辽宁绒山羊皮肤毛囊mir-1298-5p靶基因预测及表达载体构建
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摘要
探讨辽宁绒山羊皮肤毛囊不同发育时期mir-1298-5p的调控作用及其与靶基因的潜在关系,为mir-1298-5p与其靶基因对皮肤毛囊发育作用提供理论依据。以辽宁绒山羊皮肤毛囊组织为材料,通过mi RNA的分离、表达、靶基因预测筛选及靶位点3'UTR表达载体构建,采用RT-PCR进行表达检测,在线靶基因预测软件预测mir-1298-5p的靶基因,并对其靶基因TGF-βR1的靶位点进行克隆测序、缺失突变。结果表明:靶基因预测发现TGF-βR1的3'UTR存在mir-1298-5p种子区结合位点,且mir-1298-5p及其靶基因TGF-βR1均在皮肤毛囊不同发育时期呈现差异性表达。mir-1298-5p对皮肤毛囊周期性发育可能起到一定的调控作用,并成功构建了TGF-βR1 3'UTR表达载体,为后期过表达转染体系的建立和功能基因的验证奠定了基础。
We aim to explore the regulation of mir-1298-5p in skin hair follicles which were at different development stages,and the potential relationship between mir-1298-5p and its target gene,so as to provide theoretical data for skin hair follicles development.Skin hair follicles from Liaoning cashmere goat were used as materials.Through miRNA isolation and expression,target gene prediction and screen,and construction of 3'UTR expression vector,the target gene of mir-1298-5p was predicted,sequencing and knocking down mutation of the target site were conducted.Results showed that 3'UTR of TGF-βR1 presented a binding site of mir-1298-5p,and mir-1298-5p and TGF-βR1 presented differential expression during different development stages of skin hair follicles.So mir-1298-5p might play an important role in periodic development of skin hair follicles.In addition,3'UTR expression vector of TGF-βR1 was successfully constructed,laying a foundation for the construction of over expression transfection system and functional verification of target gene.
We aim to explore the regulation of mir-1298-5p in skin hair follicles which were at different development stages,and the potential relationship between mir-1298-5p and its target gene,so as to provide theoretical data for skin hair follicles development.Skin hair follicles from Liaoning cashmere goat were used as materials.Through miRNA isolation and expression,target gene prediction and screen,and construction of 3'UTR expression vector,the target gene of mir-1298-5p was predicted,sequencing and knocking down mutation of the target site were conducted.Results showed that 3'UTR of TGF-βR1 presented a binding site of mir-1298-5p,and mir-1298-5p and TGF-βR1 presented differential expression during different development stages of skin hair follicles.So mir-1298-5p might play an important role in periodic development of skin hair follicles.In addition,3'UTR expression vector of TGF-βR1 was successfully constructed,laying a foundation for the construction of over expression transfection system and functional verification of target gene.
引文
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[2]Yuan C,Wang X L,Geng R Q,et al.Discovery of cashmere goat(Capra hircus)micro RNAs in skin and hair follicles by Solexa sequencing[J].BMC Genomics,2013,14(1):1-10.
[3]Liu Z H,Xiao H M,Li H P,et al.Identification of Conserved and Novel micro RNAs in Cashmere Goat Skin by Deep Sequencing[J].PLOS One,2012,7(12):1-4.
[4]Sacco L D,Masotti A.Recent Insights and Novel Bioinformatics Tools to Understand the Role of Micro RNAs Binding to 5′Untranslated Region[J].International Journal of Molecular Sciences,2012,14(1):480-495.
[5]Sood P,Krek A,Zavolan M,et al.Cell-type-specific signatures of micro RNAs on target m RNA expression[J].Proc Natl Acad Sci USA,2006,103(8):2746-2751.
[6]Agarwal V,Bell G W,Nam J W,et al.Predicting effective micro RNA target sites in mammalian m RNAs[J].Elife Sciences,2015,4:1-3.
[7]Lebrin F,Deckers M,Bertolino P,et al.TGF-βreceptor function in the endothelium[J].Cardiovasc Res,2005,65(3):599-608.
[8]James J M,Nalbandian A,Mukouyama Y S.TGF-βsignaling is required for sprouting lymphangiogenesis during lymphatic network development in the skin[J].Development,2013,140(18):3903-3914.
[9]Liu X,Song L,Liu J,et al.mi R-18b inhibits TGF-β1-induced differentiation of hair follicle stem cells into smooth muscle cells by targeting SMAD2[J].Biochemical&Biophysical Research Communicatins,2013,438(3):551-6.
[10]Larsson J,Goumans M J,Sj?strand L J,et al.Abnormal angiogenesis but intact hematopoietic potential in TGF-beta type I receptor-deficient mice[J].EMBO J,2001,20(7):1663-1673
[11]Gao Y,Duran S,Lydon J P.et al.Constitutive activation of transforming growth factor beta receptor 1 in the mouse uterus impairs uterine morphology and function[J].Biology of Reproduction,2015,92(2):34.
[12]Gallo E M,Loch D C,Habashi J P,et al.Angiotensin II–dependent TGF-βsignaling contributes to Loeys-Dietz syndrome vascular pathogenesis[J].The journal of Clinical Investigation.2014,124(1):448-460.
[13]Foitzik K,Lindner G,Mueller-Roever S,et al.Control of murine hair follicle regression(catagen)by TGF-beta 1 in vivo[J].FASEB J,2000,14(5):752-760.
[14]Shen Y,Zhang Y,Liu N,et al.Let-7b promotes alpaca hair growth via transcriptional repression of TGFβR I[J].Gene,2015,577(1):32-36.
[15]Tsuji Y,Denda S,Soma T,et al.Apotential suppressor of TGF-beta delays catagen progression in hair follicles[J].Invest Dermatol Symp Proc,2003,8(1):65-68.
[16]Joan M.TGFβsignalling in context[J].Nat Rev Mol Cell Biol,2012,13(10):616–630.
[17]Wang L K,Hsiao T H,Hong T M,et al.Micro RNA-133a Suppresses Multiple Oncogenic Membrane Receptors and Cell Invasion in Non-Small Cell Lung Carcinoma[J].PLOS One,2014,9(5):1-11.