猪STC-1 siRNA设计及其基因沉默效率检测
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摘要
根据猪肌肉斯钙素(STC)mRNA序列设计3对小干扰RNA(siRNA)、阴性对照siRNA,转染PK15细胞后,通过荧光观察转染效率和基因表达效率,利用实时荧光定量PCR(QRT-PCR)及Western blot测定细胞内siRNA基因沉默后STC-1siRNA及蛋白的表达水平。结果表明:3个STC-1siRNA转染细胞后均有较高的荧光信号;STC-132细胞组STC-1mRNA和STC-1蛋白相对表达水平都最低,该组与其他组差异极显著。结果说明成功设计出STC-1siRNA序列,为后续通过调控STC-1表达研究其在肌肉发育中的功能奠定了基础。
Three pairs of STC-1 siRNA and a pair of negative control siRNA were designed based on STC-1 mRNA sequence.After transfection of PK15 cell,efficiency of transfection and gene expression was observed by fluorescent technique,and QRT-PCR and western blot were used to determine siRNA in cells that gene silencing on STC-1 siRNA and protein expression level.The results showed that three siRNA on STC-1 had a higher fluorescence signal after the transfection cells;STC-1 mRNA and STC-1 protein expression levels were significantly lower in STC-132 cells than those in other groups(P<0.01).The study suggested that the successfully designed STC-1 siRNA sequences can provide a foundation for subsequent control on STC-1 expression in the function of muscle development.
Three pairs of STC-1 siRNA and a pair of negative control siRNA were designed based on STC-1 mRNA sequence.After transfection of PK15 cell,efficiency of transfection and gene expression was observed by fluorescent technique,and QRT-PCR and western blot were used to determine siRNA in cells that gene silencing on STC-1 siRNA and protein expression level.The results showed that three siRNA on STC-1 had a higher fluorescence signal after the transfection cells;STC-1 mRNA and STC-1 protein expression levels were significantly lower in STC-132 cells than those in other groups(P<0.01).The study suggested that the successfully designed STC-1 siRNA sequences can provide a foundation for subsequent control on STC-1 expression in the function of muscle development.
引文
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[2]JEPSEN M R,KLOVERPRIS S,MIKKELSEN J H,et al.Stanniocalcin-2inhibits mammalian growth by proteolytic inhibition of the insulin-like growth factor axis[J].J Biol Chem,2015,290(6):3430-3439.
[3]TAKEI Y,YAMAMOTO H,SATO T,et al.Stanniocalcin 2is associated with ectopic calcification in alphaklotho mutant mice and inhibits hyperphosphatemia-induced calcification in aortic vascular smooth muscle cells[J].Bone,2012,50(4):998-1005.
[4]ZAICHICK V,ZAICHICK S.The Ca,Cl,Mg,Na,and P mass fractions in human bone affected by Ewing’s sarcoma[J].Biol Trace Elem Res,2014,159(1/2/3):32-38.
[5]OLSEN H S,CEPEDA M A,ZHANG Q Q,et al.Human stanniocalcin:apossible hormonal regulator of mineral metabolism[J].Proc Natl Acad Sci USA,1996,93(5):1792-1796.
[6]MARTINEZ-HERVAS S,MANSEGO M L,DE MARCO G,et al.Polymorphisms of the UCP2gene are associated with body fat distribution and risk of abdominal obesity in Spanish population[J].Eur J Clin Invest,2012,42(2):171-178.
[7]鞠辉明,赵如梦,于珊,等.小鼠生长激素(GH)shRNA可控表达载体构建及其基因沉默效率[J].农业生物技术学报,2015,23(12):1611-1616.
[8]王怡,刘伟,赵鸿雁,等.镉对大鼠成骨细胞OPG/RANKL表达的影响[J].扬州大学学报(农业与生命科学版),2014,35(1):9-12.
[9]王棋文,王涛,王怡,等.Beclin-1复合物在镉诱导PC12细胞自噬中的作用[J].扬州大学学报(农业与生命科学版),2015,36(3):10-12.
[10]祁钰钰,杨跃飞,杨开典,等.小鼠MSTN shRNA设计及基因沉默效率检测[J].扬州大学学报(农业与生命科学版),2015,36(3):5-8.
[11]陈维娜,朱广瑾.斯钙素的研究进展[J].生物科学进展,2008,39(3):225-228.
[12]LEE J H,JANG H,LEE S M,et al.ATP-citrate lyase regulates cellular senescence via an AMPK-and p53-dependent pathway[J].Febs J,2015,282(2):361-371.
[13]PARK S H,HO W K,JEON J H.AMPK regulates K(ATP)channel trafficking via PTEN inhibition in leptintreated pancreaticβ-cells[J].Biochem Biophys Res Commun,2013,440(4):539-544.
[14]PAN J S,HUANG L,BELOUSOVA T,et al.Stanniocalcin-1inhibits renal ischemia/reperfusion injury via an AMP-activated protein kinase-dependent pathway[J].J Am Soc Nephrol,2015,26(2):364-378.
[15]SHEIKH-HAMAD D.Mammalian stanniocalcin-1activates mitochondrial antioxidant pathways:new paradigms for regulation of macrophages and endothelium[J].Am J Physiol Renal Physiol,2010,298(2):248-254.
[16]WONG C K,HO M A,WAGNER G F.The co-localization of stanniocalcin protein,mRNA and kidney cell markers in the rat kidney[J].J Endocrinol,1998,158(2):183-189.
[17]MELLO C C,CONTE D J R.Revealing the world of RNA interference[J].Nature,2004,431(7006):338-342.
[18]CULLEN B R.Induction of stable RNA interference in mammalian cells[J].Gene Ther,2006,13(6):503-508.
[19]WANG Y L,HUANG L P,ABDELRAHIM M,et al.Stanniocalcin-1suppresses superoxide generation in macrophages through induction of mitochondrial UCP2[J].J Leukocyte Blot,2009,86(4):981-988.
[20]FANG Z,TIAN Z,LUO K,et al.Clinical significance of stanniocalcin expression in tissue and serum of gastric cancer patients[J].Chin J Cancer Res,2014,26(5):602-610.
[21]MIYAZAKI S,MANSEGO M L,DE MARCO G,et al.Anti-VEGF antibody therapy induces tumor hypoxia and stanniocalcin 2expression and potentiates growth of human colon cancer xenografts[J].Int J Cancer,2014,135(2):295-307.
[22]JEPSEN M R,KLOVERPRIS S,MIKKELSEN J H,et al.Stanniocalcin-2inhibits mammalian growth by proteolytic inhibition of the insulin-like growth factor axis[J].J Biol Chem,2015,290(6):3430-3439.