Role of long non-coding RNA in osteoblast differentiation of C3H10T1/2 cells induced by bone morphogenetic protein-2

doi:10.3969/j.issn.2095-4344.2014.20.020

Abstract

Abstract:

BACKGROUND: Long non-coding RNAs (lncRNAs) have became the hot topic in current studies and play an important role in the tumorigenesis. However, lncRNAs involved in the osteoblast differentiation remain poorly reported.
OBJECTIVE: To investigate the role of human LncRNAs in osteogenic differentiation of C3H10T1/2 cells induced by bone morphogenetic protein-2 and explore action mechanism.
METHODS: The induction of bone morphogenetic protein-2 was validated by alkaline phosphatase staining and the expression of corresponding genes was detected. The lncRNA expression profile was analyzed using the Arraystar lncRNA array in C3H10T1/2 MSCs undergoing early osteoblast differentiation. The expression with or without bone morphogenetic protein-2 induction was compared with high-flux sequencing, and the down-regulated genes were screened. The effect of lncRNA overexpression on osteogenic differentiation of C3H10T1/2 cells induced by bone morphogenetic protein-2 was observed.
RESULTS AND CONCLUSION: The bone morphogenetic protein-2 induced C3H10T1/2 cells led to increased alkaline phosphatase activity. After 72 hours of bone morphogenetic protein-2 induction, alkaline phosphatase,
Id1,osteocalcin, Runx2, sp7 expression were increased (P < 0.05). There were 24 down-regulated lncRNAs identified between bone morphogenetic protein-2 treated and untreated groups, the decrease of expression was 1.5 folds, and among them, only AK035085 contained intron. Compared with control group with no AK03508 expression, over-expression lncRNA AK035085 decreased the expression of alkaline phosphatase, Id1, osteocalcin, Runx2, sp7 (P < 0.05). Experimental findings indicate that bone morphogenetic protein-2 induces osteogenic differentiation of C3H10T1/2 cells and AK035085 inhibits the osteogenic differentiation.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: bone morphogenetic proteins, mesenchymal stem cells, tumor, alkaline phosphatase, osteoblasts

CLC Number:

R318

Cheng Chen, Gao Yan, Li Jing, Pan Qiu-hui. Role of long non-coding RNA in osteoblast differentiation of C3H10T1/2 cells induced by bone morphogenetic protein-2[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(20): 3223-3229.

Figures/Tables 3

2.1 骨形态发生蛋白2诱导C3H10T1/2细胞的碱性磷酸酶活性变化骨髓间充质干细胞向成骨细胞分化受多种细胞因子和生长因子的调节，其中研究最为广泛的是骨形态发生蛋白2，是转录生长因子家族的成员。碱性磷酸酶活性是成骨分化早期指标。骨形态发生蛋白2诱导C3H10T1/2细胞7 d后进行碱性磷酸酶染色。发现相对于未经骨形态发生蛋白2诱导的C3H10T1/2细胞，诱导后的细胞碱性磷酸酶染色明显。此结果暗示骨形态发生蛋白2诱导C3H10T1/2细胞进入成骨早期分化过程(图1)。 2.2 骨形态发生蛋白2诱导C3H10T1/2细胞72 h后定量检测成骨相关指标骨形态发生蛋白2诱导细胞72 h之后，采用荧光定量PCR的方法，检测几个经典的成骨指标。结果发现，与未经骨形态发生蛋白2处理的对照组相比，骨形态发生蛋白2诱导72 h后C3H10T1/2细胞的成骨指标碱性磷酸酶、Id1、骨钙素、Runx2、sp7表达上升(P < 0.05，图2)。 2.3 芯片结果未诱导的C3H10T1/2细胞与骨形态发生蛋白2(100 g/L)诱导细胞芯片杂交后结果比较，其中下降达1.5倍的长链非编码RNAs有24条，差异有显著性意义(P < 0.05)，见图3，4。 2.4 长链非编码RNA AK035085过表达影响骨形态发生蛋白2诱导成骨分化 24条表达下降的长链非编码RNA中除了AK035085有内含子外，其余均无内含子。针对AK035085构建质粒，转染到细胞中，定量检测AK035085在细胞中的表达含量，发现与对照组相比，上升了210倍。100 μg/L骨形态发生蛋白2诱导72 h，进行RT-PCR反应。与未过表达AK035085的对照组比较，观察成骨相关指标碱性磷酸酶、Id1、骨钙素、Runx2、sp7基因，发现这些基因表达下降(P < 0.05)，见图5。将AK035085过表达的C3H10T1/2细胞用骨形态发生蛋白2(100 μg/L)诱导7 d，用成骨分化经典指标碱性磷酸酶染色观察成骨分化水平。与未过表达AK035085对照组相比，骨形态发生蛋白2诱导时碱性磷酸酶活性降低(图6)。"

