Differential expression of microRNAs related to apoptosis in human osteoblasts induced by sodium fluoride

doi:10.3969/j.issn.2095-4344.0131

Abstract

Abstract:

BACKGROUND: Long-term excessive intake of fluoride, especially through drinking water, can cause chronic fluorosis of bone. The disease can lead to bone damage and deformity, and is difficult to recover. Unfortunately, we have not developed a noninvasive or minimally invasive method for its early diagnosis.
OBJECTIVE: To observe the expression of apoptosis-related miRNAs under the action of excessive fluorine in human osteoblasts.
METHODS: The fluorine model was established in the human osteoblasts by cultured with 20 and 40 mg/L sodium fluoride for 24 and 48 hours, respectively. The expression levels of apoptosis-related miRNAs were determined by PCR array.
RESULTS AND CONCLUSION: After 24-hour treatment of sodium fluoride, 48 kinds of miRNAs were upregulated and 4 ones were down-regulated in the osteoblasts. After 48-hour treatment of sodium fluoride, 21 kinds of miRNAs were upregulated and 2 ones were down-regulated. It showed that nine up-regulated miRNAs and one down-regulated miRNA were same in two periods. The 10 miRNAs are selected for target gene analysis on bioinformatics software that refer to the effect of anti-apoptosis and pro-apoptosis, which is of great significance for the early identification of skeletal fluorosis.

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

Key words: Sodium Fluoride, Apoptosis, MicroRNAs, Tissue Engineering

CLC Number:

R318

Deng Qiang1, Zhang Ya-lou2, Zhou Yangjunjie1, Ma Chuang1, Sheng Wei-bin1. Differential expression of microRNAs related to apoptosis in human osteoblasts induced by sodium fluoride[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(8): 1149-1154.

Figures/Tables 3

2.2　microRNA表达差异分析 2.2.1 对照组样本与24 h处理组miRNAs差异表达分析将24 h处理组和对照组miRNA的表达量丰度进行比较后共得到52个显著差异表达miRNA(48条miRNA表达上调，4 条miRNA表达下调)。见表1。 2.2.2 对照组样本与48 h处理组miRNAs差异表达将 48 h处理组和对照组miRNA的表达量丰度进行比较后共得到23个显著差异表达miRNA(21条miRNA表达上调，2条miRNA表达下调)。见表2。 2.2.3 两个时间点同时差异表达的miRNA 进一步筛选后，得到24 h和48 h共同上调miRNA有9条，共同下调miRNA有1条。分别为上调的hsa-miR-101-3p， hsa-miR-125b-5p，hsa-miR-153-3p，hsa-miR-17-5p，hsa-miR-20a-5p，hsa-miR-21-5p，hsa-miR-365b-3p，hsa-miR-708-5p，hsa-miR-9-5p，以及下调的hsa-miR-451a。筛选出来的10条miRNA进行的功能分类见表3。 2.2.4 差异表达miRNA靶基因预测采用4种网上的生物信息学软件预测靶基因：①miRbase: (http://mirbase. org/ index.shtml)、②Tarbase: (http://microrna.gr/tarbase/)、③miRecords: (http://mirecords.biolead.org/)、④targetScan: (http://www.targetscan.org/)。由于第4种软件预测的假阳性率较低，以它为基础，与其他软件相印证，同时预测出的靶基因数量较多(可达数百种)，选出评分高的前10个靶基因如下： (1)miR-365b-3p的靶基因预测：NCR3LG1，NFIA，NUFIP2，DCP2，PPP2CB，ARL5A， SH3PXD2A， FAM60A，INO80D，SMARCC1。查阅CELL SIGNALING TECHNOLOGY网站(https://www.cst-c.com.cn)，发现NFI-C属于核因子I(NFI)家族，NFI-C表达于各种组织中而且调节转化生长因子β依赖的牙齿发育和毛囊循环，NFI-C 直接抑制FoxF1转录[8]；PPP2CB (Protein Phosphatase 2 Catalytic Subunit Beta)与胰岛素样生长因子1受体信号通路和mTOR通路有关；SMARCC1目前仅发现与小鼠胚胎发育相关[9]；余下的基因未见信号通路资料。 (2)miR-708-5p的靶基因预测：FOXJ3，SESN3，AGPAT3，CBX5，NUFIP2，PPIF，ZBTB7A，TTC17，SORT1，TUB。查阅CELL SIGNALING TECHNOLOGY网站，发现：ZBTB7A基因属于POK(POZ和Kruppel)/ ZBTB(锌指和BTB)家族参与Notch信号通路，通过抑制前凋亡基因凋亡因子前体Bim促进细胞生存[10-11]；余下基因未见信号通路资料。 (3)miR-101-3p的靶基因预测：CEP350，RC3H2，TNKS2，RBBP7，PHACTR2，PIP5K1C，IGF2R，SOX11，AKAP11，C10orf12。查阅CELL SIGNALING TECHNOLOGY网站，发现：PIP5K1C与嗜酸性粒细胞中的CCR3免疫信号通路和磷脂酶D信号通路有关[12]；IGF2R属于胰岛素样生长因子Ⅱ受体[13]；余下基因未见信号通路资料。 (4)miR-125b-5p的靶基因预测：MFHAS1，SEMA4C，RORA，FAM118A，KPNA6，ZC3H7B，CDK16，CHAMP1，RC3H2，JARID2。查阅CELL SIGNALING TECHNOLOGY网站，发现：RORA与昼夜节奏和天然免疫信号有关[14]；CDK16调节细胞周期，可以与Jun基因相互作用[15]；JARID2是一种核蛋白在可以降低细胞分化，JARID2是多梳抑制复合物2(PRC2)的一个附件，通过组蛋白H3的甲基化抑制靶基因的表达[16]；余下基因未见信号通路资料。 (5)miR-17-5p的靶基因预测：MAP3K9，RGMB，EFCAB14，ARAP2，ZNF148，TBC1D9，SUV420H1，ANKRD13C，ARNTL2，STAT3。丝裂原活化蛋白激酶激酶激酶9(MAP3K9)是MAPK信号通路中最上游的激酶分子，可以接受细胞膜受体的多种信号刺激，激活后磷酸化下游基因，调控细胞增殖分化[17]；STAT3是一种重要的具有抗凋亡作用[18]；余下基因未见信号通路资料。 (6)miR-20a-5p的靶基因预测：GINM1，RUFY2，C2CD2，TNKS2，PTPN4，RORA，EFCAB14，RASL11B，ZNF367，SIK1。RORA功能同上miR-125b-5p；余下基因未见信号通路资料。 (7)miR-21-5p的靶基因预测：GATAD2B，YOD1，KLHL15，FRS2，ADNP，BTG2，HNRNPU，KPNA4，RHOB，AGO4。FRS2(成纤维细胞生长因子受体底物2)参与细胞外碱性成纤维生长因子受体信号传递[19]；活性依赖性神经保护蛋白(activity-dependent neuroprotective protein，ADNP)调控抑癌基因P53[20]；hnRNPU也被称为脚手架附着因子A，对细胞内活动有广泛作用，包括与RNA聚合酶Ⅱ形成剪切复合体调节核内mRNA的转录，调节pre-mRNA的加工以及对细胞内mRNA翻译过程起作用等，还可以调节Caspase 9a和Caspase 9b的剪切比例影响细胞的凋亡过程[21]；RhoB 的功能包括调节细胞形状，迁移和黏附[22]；AGO4属于Argonauta家族，是与高尔基体和内质网膜相关的蛋白质，除了在RNAi现象中担当重任外，其参与细胞周期进程的调控，影响细胞增殖和发育[23]；"

