Differential Expression Pattern of linc-ROR Spliced Variants in Pluripotent and Non-Pluripotent Cell Lines

Document Type : Original Article


1 Molecular Genetics Department, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

2 Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK

3 Department of Biomolecular Sciences, University of Urbino, Via Saffi Urbino, Italy

4 Cardiogenetic Research Center, Rajaie Cardiovascular Medical and Research Center, Iran University of Medical Sciences, Tehran, Iran

5 Department of Biology, Science and Research branch, Islamic Azad University, Tehran, Iran


Objective: The human large intergenic non-coding RNA-regulator of reprogramming program (linc-ROR) is known as a
stem cell specific linc-RNA. linc-ROR counteracts differentiation via sequestering microRNA-145 (miR-145) that targets OCT4 transcript. Despite the research on the expression and function, the exact structure of Linc-ROR transcripts is not clear. Considering the contribution of alternative splicing in transcripts structures and function, identifying different spliced variants of linc-ROR is necessary for further functional analyses. We aimed to find the alternatively spliced transcripts of linc-ROR and investigate their expression pattern in stem and cancer cell lines and during neural differentiation of NT2 cells as a model for understanding linc-ROR role in stem cell and differentiation.
Materials and Methods: In this experimental study, linc-ROR locus was scanned for identifying novel exons. Different primer sets were used to detect new spliced variants by reverse transcription polymerase chain reaction (RT-PCR) and direct sequencing. Quantitative PCR (qPCR) and RT-PCR were employed to profile expression of linc-ROR transcripts in different cell lines and during neural differentiation of stem cells.
Results: We could discover 13 novel spliced variants of linc-ROR harboring unique array of exons. Our work uncovered
six novel exons, some of which were the product of exonized transposable elements. Monitoring expression profile of the linc-ROR spliced variants in a panel of pluripotent and non-pluripotent cells exhibited that all transcripts were primarily expressed in pluripotent cells. Moreover, the examined linc-ROR spliced variants showed a similar downregulation during neural differentiation of NT2 cells.
Conclusion: Altogether, our data showed despite the difference in the structure and composition of exons, various spliced variants of linc-ROR showed similar expression pattern in stem cells and through differentiation.


