pSilencer 3.1-Sirt3基因RNA干扰表达载体的构建及鉴定
|
摘要
目的构建Sirt3基因的RNA干扰载体,并在真核细胞中进行pSilencer 3.1-Sirt3的功能鉴定。方法利用体外DNA合成技术及液相色谱纯化技术合成Sirt3干扰序列,通过酶切将干扰序列连接至pSilencer 3.1载体中,将阳性克隆载体转入感受态大肠杆菌细胞后,筛选阳性克隆,通过第二代体外DNA测序技术进行序列测定。将测序正确的克隆用于Western blot实验和real time PCR实验,以对其在真核细胞中的正确表达和内源性Sirt3的干涉效果进行鉴定。结果 PCR扩增得到序列测定结果与干扰序列完全一致。构建的pSilencer 3.1-Sirt3质粒在AGS和SGC-7901细胞中进行基因的转染,pSilencer3.1-Sirt3能够有效抑制内源性Sirt3的mRNA水平和蛋白质水平的表达。结论成功构建获得Sirt3干扰载体,并有效降低Sirt3的mRNA和蛋白质水平的表达,为今后Sirt3的功能研究提供良好的基础。
Objective To construct the Sirt3 interference vector,and verify its biological function in mammal cells. Methods The Sirt3 interference sequence was synthesized by in vitro DNA synthetic technology and liquid chromatogram purification assay. The fragments were inserted into p Silencer 3.1 by digestion and DNA ligation. The resulted plasmid was transformed into competent Escherichia coli cells. The positive clone was selected and sequenced with the second DNA sequencing technology. The correct plasmid was used to observe its expression in eukaryotic cells and the invention efficacy of endogenous Sirt3 by Western blot and real time PCR assays.Results The sequence of the p Silencer 3.1-Sirt3 was consistent with that of the Sirt3 interference sequence. After the p Silencer 3.1-Sirt3 vector was transfected into AGS and SGC-7901 cells,the endogenous Sirt3 mRNA and protein expression was inhibited. Conclusion The construction of p Silencer 3.1-Sirt3 vector is successfully obtained. The vector of p Silencer 3.1-Sirt3 can efficiently decrease the mRNA and protein expression of Sirt3. The results may provide a basis for the further studies on the roles of Sirt3.
Objective To construct the Sirt3 interference vector,and verify its biological function in mammal cells. Methods The Sirt3 interference sequence was synthesized by in vitro DNA synthetic technology and liquid chromatogram purification assay. The fragments were inserted into p Silencer 3.1 by digestion and DNA ligation. The resulted plasmid was transformed into competent Escherichia coli cells. The positive clone was selected and sequenced with the second DNA sequencing technology. The correct plasmid was used to observe its expression in eukaryotic cells and the invention efficacy of endogenous Sirt3 by Western blot and real time PCR assays.Results The sequence of the p Silencer 3.1-Sirt3 was consistent with that of the Sirt3 interference sequence. After the p Silencer 3.1-Sirt3 vector was transfected into AGS and SGC-7901 cells,the endogenous Sirt3 mRNA and protein expression was inhibited. Conclusion The construction of p Silencer 3.1-Sirt3 vector is successfully obtained. The vector of p Silencer 3.1-Sirt3 can efficiently decrease the mRNA and protein expression of Sirt3. The results may provide a basis for the further studies on the roles of Sirt3.
引文
[1]Koentges C,Bode C,Bugger H.SIRT3 in cardiac physiology and disease[J].Front Cardiovasc Med,2016,3:38.
[2]Ashraf N,Zino S,Macintyre A,et al.Altered sirtuin expression is associated with node-positive breast cancer[J].Br J Cancer,2006,95(8):1056-1061.
[3]Yamamoto H,Schoonjans K,Auwerx J.Sirtuin functions in health and disease[J].Mol Endocrinol,2007,21(8):1745-1755.
[4]Michan S,Sinclair D.Sirtuins in mammals:insights into their biological function[J].Biochem J,2007,404(1):1-13.
[5]Kim HS,Patel K,Muldoon-Jacobs K,et al.SIRT3 is a mitochondria-localized tumor suppressor required for maintenance of mitochondrial integrity and metabolism during stress[J].Cancer Cell,2010,17(1):41-52.
[6]Lu Y,Wang YD,Wang XY,et al.SIRT3 in cardiovascular diseases:Emerging roles and therapeutic implications[J].Int J Cardiol,2016,220:700-705.
[7]Hirschey MD,Shimazu T,Goetzman E,et al.SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation[J].Nature,2010,464(7285):121-125.
[8]Hebert AS,Dittenhafer-Reed KE,Yu W,et al.Calorie restriction and SIRT3 trigger global reprogramming of the mitochondrial protein acetylome[J].Mol Cell,2013,49(1):186-199.
[9]Chen J,Wang A,Chen Q.Sir T3 and p53 deacetylation in aging and cancer[J].J Cell Physiol,2016.
[10]Yang W,Nagasawa K,Munch C,et al.Mitochondrial sirtuin network reveals dynamic SIRT3-dependent deacetylation in response to membrane depolarization[J].Cell,2016,167(4):985-1000 e21.
[11]Winnik S,Auwerx J,Sinclair DA,et al.Protective effects of sirtuins in cardiovascular diseases:from bench to bedside[J].Eur Heart J,2015,36(48):3404-12.
