Sirt1基因稳定低表达的LMH细胞系构建
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摘要
为构建Sirt1基因低表达的LMH细胞系,根据鸡Sirt1基因的3′UTR序列设计gSmiR30、gSmiR70和gSmiR91 3个miRNAs,以质粒pLNCX-gSmiRn(n为30、70或91)分别过表达这3个miRNAs 48 h后,依次检测其对CHO-K1细胞中报告载体psiCHECH2-gSirt1-UTR672所表达的荧光素酶活性的影响以及对LMH细胞中Sirt1基因表达的干扰情况;然后选用干扰效果较好的pLNCX-gSmiR30包装的反转录病毒感染LMH细胞,以终浓度1μg·mL~(-1)的G418筛选稳定过表达gSmiR30的LMH-gSmiR30细胞系,荧光定量PCR和Western blot检测Sirt1基因的表达。结果表明:人工设计的3个miRNAs可抑制含靶位点的海肾荧光素酶活性在97%以上;且LMH细胞中Sirt1的mRNA表达水平均能被这3个miRNAs下调40%以上;筛选得到的LMH-gSmiR30细胞中绿色荧光蛋白的表达率在99%以上,细胞内Sirt1基因的mRNA水平下降75%, SIRT1蛋白也明显减少。这一研究成功获得了Sirt1基因低表达的LMH-gSmiR30细胞系,为进一步研究Sirt1基因在鸡肝脏中的功能提供了一种很好的细胞模型。
To generate the LMH cell line with low Sirt1 expression, three miRNAs(gSmiR30, gSmiR70 and gSmiR91) were designed targeting to the 3'UTR sequence of chicken Sirt1 gene. The luciferase activity was assessed after pLNCX-gSmiRn(n: 30, 70 or 91) plasmids expressing the three miRNAs were co-transfected with the reporter vector psiCHECH2-gSirt1-UTR672 into CHO-K1 for 48 hours. Sirt1 gene expression was also detected to screen for the best effective sequence in LMH cells. Then, LMH cells were infected with the retroviral particles packaged with pLNCX-gSmiR30 with the best interference effect. LMH-gSmiR30 cells stably overexpressing gSmiR30 were screened with 1 μg·mL~(-1) G418. The expression of Sirt1 gene was detected by RT-PCR and Western blot. The results showed that the three miRNAs could inhibit the activity of luciferase containing target sites by more than 97%. Sirt1 mRNA was also inhibited about 40% after three miRNAs were separately transfected in to LMH cells for 48 h. The expression ratio of the green fluorescent protein(GFP) was more than 99% in LMH-gSmiR30 after G418 resistance screening. Importantly, Sirt1 expression was significantly knocked down in LMH-gSmiR30 cell lines, as indicated by the markedly decreased levels of Sirt1 mRNA about 75% and protein expression. These results indicated that the LMH cells with low Sirt1 expression were successfully obtained, which provided a good cellular model for further study of the the Sirt1 gene function in chicken liver.
To generate the LMH cell line with low Sirt1 expression, three miRNAs(gSmiR30, gSmiR70 and gSmiR91) were designed targeting to the 3'UTR sequence of chicken Sirt1 gene. The luciferase activity was assessed after pLNCX-gSmiRn(n: 30, 70 or 91) plasmids expressing the three miRNAs were co-transfected with the reporter vector psiCHECH2-gSirt1-UTR672 into CHO-K1 for 48 hours. Sirt1 gene expression was also detected to screen for the best effective sequence in LMH cells. Then, LMH cells were infected with the retroviral particles packaged with pLNCX-gSmiR30 with the best interference effect. LMH-gSmiR30 cells stably overexpressing gSmiR30 were screened with 1 μg·mL~(-1) G418. The expression of Sirt1 gene was detected by RT-PCR and Western blot. The results showed that the three miRNAs could inhibit the activity of luciferase containing target sites by more than 97%. Sirt1 mRNA was also inhibited about 40% after three miRNAs were separately transfected in to LMH cells for 48 h. The expression ratio of the green fluorescent protein(GFP) was more than 99% in LMH-gSmiR30 after G418 resistance screening. Importantly, Sirt1 expression was significantly knocked down in LMH-gSmiR30 cell lines, as indicated by the markedly decreased levels of Sirt1 mRNA about 75% and protein expression. These results indicated that the LMH cells with low Sirt1 expression were successfully obtained, which provided a good cellular model for further study of the the Sirt1 gene function in chicken liver.