References

[1] Pramanik S,Saha D.The genetic influence in fluorosis. Environ Toxicol Pharmacol.2017;56:157-162.

[2] Dec K, ?ukomska A, Maciejewska D,et al.The Influence of Fluorine on the Disturbances of Homeostasis in the Central Nervous System. Biol Trace Elem Res.2017;177(2):224-234.

[3] Brookes SJ, Barron MJ, Dixon MJ,et al. The Unfolded Protein Response in Amelogenesis and Enamel Pathologies. Front Physiol.2017,8:653

[4] Goodarzi F, Mahvi AH, Hosseini M,et al.The prevalence of dental fluorosis and exposure to fluoride in drinking water: A systematic review. J Dent Res Dent Clin Dent Prospects.2016; 10(3):127-135.

[5] 万桂敏,莫志亚,刘忠杰,等.地方性氟中毒患者多项检验指标的测定及分析[J].中国地方病学杂志,2001,20(2):137-139.

[6] 张亚楼,孙小娜,冯树梅,等.过量氟引起成骨细胞内质网应激信号通路的基因差异表达[J].重庆医学,2014,43(33):4425-4427.

[7] Daiwile AP,Sivanesan S,Izzotti A,et al.Noncoding RNAs: Possihle Players in the Development of Fluorosis. Biomed Res Int. 2015;2015:274852.

[8] Nilsson J,Helou K,Kovács A,et al. Nuclear Janus-activated kinase 2/nuclear factor 1-C2 suppresses tumorigenesis and epithelial-to-mesenchymal transition by repressing Forkhead box F1.Cancer Res.2010;70,2020-2029.

[9] Ho L, Crabtree GR.Chromatin remodelling during development. Nature.https://www.ncbi.nlm.nih.gov/ pubmed/201109912010;463(7280): 474-484.

[10] Liu XS, Haines JE, Mehanna EK,et al.ZBTB7A acts as a tumor suppressor through the transcriptional repression of glycolysis. Genes Dev.2014;28(17):1917-1928.

[11] Liu XS, Liu Z, Gerarduzzi C, et al. Somatic human ZBTB7A zinc finger mutations promote cancer progression. Oncogene. 2016;35(23):3071-3078.