References

[1] Morceau F, Chateauvieux S, Diederich M,et al. Long and short non-coding RNAs as regulators of hematopoietic differentiation. Int J Mol Sci. 2013;14(7):14744-14770.
[2] Kung JT, Colognori D, Lee JT. Long noncoding RNAs: past, present, and future. Genetics. 2013;193(3):651-669.
[3] Wilusz JE,Sunwoo H,Spector DL.Long noncoding RNAs: functional surprises from the RNA world. Genes Dev. 2009; 23(13):1494-1504.
[4] Huttenhofer A,Schattner P,Polacek N. Non-coding RNAs: hope or hype? Trends Genet. 2005;21(5):289-297.
[5] Gong C, Maquat LE. lncRNAs transactivate STAU1- mediated mRNA decay by duplexing with 3' UTRs via Alu elements. Nature. 2011;470(7333): 284-288.
[6] Wutz A, Rasmussen TP, Jaenisch R. Chromosomal silencing and localization are mediated by different domains of Xist RNA. Nat Genet. 2002;30(2):167-174.
[7] Clemson CM, Hutchinson JN, Lawrence JB,et al. An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles. Mol Cell. 2009; 33(6):717-726.
[8] Gupta RA, Shah N, Chang HY,et al. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature. 2010;464(7291):1071-1076.
[9] Tsai MC, Manor O, Chang HY,et al. Long noncoding RNA as modular scaffold of histone modification complexes. Science. 2010;329(5992):689-693.
[10] Sone M, Hayashi T, Tarui H, et al. The mRNA -like noncoding RNA Gomafu constitutes a novel nuclear domain in a subset of neurons.J Cell Sci. 2007;120(15):2498-2506.
[11] Wozney JM, Rosen V, Celeste AJ, et al. Novel regulators of bone formation: molecular clones and activities. Science. 1988;242:1528-1534.
[12] Makhjani NS, Bischoff DS, Yamaguchi DT. Regulation of proliferation and migration in retinoic acid treated C3H10T1/2 cells by TGF-beta is forms. J Cell Physiol. 2005;202(1):304-313.
[13] Date T, Doiguchi Y, Shindo H,et al. Bone morphogenetic protein-2 induces differentiation of multipotent C3H10T1/2 cells into osteoblasts, chondrocytes, and adipocytes in vivo and in vitro. J Orthop Sci. 2004; 9(5):503-508.
[14] Zuo C, Wang Z，Cui L,et al. Expression profiling of lncRNAs in C3H10T1/2 mesenchymal stem cells undergoing early osteoblast differentiation. Mol Med Rep. 2013;8(2):463-467.
[15] Perez DS, Hoage TR, Smith DI.et al. Long abundantly expressed non-coding transcripts are altered in cancer. Hum Mol Genet. 2008;17:642-655.
[16] Silva JM, Perez DS, Smith DI,et al. Identification of long stress-induced non-coding transcripts that have altered expression in cancer. Genomics. 2010;95(6):355-362.
[17] Prensner JR, Iyer MK, Chinnaiyan AM,et al. Transcriptome sequencing across a prostate cancer cohort identifies PCAT-1, an unannotated lincRNA implicated in disease progression. Nat Biotechnol. 2011;29:742-749.
[18] Yang F, Zhang L, Sun SH, et al. Long noncoding RNA high expression in hepatocellular carcinoma facilitates tumor growth through enhancer of zeste homolog 2 in humans. Hepatology.2011;54:1679-1689.
[19] Yu G,Yao W, Ye Z,et al. LncRNAs expression signatures of renal clear cell carcinoma revealed by microarray. PLoS One. 2012;7:e42377.
[20] Nagano T, Fraser P. No-nonsense functions for long non- coding RNAs. Cell. 2011;145:178-181.
[21] Guttmann M, Rinn JL. Modular regulatory principles of large non-coding RNAs. Nature. 2012; 482: 339-346.
