The expression of long non-coding RNA AK089560 in mesenchymal stem cells undergoing osteogenic and adipogenic differentiation

doi:10.3969/j.issn.2095-4344.2014.23.021

Abstract

Abstract:

BACKGROUND: Recent studies have found that stem cell pluripotency and differentiation is regulated by many long non-coding RNAs (LncRNAs). The expression and effect of LncRNA AK089560 during differentiation of stem cells is unclear.

OBJECTIVE: To investigate the expression of LncRNA AK089560 in mesenchymal stem cells C3H10T1/2 undergoing osteogenic and adipogenic differentiation.

METHODS: Osteogenic differentiation of mesenchymal stem cells C3H10T1/2 was induced by recombinant human bone morphogenetic protein-2 and evaluated using alkaline phosphatase staining. The adipogenic differentiation of mesenchymal stem cells C3H10T1/2 was induced by three factors (dexamethasone, indomethacin and insulin) and evaluated by oil red O staining. The dynamical expression of LncRNA AK089560 was detected by qRT-PCR assay. The AK089560 secondary structure was predicted using RNAfold software. The relationship between AK089560 and neighboring protein-coding genes was analyzed using UCSC genome browser and visualized by fancyGENE online software.

RESULTS AND CONCLUSION: Over 70% of C3H10T1/2 cells were positive for alkaline phosphatase after osteogenic induction and more than 80% of the cells positive for oil red O staining after adipogenic induction. qRT-PCR results showed that the expression of LncRNA AK089560 at days 2, 4, 6 of both osteogenic and adipogenic differentiation was significantly decreased compared with the control group (P < 0.05). Bioinformatics analysis showed that there was a stem-loop structure for AK089560 and sense overlap relationship between AK089560 and protein-encoding gene Sema3a. These findings indicate that LncRNA AK089560 expression is reduced during osteogenic differentiation and adipogenic differentiation, showing that AK089560 may be involved in regulating the multi-directional differentiation of stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: cell differentiation, mesenchymal stem cells, bone morphogenetic proteins

CLC Number:

R394.2

Zuo Chang-qing, Lu Han-yun, Zhong Yue-chun, Wang Zong-gui, Dai Zhong, Liu Yu-yu, Wu Tie. The expression of long non-coding RNA AK089560 in mesenchymal stem cells undergoing osteogenic and adipogenic differentiation[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(23): 3732-3738.

Figures/Tables 3

2.1 间质干细胞C3H10T1/2早期成骨分化鉴定结果碱性磷酸酶(ALP)是早期成骨分化的标志，其表达与成骨细胞的分化、成熟呈正相关。在实验中，实验用rhBMP 2诱导间质干细胞C3H10T1/2成骨定向分化。质量浓度300 μg/L rhBMP 2诱导分化6 d后，进行碱性磷酸酶染色，结果显示：成骨诱导分化组可见70%以上细胞染色阳性，细胞呈紫蓝色，而对照组细胞未见明显变化，见图1，这提示rhBMP 2成功诱导间质干细胞C3H10T1/2早期成骨分化。 2.2 间质干细胞C3H10T1/2成脂分化鉴定结果间质干细胞C3H10T1/2经地塞米松、吲哚美辛、胰岛素三因子(DII)联合诱导成脂分化，在分化的第2天，细胞形态明显变圆，细胞胞浆内开始出现少量细小脂滴，随着时间延长，脂滴数量明显增多，脂滴明显增大。倒置显微镜下可见晶莹透亮的小圆形脂滴。分化到8 d时，胞内的小脂滴逐渐融合成折光性较强的大脂滴，而对照组无明显变化，细胞内没有或极少脂滴出现。油红O染色结果显示：80%以上细胞呈亮红色，对照组无明显变化。提示C3H10T1/2细胞在三因子作用8 d能有效定向诱导成脂分化，见图2。 2.3 qRT-PCR鉴定LncRNA AK089560在成骨分化与成脂分化中时序表达模式实验前期通过长链非编码RNA芯片筛选发现AK089560在成骨分化中下调表达。实验进一步采用qRT-PCR法鉴定LncRNA AK089560在成骨分化与成脂分化中时序表达模式。结果显示AK089560的表达值在C3H10T1/2细胞成骨分化的第2，4，6天明显下降，与对照组相比较差异具有显著性意义(P < 0.05)；在C3H10T1/2细胞成脂分化的第2，4，6天，AK089560的表达量同样明显下降，与对照组相比较差异有显著性意义(P < 0.05)。见图3。 2.4 AK089560编码蛋白性能鉴定根据CPC软件预测分析结果，AK089560序列的蛋白编码得分为-0.961223；说明此序列不能编码蛋白质，属于长链非编码RNA。 2.5 AK089560二级结构预测 LncRNA和microRNA除了碱基数长度的差异外，近期的研究表明lncRNA具有保守的二级结构，可以与蛋白质、DNA和RNA发生相互作用，因此对生物过程的调控比microRNA更加复杂多样。通过采用RNAflod软件进行AK089560结构预测，实验发现AK089560具有非常复杂的结构特点，包含有多个茎环结构。见图4。"