  1. Fort V, Khelifi G, Hussein SM. Long non-coding RNAs and transposable elements: a functional relationship. Biochim Biophys Acta Mol Cell Res. 2021; 1868(1): 118837.
  2. Statello L, Guo CJ, Chen LL, Huarte M. Gene regulation by long non-coding RNAs and its biological functions. Nat Rev Mol Cell Biol. 2021; 22(2): 96-118.
  3. Szcześniak MW, Makałowska I. lncRNA-RNA interactions across the human transcriptome. PLoS One. 2016; 11(3): e0150353.
  4. Yang Y, Chen Q, Piao HY, Wang B, Zhu GQ, Chen EB, et al. HNRNPAB- regulated lncRNA-ELF209 inhibits the malignancy of hepatocellular carcinoma. Int J Cancer. 2020; 146(1): 169-180.
  5. Xue Z, Hennelly S, Doyle B, Gulati AA, Novikova IV, Sanbonmatsu KY, et al. A G-rich motif in the lncRNA braveheart interacts with a zinc-finger transcription factor to specify the cardiovascular lineage. Mol Cell. 2016; 64(1): 37-50.
  6. Wang C, Wang L, Ding Y, Lu X, Zhang G, Yang J, et al. LncRNA structural characteristics in epigenetic regulation. Int J Mol Sci. 2017; 18(12): 2659.
  7. Militello G, Weirick T, John D, Döring C, Dimmeler S, Uchida S. Screening and validation of lncRNAs and circRNAs as miRNA sponges. Brief Bioinform. 2017; 18(5): 780-788.
  8. Chen X, Yan CC, Zhang X, You ZH. Long non-coding RNAs and complex diseases: from experimental results to computational models. Brief Bioinform. 2017; 18(4): 558-576.
  9. Meseure D, Vacher S, Lallemand F, Alsibai KD, Hatem R, Chemlali W, et al. Prognostic value of a newly identified MALAT1 alternatively spliced transcript in breast cancer. Br J Cancer. 2016; 114(12): 1395-1404.
  10. Malakootian M, Azad FM, Naeli P, Pakzad M, Fouani Y, Bajgan ET, et al. Novel spliced variants of OCT4, OCT4C and OCT4C1, with distinct expression patterns and functions in pluripotent and tumor cell lines. Eur J Cell Biol. 2017; 96(4): 347-355.
  11. Azad FM, Malakootian M, Mowla SJ. lncRNA PSORS1C3 is regulated by glucocorticoids and fine-tunes OCT4 expression in nonpluripotent cells. Sci Rep. 2019; 9 : 8370.
  12. Fico A, Fiorenzano A, Pascale E, Patriarca EJ, Minchiotti G. Long non-coding RNA in stem cell pluripotency and lineage commitment: functions and evolutionary conservation. Cell Mol Life Sci. 2019; 76(8): 1459-1471.
  13. Ransohoff JD, Wei Y, Khavari PA. The functions and unique features of long intergenic non-coding RNA. Nat Rev Mol Cell Biol. 2018; 19(3): 143-157.
  14. Loewer S, Cabili MN, Guttman M, Loh Y-H, Thomas K, Park IH, et al. Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells. Nat Genet. 2010; 42(12): 1113-1117.
  15. Wang Y, Xu Z, Jiang J, Xu C, Kang J, Xiao L, et al. Endogenous miRNA sponge lincRNA-RoR regulates Oct4, Nanog, and Sox2 in human embryonic stem cell self-renewal. Dev Cell. 2013; 25(1): 69- 80.
  16. Sahebi R, Malakootian M, Balalaee B, Shahryari A, Khoshnia M, Abbaszadegan MR, et al. Linc-ROR and its spliced variants 2 and 4 are significantly up-regulated in esophageal squamous cell carcinoma. Iran J Basic Med Sci. 2016; 19(10): 1131-1135.
  17. Chen W, Wang H, Liu Y, Xu W, Ling C, Li Y, et al. Linc-RoR promotes proliferation, migration, and invasion via the Hippo/YAP pathway in pancreatic cancer cells. J Cell Biochem. 2020; 121(1): 632-641.
  18. Yu X, Ding H, Shi Y, Yang L, Zhou J, Yan Z, et al. Downregulated expression of linc-ROR in gastric cancer and its potential diagnostic and prognosis value. Dis Markers. 2020; 2020: 7347298.
  19. Li X, Chen W, Jia J, You Z, Hu C, Zhuang Y, et al. The long noncoding RNA-RoR promotes the tumorigenesis of human colorectal cancer by targeting miR-6833-3p Through SMC4. Onco Targets Ther. 2020; 13: 2573-2581.
  20. Lou Y, Jiang H, Cui Z, Wang L, Wang X, Tian T. Linc-ROR induces epithelial-to-mesenchymal transition in ovarian cancer by increasing Wnt/β-catenin signaling. Oncotarget. 2017; 8(41): 69983- 69994.