引文
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[19] 崔佣楸,王元红,张谦,等.小反刍兽疫病毒非结构蛋白C的原核表达及多克隆抗体制备 [J].扬州大学学报(农业与生命科学版),2018,39(2):65-69.
[20] EMI N,FRIEDMANN T,YEE J K.Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis virus [J].J Virol,1991,65(3):1202-1207.
[2] BORDONE L,GUARENTE L.Calorie restriction,SIRT1 and metabolism:understanding longevity [J].Nat Rev Mol Cell Biol,2005,6(4):298-305.
[3] SCHIEDEL M,ROBAA D,RUMPF T,et al.The current state of NAD(+)-dependent histone deacetylases (sirtuins) as novel therapeutic targets [J].Med Res Rev,2018,38(1):147-200.
[4] YOSHINO J,MILLS K F,YOON M J,et al.Nicotinamide mononucleotide,a key NAD(+) intermediate,treats the pathophysiology of diet-and age-induced diabetes in mice [J].Cell Metab,2011,14(4):528-536.
[5] PURUSHOTHAM A,XU Q,LU J,et al.Hepatic deletion of SIRT1 decreases hepatocyte nuclear factor 1alpha/farnesoid X receptor signaling and induces formation of cholesterol gallstones in mice [J].Mol Cell Biol,2012,32(7):1226-1236.
[6] RODGERS J T,LERIN C,HAAS W,et al.Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1 [J].Nature,2005,434(7029):113-118.
[7] LIU Y,DENTIN R,CHEN D,et al.A fasting inducible switch modulates gluconeogenesis via activator/coactivator exchange [J].Nature,2008,456(7219):269-273.
[8] PONUGOTI B,KIM D H,XIAO Z,et al.SIRT1 deacetylates and inhibits SREBP-1C activity in regulation of hepatic lipid metabolism [J].J Biol Chem,2010,285(44):33959-33970.
[9] ANDRADE J M,PARAISO A F,DE OLIVEIRA M V,et al.Resveratrol attenuates hepatic steatosis in high-fat fed mice by decreasing lipogenesis and inflammation [J].Nutrition,2014,30(7/8):915-919.
[10] GARIANI K,MENZIES K J,RYU D,et al.Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice [J].Hepatology,2016,63(4):1190-1204.
[11] PURUSHOTHAM A,SCHUG T T,XU Q,et al.Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation [J].Cell Metab,2009,9(4):327-338.
[12] MCBRIDE J L,BOUDREAU R L,HARPER S Q,et al.Artificial miRNAs mitigate shRNA-mediated toxicity in the brain:implications for the therapeutic development of RNAi [J].Proc Natl Acad Sci USA,2008,105(15):5868-5873.
[13] FIRE A,XU S,MONTGOMERY M K,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J].Nature,1998,391(6669):806-811.
[14] BRUMMELKAMP T R,BERNARDS R,AGAMI R.A system for stable expression of short interfering RNAs in mammalian cells [J].Science,2002,296(5567):550-553.
[15] KAADT E,ALSING S,CECCHI C R,et al.Efficient knockdown and lack of passenger strand activity by dicer-independent shRNAs expressed from polⅡ-driven microRNA scaffolds [J].Mol Ther Nucleic Acids,2019,14:318-328.
[16] 宋红芹,姜翠翠,张鑫宇,等.巨噬细胞特异的人工miRNA表达载体的构建与鉴定 [J].细胞与分子免疫学杂志,2015,31(4):504-506,510.
[17] LAGOS-QUINTANA M,RAUHUT R,YALCIN A,et al.Identification of tissue-specific microRNAs from mouse [J].Curr Biol,2002,12(9):735-739.
[18] ZENG Y,WAGNER E J,CULLEN B R.Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells [J].Mol Cell,2002,9(6):1327-1333.
[19] 崔佣楸,王元红,张谦,等.小反刍兽疫病毒非结构蛋白C的原核表达及多克隆抗体制备 [J].扬州大学学报(农业与生命科学版),2018,39(2):65-69.
[20] EMI N,FRIEDMANN T,YEE J K.Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis virus [J].J Virol,1991,65(3):1202-1207.
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