References

[1] Huang J, Zhou N, Watabe K, et al. Long non-coding RNA UCA1 promotes breast tumor growth by suppression of p27 (Kip1). Cell Death Dis. 2014;5:e1008.
[2] Yang F, Zhang L, Huo XS, et al.Long noncoding RNA high expression in hepatocellular carcinoma facilitates tumor growth through enhancer of zeste homolog 2 in humans. Hepatology. 2011;54(5):1679-1689.
[3] Massone S, Vassallo I, Fiorino G, et al. 17A, a novel non-coding RNA, regulates GABA B alternative splicing and signaling in response to inflammatory stimuli and in Alzheimer disease. Neurobiol Dis. 2011;41(2):308-317.
[4] Holden T, Nguyen A, Lin E, et al. Exploratory bioinformatics study of lncRNAs in Alzheimer's disease mRNA sequences with application to drug development. Comput Math Methods Med. 2013: 579136.
[5] Ghosal S, Das S, Chakrabarti J. Long noncoding RNAs: new players in the molecular mechanism for maintenance and differentiation of pluripotent stem cells. Stem Cells Dev. 2013; 22(16):2240-2253.
[6] Hu W, Alvarez-Dominguez JR, Lodish HF. Regulation of mammalian cell differentiation by long non-coding RNAs. EMBO Rep. 2012;13(11):971-983.
[7] Sheik Mohamed J, Gaughwin PM, Lim B, et al. Conserved long noncoding RNAs transcriptionally regulated by Oct4 and Nanog modulate pluripotency in mouse embryonic stem cells. RNA. 2010;16(2):324-337.
[8] Ng SY, Bogu GK, Soh BS, et al. The long noncoding RNA RMST interacts with SOX2 to regulate neurogenesis. Mol Cell. 2013;51(3):349-359.
[9] Klattenhoff CA, Scheuermann JC, Surface LE, et al. Braveheart, a long noncoding RNA required for cardiovascular lineage commitment. Cell. 2013;152(3): 570-583.
[10] Guttman M, Donaghey J, Carey BW, et al. lincRNAs act in the circuitry controlling pluripotency and differentiation. Nature. 2011;477(7364):295-300.
[11] Zuo C, Wang Z, Lu H, et al. Expression profiling of lncRNAs in C3H10T1/2 mesenchymal stem cells undergoing early osteoblast differentiation. Mol Med Rep. 2013;8(2): 463-467.
[12] Cheng SL, Shao JS, Charlton-Kachigian N, et al. MSX2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors. J Biol Chem. 2003;278(46):45969-45977.
[13] Kong L, Zhang Y, Ye ZQ, et al. CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine. Nucleic Acids Res. 2007;35(Web Server issue):W345-W349.
[14] Hofacker IL. RNA secondary structure analysis using the Vienna RNA package. Curr Protoc Bioinformatics. 2009; Chapter 12:Unit12.2.
[15] Reuter JS, Mathews DH. RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics. 2010;11:129.
[16] Fujita PA, Rhead B, Zweig AS, et al.The UCSC Genome Browser database: update 2011. Nucleic Acids Res. 2011; 39(Database issue):D876-882.
[17] Rambaldi D, Ciccarelli FD. FancyGene: dynamic visualization of gene structures and protein domain architectures on genomic loci. Bioinformatics. 2009;25(17):2281-2282.
[18] Lin GL, Hankenson KD. Integration of BMP, Wnt, and notch signaling pathways in osteoblast differentiation. J Cell Biochem. 2011;112(12):3491-3501.
[19] Abdallah BM, Kassem M. New factors controlling the balance between osteoblastogenesis and adipogenesis. Bone. 2012; 50(2):540-545.
[20] Chau JF, Leong WF, Li B. Signaling pathways governing osteoblast proliferation, differentiation and function. Histol Histopathol. 2009;24(12):1593-1606.
[21] James AW. Review of signaling pathways governing msc osteogenic and adipogenic differentiation. Scientifica (Cairo). 2013;2013:684736.
[22] Kawai M, Rosen CJ. The IGF-I regulatory system and its impact on skeletal and energy homeostasis. J Cell Biochem. 2010;111(1):14-19.
[23] Zamani N and Brown CW: Emerging roles for the transforming growth factor-{beta} superfamily in regulating adiposity and energy expenditure. Endocr Rev. 2010;32(3): 387-403.
[24] Luzi E, Marini F, Sala SC, et al.Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor. J Bone Miner Res. 2008;23(2):287-295.
[25] Kim YJ, Bae SW, Yu SS, et al.miR-196a regulates proliferation and osteogenic differentiation in mesenchymal stem cells derived from human adipose tissue. J Bone Miner Res. 2009;24(5):816-825.