[12] Sengelaub CA, Navrazhina K, Ross JB,et al. PTPRN2 and PLCβ1 promote metastatic breast cancer cell migration through PI(4,5)P2-dependent actin remodeling. EMBO J. 2016;35(1):62-76.

[13] Puxbaum V, Nimmerfall E, Bäuerl C,et al. M6P/IGF2R modulates the invasiveness of liver cells via its capacity to bind mannose 6-phosphate residues. J Hepatol.2012; 57(2):337-343.

[14] Castro G, Liu X, Ngo K,et al.RORγt and RORα signature genes in human Th17 cells. PLoS One. 2017;12(8): e0181868.

[15] Wang Y, Qin X, Guo T,et al.Up-regulation of CDK16 by multiple mechanisms in hepatocellular carcinoma promotes tumor progression.J Exp Clin Cancer Res.2017;36(1):97.

[16] Cloos PA,Christensen J,Agger K,et al. Erasing the methyl mark: histone demethylases at the center of cellular differentiation and disease. Genes Dev.2008;22(9): 1115-1140.

[17] Cai P, Yang T, Jiang X,et al. Role of miR-15a in intervertebral disc degeneration through targeting MAP3K9.Biomed Pharmacother.2017;87:568-574.

[18] Xiong M,Wang L,Yu HL,et al.Ginkgetin exerts growth inhibitory and apoptotic effects on osteosarcoma cells through inhibition of STAT3 and activation of caspase-3/9. Oncol Rep. 2016;35(2):1034-1040.

[19] Wang HF, Zhang YY,Zhuang HW,et al.MicroRNA-613 attenuates the proliferation, migration and invasion of Wilms' tumor via targeting FRS2. Eur Rev Med Pharmacol Sci.2017; 21(15):3360-3369.

[20] Castorina A, Giunta S, Scuderi S,et al.Involvement of PACAP/ADNP signaling in the resistance to cell death in malignant peripheral nerve sheath tumor (MPNST) cells. J Mol Neurosci.2012;48(3):674-683.

[21] Ye J, Beetz N, O'Keeffe S,et al.hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function. Proc Natl Acad Sci U S A.2015; 112(23):E3020-9.

[22] Pronk MCA, van Bezu JSM, van Nieuw Amerongen GP,et al. RhoA, RhoB and RhoC differentially regulate endothelial barrier function. Small GTPases. 2017 Sep 26:1-19.

[23] Modzelewski AJ, Holmes RJ, Hilz S,et al. AGO4 regulates entry into meiosis and influences silencing of sex chromosomes in the male mouse germline.Dev Cell.2012; 23(2):251-264.

[24] Michot P, Fritz S, Barbat A,et al. A missense mutation in PFAS (phosphoribosylformylglycinamidine synthase) is likely causal for embryonic lethality associated with the MH1 haplotype in Montbéliarde dairy cattle.J Dairy Sci.2017;100(10): 8176-8187.

[25] Tian Y,Nan Y,Han L,et al. MicroRNA miR-451 downregulates the PI3K/AKT pathway through CAB39 in human glioma. Int J Oncol.2012;40(4):1105-1112.

[26] Lü M, Ding K, Zhang G,et al. MicroRNA-320a sensitizes tamoxifen-resistant breast cancer cells to tamoxifen by targeting ARPP-19 and ERRγ.Sci Rep.2015;5:8735.

[27] Xin Fu, Fang-Nan Xie,et al. High-Dose Fluoride Impairs the Properties of Human Embryonic Stem Cells via JNK Signaling. PLOS ONE.2016;0148819.

[28] Seo KW, Lee SJ, Ye BH,et al. Mechanical stretch enhances the expression and activity of osteopontin and MMP-2 via the Akt1/AP-1 pathways in VSMC.J Mol Cell Cardiol.2015;85: 13-24.

[29] 徐嘉殉,裴俊瑞,孙殿军.微小RNA在氟骨症发病机制研究中的作用[J].中华地方病学杂志,2017,36(6): 460-463.

[30] 康夏,康菲,杨波,等.微小RNA-451靶向调节钙结合蛋白39对MSCs成骨分化的促进效应[J].中国修复重建外科杂志,2013, 27(9):1122-1127.

[31] Sugatani T,Vacher J,Hruska KA.A microRNA expression signature of osteoclastogenesis.Blood. 2011;117(13): 3648-3657.

[32] Jones SW,Watkins G,Le Good N,et al.The identification of differentially expressed microRNA in osteoarthritic tissue that modulate the production of TNF-alpha and MMP13. Osteoarthritic Cartilage.2009;17(4):464-472.

[33] Yeliz Yuva-Aydemir, Alfred Simkin, et al.MicroRNA-9 Functional evolution of a conserved small regulatory RNA. Landes Bioscience.2011;8(4):557-564.

[34] Xiao-su Zhao,Yi-nuo Wang,et al.miR-153-3p,a new bio-target,is involved in the pathogenesis of acute graft-versus-host disease via inhibition of indoleamine-2,3- dioxygenase.Oncotarget.2016;7(30):48321-48334.