  21. Peng Wx, Huang Jg, Yang L, Gong Ah, Mo YY. Linc-RoR promotes MAPK/ERK signaling and confers estrogen-independent growth of breast cancer. Mol Cancer. 2017; 16: 1-11.
  22. Baharvand H, Ashtiani SK, Taee A, Massumi M, Valojerdi MR, Yazdi PE, et al. Generation of new human embryonic stem cell lines with diploid and triploid karyotypes. Dev Growth Differ. 2006; 48(2): 117-128.
  23. Totonchi M, Taei A, Seifinejad A, Tabebordbar M, Rassouli H, Farrokhi A, et al. Feeder-and serum-free establishment and expansion of human induced pluripotent stem cells. Int J Dev Biol. 2009; 54(5): 877-886.
  24. Malakootian M, Azad FM, Fouani Y, Bajgan ET, Saberi H, Mowla SJ. Anti-differentiation non-coding RNA, ANCR, is differentially expressed in different types of brain tumors. J Neurooncol. 2018; 138(2): 261-270.
  25. Chen LL, Yang L. ALUternative regulation for gene expression. Trends Cell Biol. 2017; 27(7): 480-490.
  26. Atlasi Y, Mowla SJ, Ziaee SA, Gokhale PJ, Andrews PW. OCT4 spliced variants are differentially expressed in human pluripotent and nonpluripotent cells. Stem Cells. 2008; 26(12): 3068-3074.
  27. Bush SJ, Chen L, Tovar-Corona JM, Urrutia AO. Alternative splicing and the evolution of phenotypic novelty. Philos Trans R Soc Lond B Biol Sci. 2017; 372(1713): 20150474.
  28. Rodriguez JM, Pozo F, di Domenico T, Vazquez J, Tress ML. An analysis of tissue-specific alternative splicing at the protein level. PLoS Comput Biol. 2020; 16(10): e1008287.
  29. Poursani EM, Mehravar M, Soltani BM, Mowla SJ. OCT4B2, a novel alternative spliced variant of OCT4, is significantly upregulated under heat-stress condition and downregulated in differentiated cells. Tumour Biol. 2017; 39(10): 1010428317724280.
  30. Mirzadeh Azad F, Malakootian M, Mowla SJ. The regulatory effect of lncRNA PSORS1C3 on different variants of OCT4 in nonpluripotent cells. J Cell Mol Med. 2019; 11(1): 8-13.
  31. Arun G, Aggarwal D, Spector DL. MALAT1 long non-coding RNA: functional implications. Noncoding RNA. 2020; 6(2): 22.
  32. Johnson R, Guigó R. The RIDL hypothesis: transposable elements as functional domains of long noncoding RNAs. RNA. 2014; 20(7): 959-976.
  33. Babaian A, Mager DL. Endogenous retroviral promoter exaptation in human cancer. Mob DNA. 2016; 7: 24.
  34. Bejerano G, Lowe CB, Ahituv N, King B, Siepel A, Salama SR, et al. A distal enhancer and an ultraconserved exon are derived from a novel retroposon. Nature. 2006; 441(7089): 87-90.
  35. Zeng L, Pederson SM, Cao D, Qu Z, Hu Z, Adelson DL, et al. Genome- wide analysis of the association of transposable elements with gene regulation suggests that alu elements have the largest overall regulatory impact. J Comput Biol. 2018; 25(6): 551-562.
  36. Kelley D, Rinn J. Transposable elements reveal a stem cell-specific class of long noncoding RNAs. Genome Biol. 2012; 13(11): R107.
  37. Feng L, Shi L, Lu Yf, Wang B, Tang T, Fu Wm, et al. Linc-ROR promotes osteogenic differentiation of mesenchymal stem cells by functioning as a competing endogenous RNA for miR-138 and miR-145. Mol Ther Nucleic Acids. 2018; 11: 345-353.
  38. Hou P, Zhao Y, Li Z, Yao R, Ma M, Gao Y, et al. LincRNA-ROR induces epithelial-to-mesenchymal transition and contributes to breast cancer tumorigenesis and metastasis. Cell Death Dis. 2014; 5(6): e1287-e1287.
  39. Taheri Bajgan E, Gholipour A, Faghihi M, Mowla SJ, Malakootian M. Linc-ROR has a Potential ceRNA Activity for OCT4A by Sequestering miR-335-5p in the HEK293T Cell Line. Biochem Genet. 2022; 60(3): 1007-1024.
  40. Chen J, Weiss W. Alternative splicing in cancer: implications for biology and therapy. Oncogene. 2015; 34: 1-14.