[26] Li Z, Hassan MQ, Volinia S, et al. A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci U S A. 2008;105(37):13906-13911.
[27] Huang J, Zhao L, Xing L, et al. MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation. Stem Cells. 2010;28(2):357-364.
[28] Bork S, Horn P, Castoldi M, et al. Adipogenic differentiation of human mesenchymal stromal cells is down-regulated by microRNA-369-5p and up-regulated by microRNA-371. J Cell Physiol. 2011;226(9):2226-2234.
[29] Pang WJ, Lin LG, Xiong Y, et al. Knockdown of PU.1 AS lncRNA inhibits adipogenesis through enhancing PU.1 mRNA translation. J Cell Biochem. 2013;114(11):2500-2512.
[30] Sun L, Goff LA, Trapnell C, et al. Long noncoding RNAs regulate adipogenesis. Proc Natl Acad Sci U S A. 2013; 110(9):3387-3392.
[31] Zhu L, Xu PC. Downregulated LncRNA-ANCR promotes osteoblast differentiation by targeting EZH2 and regulating Runx2 expression. Biochem Biophys Res Commun. 2013; 432(4):612-617.
[32] Yang Z, Bian C, Zhou H, et al. MicroRNA hsa-miR-138 inhibits adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells through adenovirus EID-1. Stem Cells Dev. 2010;20(2):259-267.
[33] Eskildsen T, Taipaleenmaki H, Stenvang J, et al. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo. Proc Natl Acad Sci U S A. 2011;108(15):6139-6144.
[34] Kretz M, Webster DE, Flockhart RJ, et al. Suppression of progenitor differentiation requires the long noncoding RNA ANCR. Genes Dev. 2012;26(4):338-343.
[35] Lindberg J, Lundeberg J. The plasticity of the mammalian transcriptome. Genomics. 2010;95(1):1-6.
[36] Wilusz JE, Sunwoo H, Spector DL. Long noncoding RNAs: functional surprises from the RNA world. Genes Dev. 2009; 23(13):1494-1504.
[37] Ponting CP, Oliver PL, Reik W. Evolution and functions of long noncoding RNAs. Cell. 2009;136(4):629-641.
[38] Jiang J, Jing Y, Cost GJ, et al. Translating dosage compensation to trisomy 21. Nature. 2013; 500(7462): 296-300.
[39] Watts R, Johnsen VL, Shearer J, et al. Myostatin-induced inhibition of the long noncoding RNA Malat1 is associated with decreased myogenesis. Am J Physiol Cell Physiol. 2013;304(10):C995-1001.
[40] Luo M, Li Z, Wang W, et al. Upregulated H19 contributes to bladder cancer cell proliferation by regulating ID2 expression. FEBS J. 2013;280(7):1709-1716.
[41] Phermthai T, Suksompong S, Tirawanchai N, et al. Epigenetic analysis and suitability of amniotic fluid stem cells for research and therapeutic purposes. Stem Cells Dev. 2013; 22(9):1319-1328.
[42] Meola N, Pizzo M, Alfano G, et al. The long noncoding RNA Vax2os1 controls the cell cycle progression of photoreceptor progenitors in the mouse retina. RNA. 2012;18(1):111-123.
[43] Sato S, Yoshida W, Soejima H, et al. Methylation dynamics of IG-DMR and Gtl2-DMR during murine embryonic and placental development. Genomics. 2011;98(2):120-127.
[44] Askarian-Amiri ME, Crawford J, French JD, et al. SNORD-host RNA Zfas1 is a regulator of mammary development and a potential marker for breast cancer. RNA. 2011;17(5):878-891.
[45] Szegedi K, Sonkoly E, Nagy N, et al. The anti-apoptotic protein G1P3 is overexpressed in psoriasis and regulated by the non-coding RNA, PRINS Exp Dermatol. 2010;19(3): 269-278.
[46] Kim NH, Lee CH, Lee AY. H19 RNA downregulation stimulated melanogenesis in melasma. Pigment Cell Melanoma Res. 2010;23(1):84-92.
[47] Matouk I, Ayesh B, Schneider T, et al. Oncofetal splice-pattern of the human H19 gene.Biochem Biophys Res Commun. 2004;318(4):916-919.
[48] Lanz RB, Chua SS, Barron N, et al. Steroid receptor RNA activator stimulates proliferation as well as apoptosis in vivo. Mol Cell Biol. 2003;23(20):7163-7176.
[49] Katayama S, Tomaru Y, Kasukawa T, et al. Antisense transcription in the mammalian transcriptome. Science. 2005; 309(5740):1564-1566.
[50] Lempradl A, Ringrose L. How does noncoding transcription regulate Hox genes? Bioessays. 2008;30(2):110-121.
[51] Fukuda T, Takeda S, Xu R, et al. Sema3A regulates bone-mass accrual through sensory innervations. Nature. 2013;497(7450):490-493.
[52] Hayashi M, Nakashima T, Taniguchi M, et al. Osteoprotection by semaphorin 3A. Nature. 2012;485(7396):69-74.