[12]Kincaid B,Bossy-Wetzel E.Forever young:SIRT3 a shield against mitochondrial meltdown,aging,and neurodegeneration[J].Front Aging Neurosci,2013,5:48.
[13]Qiu X,Brown K,Hirschey MD,et al.Calorie restriction reduces oxidative stress by SIRT3-mediated SOD2 activation[J].Cell Metab,2010,12(6):662-667.
[14]Chen Y,Zhang J,Lin Y,et al.Tumour suppressor SIRT3 deacetylates and activates manganese superoxide dismutase to scavenge ROS[J].EMBO Rep,2011,12(6):534-541.
[15]Ahn BH,Kim HS,Song S,et al.A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis[J].Proc Natl Acad Sci U S A,2008,105(38):14447-14452.
[16]Rahman M,Nirala NK,Singh A,et al.Drosophila Sirt2/mammalian SIRT3 deacetylates ATP synthase beta and regulates complex V activity[J].J Cell Biol,2014,206(2):289-305.
[17]Sundaresan NR,Gupta M,Kim G,et al.Sirt3 blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant defense mechanisms in mice[J].J Clin Invest,2009,119(9):2758-2771.
[18]Peserico A,Chiacchiera F,Grossi V,et al.A novel AMPK-dependent Fox O3A-SIRT3 intramitochondrial complex sensing glucose levels[J].Cell Mol Life Sci,2013,70(11):2015-2029.
[19]Ansari A,Rahman MS,Saha SK,et al.Function of the SIRT3mitochondrial deacetylase in cellular physiology,cancer,and neurodegenerative disease[J].Aging Cell,2017,16(1):4-16.
[20]Finley LW,Carracedo A,Lee J,et al.SIRT3 opposes reprogramming of cancer cell metabolism through HIF1alpha destabilization[J].Cancer Cell,2011,19(3):416-428.
[2]Ashraf N,Zino S,Macintyre A,et al.Altered sirtuin expression is associated with node-positive breast cancer[J].Br J Cancer,2006,95(8):1056-1061.
[3]Yamamoto H,Schoonjans K,Auwerx J.Sirtuin functions in health and disease[J].Mol Endocrinol,2007,21(8):1745-1755.
[4]Michan S,Sinclair D.Sirtuins in mammals:insights into their biological function[J].Biochem J,2007,404(1):1-13.
[5]Kim HS,Patel K,Muldoon-Jacobs K,et al.SIRT3 is a mitochondria-localized tumor suppressor required for maintenance of mitochondrial integrity and metabolism during stress[J].Cancer Cell,2010,17(1):41-52.
[6]Lu Y,Wang YD,Wang XY,et al.SIRT3 in cardiovascular diseases:Emerging roles and therapeutic implications[J].Int J Cardiol,2016,220:700-705.
[7]Hirschey MD,Shimazu T,Goetzman E,et al.SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation[J].Nature,2010,464(7285):121-125.
[8]Hebert AS,Dittenhafer-Reed KE,Yu W,et al.Calorie restriction and SIRT3 trigger global reprogramming of the mitochondrial protein acetylome[J].Mol Cell,2013,49(1):186-199.
[9]Chen J,Wang A,Chen Q.Sir T3 and p53 deacetylation in aging and cancer[J].J Cell Physiol,2016.
[10]Yang W,Nagasawa K,Munch C,et al.Mitochondrial sirtuin network reveals dynamic SIRT3-dependent deacetylation in response to membrane depolarization[J].Cell,2016,167(4):985-1000 e21.
[11]Winnik S,Auwerx J,Sinclair DA,et al.Protective effects of sirtuins in cardiovascular diseases:from bench to bedside[J].Eur Heart J,2015,36(48):3404-12.
[12]Kincaid B,Bossy-Wetzel E.Forever young:SIRT3 a shield against mitochondrial meltdown,aging,and neurodegeneration[J].Front Aging Neurosci,2013,5:48.
[13]Qiu X,Brown K,Hirschey MD,et al.Calorie restriction reduces oxidative stress by SIRT3-mediated SOD2 activation[J].Cell Metab,2010,12(6):662-667.
[14]Chen Y,Zhang J,Lin Y,et al.Tumour suppressor SIRT3 deacetylates and activates manganese superoxide dismutase to scavenge ROS[J].EMBO Rep,2011,12(6):534-541.
[15]Ahn BH,Kim HS,Song S,et al.A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis[J].Proc Natl Acad Sci U S A,2008,105(38):14447-14452.
[16]Rahman M,Nirala NK,Singh A,et al.Drosophila Sirt2/mammalian SIRT3 deacetylates ATP synthase beta and regulates complex V activity[J].J Cell Biol,2014,206(2):289-305.
[17]Sundaresan NR,Gupta M,Kim G,et al.Sirt3 blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant defense mechanisms in mice[J].J Clin Invest,2009,119(9):2758-2771.
[18]Peserico A,Chiacchiera F,Grossi V,et al.A novel AMPK-dependent Fox O3A-SIRT3 intramitochondrial complex sensing glucose levels[J].Cell Mol Life Sci,2013,70(11):2015-2029.
[19]Ansari A,Rahman MS,Saha SK,et al.Function of the SIRT3mitochondrial deacetylase in cellular physiology,cancer,and neurodegenerative disease[J].Aging Cell,2017,16(1):4-16.
[20]Finley LW,Carracedo A,Lee J,et al.SIRT3 opposes reprogramming of cancer cell metabolism through HIF1alpha destabilization[J].Cancer Cell,2011,19(3):416-428.