring carotid artery remodeling: role of miR-29c. J Dev Orig Health Dis. 2015;6(4):342–349.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Almeida TA, Quispe-Ricalde A, Montes de Oca F, Foronda P, Hernandez MM. A high-throughput open-array qPCR gene panel to identify housekeeping genes suitable for myometrium and leiomyoma expression analysis. Gynecol Oncol. 2014;134(1):138–143.

    Article  CAS  PubMed  Google Scholar 

  55. Braconi C, Kogure T, Valeri N, et al. microRNA-29 can regulate expression of the long non-coding RNA gene MEG3 in hepatocellular cancer. Oncogene. 2011;30(47):4750–4756.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Do JT, Han DW, Gentile L, Sobek-Klocke I, Stehling M, Scholer HR. Enhanced reprogramming of Xist by induced upregulation of Tsix and Dnmt3a. Stem Cells (Dayton, Ohio). 2008;26(11):2821–2831.

    Article  CAS  Google Scholar 

  57. Wang Z, Jinnin M, Nakamura K, et al. Long non-coding RNA TSIX is upregulated in scleroderma dermal fibroblasts and controls collagen mRNA stabilization. Exp Dermatol. 2016;25(2):131–136.

    Article  CAS  PubMed  Google Scholar 

  58. Yuan JH, Yang F, Wang F, et al. A long noncoding RNA activated by TGF-beta promotes the invasion-metastasis cascade in hepatocellular carcinoma. Cancer Cell. 2014;25(5):666–681.

    Article  CAS  PubMed  Google Scholar 

  59. Shi SJ, Wang LJ, Yu B, Li YH, Jin Y, Bai XZ. LncRNA-ATB promotes trastuzumab resistance and invasion-metastasis cascade in breast cancer. Oncotarget. 2015;6(13):11652–11663.

    Article  PubMed  PubMed Central  Google Scholar 

  60. Terashima M, Tange S, Ishimura A, Suzuki T. MEG3 long non-coding RNA contributes to the epigenetic regulation of epithelial-mesenchymal transition in lung cancer cell lines. J Biol Chem. 2017;292(1):82–99.

    Article  CAS  PubMed  Google Scholar 

  61. Yu X, Zheng H, Chan MT, Wu WK. HULC: an oncogenic long non-coding RNA in human cancer. J Cell Mol Med. 2017;21(2):410–417.

    Article  CAS  PubMed  Google Scholar 

  62. Li SP, Xu HX, Yu Y, et al. LncRNA HULC enhances epithelial-mesenchymal transition to promote tumorigenesis and metastasis of hepatocellular carcinoma via the miR-200a-3p/ZEB1 signaling pathway. Oncotarget. 2016;7(27):42431–42446.

    Article  PubMed  PubMed Central  Google Scholar 

  63. Liu X, Chen H, Kong W, et al. Down-regulated long non-coding RNA-ATB in preeclampsia and its effect on suppressing migration, proliferation, and tube formation of trophoblast cells. Placenta. 2017;49:80–87.

    Article  CAS  PubMed  Google Scholar 

  64. Ulitsky I. Evolution to the rescue: using comparative genomics to understand long non-coding RNAs. Nat Rev Genet. 2016;17(10):601–614.

    Article  CAS  PubMed  Google Scholar 

  65. Orom UA, Derrien T, Guigo R, Shiekhattar R. Long noncoding RNAs as enhancers of gene expression. Cold Spring Harb Symp Quant Biol. 2010;75:325–331.

    Article  CAS  PubMed  Google Scholar 

  66. Moore A. Commissioning support. The future will be integrated. Health Serv J. 2013;123(6344):suppl 10-suppl 11.

    Google Scholar 

  67. Huang X, Yuan T, Tschannen M, et al. Characterization of human plasma-derived exosomal RNAs by deep sequencing. BMC Genomics. 2013;14:319.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Viereck J, Thum T. Circulating noncoding RNAs as biomarkers of cardiovascular disease and injury. Circ Res. 2017;120(2):381–399.

    Article  CAS  PubMed  Google Scholar 

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  1. LA-Biomed Research Institute, Torrance, CA, USA

    Tsai-Der Chuang PhD & Omid Khorram MD, PhD

  2. Omid Khorram, Department of Obstetrics and Gynecology, Harbor-UCLA Medical Center, Box 489, 1000 W. Carson St, Torrance, CA, 90502, USA

    Tsai-Der Chuang PhD & Omid Khorram MD, PhD

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Chuang, TD., Khorram, O. Expression Profiling of IncRNAs, miRNAs, and mRNAs and Their Differential Expression in Leiomyoma Using Next-Generation RNA Sequencing. Reprod. Sci. 25, 246–255 (2018). https://doi.org/10.1177/1933719117711265

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