软骨细胞SHOX基因过/低表达的差异miRNA筛选与验证
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摘要
目的建立软骨细胞(HC-α)SHOX基因过/低表达模型,筛选与未处理组差异表达miRNA,并予以验证。方法通过慢病毒过/低表达SHOX基因,用miRNA芯片筛选差异表达的miRNA。结果 SHOX过表达组与对照组有425条差异表达的miRNA;SHOX低表达组与对照组有379条差异表达的miRNA;按照表达水平差异相反的标准筛选差异miRNA,其中6条miRNA在处理组中表达水平差异相反。实时荧光定量PCR对6条miRNA(miR-126、miR-192、miR-370、miR-432、miR-3162、miR-4745)进行验证,miR-126在过表达组明显上调(P<0.05),在低表达组明显下调(P<0.05)。结论 miR-126参与SHOX介导的软骨细胞凋亡过程。
Objective To establish 2 cell models with overexpression and low expression of short stature homeo-box-containing gene(SHOX) in chondrocytes and screen the differentially expressed miRNAs in the cells. Methods Lentiviral vectors were used to establish a cell model of SHOX overexpression and a cell model of SHOX gene interference in HC-α cells. Microarray chip technique was used to screen the differentially expressed miRNAs in the 2 cell models compared with HC-α cells without genetic modification(NC group). Results Compared with the NC group,425 and 379 differentially expressed miRNAs were identified in HC-α cells with SHOX overexpression and in cells with SHOX interference,respectively. Six miRNAs were found to have opposite changes between the 2 cell models,and verification of the 6 miRNAs(miR-126,miR-192,miR-370,miR-432,miR-3162,and miR-4745) with real-time PCR confirmed that miR-126 was significantly up-regulated(P < 0. 05) in cells with SHOX overexpression and was significantly down-regulated(P < 0. 05) in cells with SHOX interference. Conclusion miR-126 is involved in SHOXmediated chondrocyte apoptosis.
Objective To establish 2 cell models with overexpression and low expression of short stature homeo-box-containing gene(SHOX) in chondrocytes and screen the differentially expressed miRNAs in the cells. Methods Lentiviral vectors were used to establish a cell model of SHOX overexpression and a cell model of SHOX gene interference in HC-α cells. Microarray chip technique was used to screen the differentially expressed miRNAs in the 2 cell models compared with HC-α cells without genetic modification(NC group). Results Compared with the NC group,425 and 379 differentially expressed miRNAs were identified in HC-α cells with SHOX overexpression and in cells with SHOX interference,respectively. Six miRNAs were found to have opposite changes between the 2 cell models,and verification of the 6 miRNAs(miR-126,miR-192,miR-370,miR-432,miR-3162,and miR-4745) with real-time PCR confirmed that miR-126 was significantly up-regulated(P < 0. 05) in cells with SHOX overexpression and was significantly down-regulated(P < 0. 05) in cells with SHOX interference. Conclusion miR-126 is involved in SHOXmediated chondrocyte apoptosis.
引文
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[14]XIE Y,ZHOU S,CHEN H,et al.Recent research on the growth plate:Advances in fibroblast growth factor signaling in growth plate development and disorders[J].J Mol Endocrinol,2014,53(1):T11-T34.DOI:10.1530/JME-14-0012.
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[2]MARCHINI A,MARTTILA T,WINTER A,et al.The short stature homeodomain protein SHOX induces cellular growth arrest and apoptosis and is expressed in human growth plate chondrocytes[J].J Biol Chem,2004,279(35):37103-37114.DOI:10.1074/jbc.M307006200.
[3]HRISTOV G,MARTTILA T,DURAND C,et al.SHOX triggers the lysosomal pathway of apoptosis via oxidative stress[J].Hum Mol Genet,2014,23(6):1619-1630.DOI:10.1093/hmg/ddt552.
[4]赵莎,钟燕,蒋耀辉,等.特发性矮身材儿童循环microRNA表达水平的研究[J].中国当代儿科杂志,2013,15(12):1104-1108.DOI:10.7499/j.issn.1008-8830.2013.12.017.ZHAO S,ZHONG Y,JIANG Y H,et al.Circulating microrna expression in children with idiopathic short stature[J].Chin J Contemp Pediatrics,2013,15(12):1104-1108.DOI:10.7499/j.issn.1008-8830.2013.12.017.
[5]MUNNS C J,HAASE H R,CROWTHER L M,et al.Expression of SHOX in human fetal and childhood growth plate[J].J Clin Endocrinol Metab,2004,89(8):4130-4135.DOI:10.1210/jc.2003-032230.
[6]BARTEL D P.MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
[7]LAINE S K,ALM J J,VIRTANEN S P,et al.MicroRNAs miR-96,miR-124,and miR-199a regulate gene expression in human bone marrow-derived mesenchymal stem cells[J].J Cell Biochem,2012,113(8):2687-2695.DOI:10.1002/jcb.24144.
[8]DUDEK K A,LAFONT J E,MARTINEZ-SANCHEZ A,et al.TypeⅡcollagen expression is regulated by tissuespecific miR-675 in human articular chondrocytes[J].J Biologic Chem,2010,285(32):24381-24387.DOI:10.1074/jbc.m110.111328.
[9]ABOUHEIF M M,NAKASA T,SHIBUYA H,et al.Silencing microRNA-34a inhibits chondrocyte apoptosis in a rat osteoarthritis model in vitro[J].Rheumatology(Oxford),2010,49(11):2054-2060.DOI:10.1093/rheumatology/keq247.
[10]LI H,XIE H,LIU W,et al.A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans[J].J Clin Invest,2009,119(12):3666-3677.DOI:10.1172/JCI39832.
[11]CHENG X W,WAN Y F,ZHOU Q,et al.MicroRNA-126inhibits endothelial permeability and apoptosis in apolipoprotein E-knockout mice fed a high-fat diet[J].Mol Med Rep,2017,16(3):3061-3068.DOI:10.3892/mmr.2017.6952.
[12]CHEN L W,XIAO J,LIN H Y,et al.MiR-126 regulates proliferation and invasion in the bladder cancer BLS cell line by targeting the PIK3R2-mediated PI3K/Akt signaling pathway[J].Onco Targets and Therapy,2016,9:5181-5193.DOI:10.2147/ott.s105198.
[13]ZHAO C,LI Y,ZHANG M,et al.miR-126 inhibits cell proliferation and induces cell apoptosis of hepatocellular carcinoma cells partially by targeting Sox2[J].Hum Cell,2015,28(2):91-99.DOI:10.1007/s13577-014-0105-z.
[14]XIE Y,ZHOU S,CHEN H,et al.Recent research on the growth plate:Advances in fibroblast growth factor signaling in growth plate development and disorders[J].J Mol Endocrinol,2014,53(1):T11-T34.DOI:10.1530/JME-14-0012.
[15]FOLDYNOVA-TRANTIRKOVA S,WILCOX W R,KREJCI P.Sixteen years and counting:the current understanding of fibroblast growth factor receptor 3(FGFR3)signaling in skeletal dysplasias[J].Hum Mutat,2012,33(1):29-41.DOI:10.1002/humu.21636.