MiR-3880对奶山羊乳腺上皮细胞OGR1基因的靶向调控作用
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摘要
为研究miR-3880对奶山羊OGR1基因的靶向调节作用,试验选用泌乳期奶山羊乳腺上皮细胞为研究材料,采用生物信息学和双荧光素酶报告载体试验,初步确定OGR1为miR-3880的靶基因。利用RT-qPCR和Western blot方法进一步研究miR-3880对OGR1的靶向作用。结果显示,miR-3880通过与OGR1基因3ˊUTR结合调控其mRNA和蛋白的表达。本试验为研究miR-3880对乳腺上皮细胞泌乳功能的调节作用提供试验基础。
In order to study the targeted regulation of miR-3880 on the OGR1 gene in dairy goats, the mammary epithelial cells of lactating dairy goats were selected as the research materials. The bioinformatics and dual luciferase reporter vector experiments were used to determine the OGR1 as the target gene of miR-3880. The targeting effect of miR-3880 on OGR1 was further investigated by RT-qPCR and western blot. The results showed that miR-3880 regulated the expression of mRNA and protein by binding to the OGR1 gene 3ˊUTR. This experiment provides a basis for studying the regulation of miR-3880 on the lactation function of mammary epithelial cells.
In order to study the targeted regulation of miR-3880 on the OGR1 gene in dairy goats, the mammary epithelial cells of lactating dairy goats were selected as the research materials. The bioinformatics and dual luciferase reporter vector experiments were used to determine the OGR1 as the target gene of miR-3880. The targeting effect of miR-3880 on OGR1 was further investigated by RT-qPCR and western blot. The results showed that miR-3880 regulated the expression of mRNA and protein by binding to the OGR1 gene 3ˊUTR. This experiment provides a basis for studying the regulation of miR-3880 on the lactation function of mammary epithelial cells.
引文
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[19] 林先滋,罗军,张犁苹,等.miR-200a对奶山羊乳腺上皮细胞乳脂合成相关基因mRNA表达的影响[J].畜牧兽医学报,2012,43(7):1 028-1 036.
[20] 王褚悦,王春梅,邵丽,等.miR-142-3p对奶山羊乳腺上皮细胞泌乳功能的影响[J].中国畜牧兽医,2015,42(7):1 823-1 829.
[21] HOU J,AN X,SONG Y,et al.Detection and comparison of microRNAs in the caprine mammary gland tissues of colostrum and common milk stages[J].BMC Genetics,2017,18:1-8.
[2] 曲波,王春梅,仇有文,等.乳用动物乳腺特异性miRNAs研究进展[J].中国乳品工业,2014,42(9):26-29.
[3] CHEN K W,HOU J X,SONG Y X,et al.Chi-miR-3031 regulates beta-casein via the PI3K/AKT-mTOR signaling pathway in goat mammary epithelial cells (GMECs)[J].BMC Veterinary Research.2018,14:1-10.
[4] LIM L P ,LAU N C,GARRETT-ENGELE P,et al.Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs[J].Nature,2005,433(7027):769-773.
[5] SHANG Y F,KANG Y J,SUN J T,et al.MiR-145-modulated SOX9-mediated hypospadias through acting on mitogen-activated protein kinase signaling pathway[J].Journal of cellular physiology,2019,234(7):10 397-10 410.
[6] KIM K S,REN J,JIANG Y,et al.GPR4 plays a critical role in endothelial cell function and mediates the effects of sphingosylphosphorylcholine[J].Faseb Journal,2005,19(7):819-823.
[7] D'SOUZA,ZHAO F,LI X,et al.OGR1/GPR68 modulates the severity of experimental autoimmune encephalomyelitis and regulates nitric oxide production by macrophages[J].Plos One,2016,11(2):e0148439.
[8] ICHIMONJI I,TOMURA H,MOGI C,et al.Extracellular acidification stimulates IL-6 production and Ca(2+) mobilization through proton-sensing OGR1 receptors in human airway smooth muscle cells[J].Am J Physiol Lung Cell Mol Physiol,2010,299(4):567-777.
[9] LUDWIG M G,VANEK M,GUERINI D,et al.Proton-sensing G-protein-coupled receptors[J].Nature,2003,425(6953):93-98.
[10] 张海强.GPR4配体溶血卵磷脂及鞘氨醇磷脂酰胆碱介导HUVEC细胞生长及其相关信号通路[D].湖南衡阳:南华大学,2007.
[11] KATO K,MORITA I.Promotion of osteoclast differentiation and activation in spite of impeded osteoblast-lineage differentiation under acidosis:effects of acidosis on bone metabolism[J].Bioscience Trends,2013,7(1):33-41.
[12] BOSIER B,DOYEN PJ,BROLET A,et al.Inhibition of the regulator of G protein signalling RGS4 in the spinal cord decreases neuropathic hyperalgesia and restores cannabinoid CB1 receptor signalling[J].British Journal of Pharmacology,2015,172(22):5 333-5 346.
[13] LAI E C.Predicting and validating microRNA targets[J].Genome Biology,2004,5 (9):1 151-1 158.
[14] YAO G,YIN M,LIAN J,et al.MicroRNA-224 is involved in transforming growth factor-beta-mediated mouse granulosa cell proliferation and granulosa cell function by targeting Smad4[J].Molecular Endocrinology,2010,24(3):540-551.
[15] GREGORY R I,CHENDRIMADA T P,COOCH N,et al.Human RISC couples microRNA biogenesis and posttranscriptional gene silencing[J].Cell,2005,123(4):631-640.
[16] AMBROS V.MicroRNA pathways in flies and worms:growth,death,fat,stress,and timing[J].Cell,2003,113(6):673-676.
[17] BARTEL D P.MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
[18] BOEHM M,SLACK F.A developmental timing microRNA and its target regulate life span in C.elegans[J].Science,2005,310(5756):1 954-1 957.
[19] 林先滋,罗军,张犁苹,等.miR-200a对奶山羊乳腺上皮细胞乳脂合成相关基因mRNA表达的影响[J].畜牧兽医学报,2012,43(7):1 028-1 036.
[20] 王褚悦,王春梅,邵丽,等.miR-142-3p对奶山羊乳腺上皮细胞泌乳功能的影响[J].中国畜牧兽医,2015,42(7):1 823-1 829.
[21] HOU J,AN X,SONG Y,et al.Detection and comparison of microRNAs in the caprine mammary gland tissues of colostrum and common milk stages[J].BMC Genetics,2017,18:1-8.