[22] Broadbent HM, Peden JF, Lorkowski S,et al. Susceptibility to coronary artery disease and diabetes is encoded by distinct，tightly linked SNPs in the ANRIL locus on chromosome 9p. Hum Mol Genet. 2008;17(6) :806-814.
[23] Srikantan V, Zou Z, Petrovics G, et al. PCGEM1, a prostate-specific gene, is overexpressed in prostate cancer.Proc Natl Acad Sci USA. 2000;97(22): 12216-12221.
[24] Zhou Y, Zhong Y, Wang Y, et al. Activation of p53 by MEG3 non-coding RNA. J Biol Chem. 2007;282(34):24731-24742.
[25] Yu W,Gius D,Onyango P,et al.Epigenetic silencing of tumor suppressor gene p15 by its antisense RNA. Nature. 2008; 451(7175):202-206.
[26] Gupta RA,Shah N,Wang KC,et al.Long non-coding RNA HOTAI Reprograms chromatin state to promote cancer metastasis.Nature. 2010;464(7291):1071-1076.
[27] Crea F, Clermont PL, Helgason CD, et al. The non-coding transcriptome as a dynamic regulator of cancer metastasis. Cancer Metastasis Rev. 2013.
[28] Subramanian M, Jones MF. Long Non-Coding RNAs Embedded in the Rb and p53 Pathways. Cancers (Basel). 2013;5(4):1655-1675.
[29] Huang T, Alvarez A, Hu B, et al. Noncoding RNAs in cancer and cancer stem cells. Chin J Cancer. 2013; 32(11):582-593.
[30] Taft RJ,Pang KC,Mercer TR,et al. Non-coding RNAs: regulators of disease. J Pathol. 2010;220(2):126-139.
[31] Cesana M, Cacchiarelli D, Legnini I, et al. A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA. Cell. 2011;147:358-369.
[32] Kretz M, Siprashvili Z, Chu C, et al. Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature. 2013;493:231-235.
[33] Kretz M, Webster DE, Flockhart RJ, et al. Suppression of progenitor differentiation requires the long non coding RNA ANCR. Genes Dev. 2012;26:338-343.
[34] Ng SY, Johnson R, Stanton LW,et al.Human long non-coding RNAs promote pluripotency and neuronal differentiation by association with chromatin modifiers and transcription factors. EMBO J. 2011;31:522-533.
[35] Huang HY, Hu LL, Tang QQ,et al. Involvement of Cytoskeleton-associated Proteins in the Commitment of C3H10T1/2 Pluripotent Stem Cells to Adipocyte Lineage Induced by BMP2/4. Biochem Biophys Res Commun. 2000; 268(3):757-762.
[36] Ogasawara T, Ohba S,Hoshi K,et al. Nanog promotes osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2 by modulating bone morphogenetic protein (BMP) signaling. J Cell Physiol. 2013;228(1):163-171.
[37] Mikami Y, Asano M, Takagi M,et al.Bone morphogenetic protein 2 and dexamethasone synergistically increase alkaline phosphatase levels through JAK/STAT signaling in C3H10T1/2 cells. J Cell Physiol. 2010;223(1):123-133.
[38] Zhu J, Shimizu E, Qin L,et al. EGFR signaling suppresses osteoblast differentiation and inhibits expression of master osteoblastic transcription factors Runx2 and Osterix. J Cell Biochem. 2011;112:1749-1760.
[39] 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.
[40] Fatica A, Bozzoni I. Long non-coding RNAs: new players in cell differentiation and development. Nat Rev Genet. 2014; 15(1):7-21.
[41] 夏天,肖丙秀,郭俊明. 长链非编码RNA的作用机制及其研究方法[J]. 遗传,2013, 35(3):269-280.
[42] Spizzo R,Almeida MI,Colombatti A,et al. Long non-coding RNAs and cancer:a new frontier of translational research. Oncogene. 2012;(43):4577-4587.
[43] A410.新的长链非编码RNA LLEST在急性白血病中作用机制的研究. 中华医学会第十二次全国血液学学术会议,2012.
[44] 连晓清,王连生. 长链非编码RNA的功能及与心血管病的研究进展[J]. 国际遗传学杂志,2012,35(5):295-300.
[45] 何烨,王科明. 长链非编码RNA在肿瘤中作用的研究及进展[J]. 现代肿瘤医学,2013,21(6):1811-1383.