RNA干扰Gas2基因对低温胁迫下罗非鱼肾脏细胞系增殖及凋亡的影响
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摘要
探讨低温胁迫下,干扰生长抑制特异性基因2(Gas2)对罗非鱼肾脏细胞系增殖和凋亡的影响。采用脂质体转染法介导Gas2短发夹RNA(short hairpin RNA, shRNA)及阴性对照短发夹RNA处理罗非鱼肾脏细胞,24 h后,以5℃/d降温速率对转染后的罗非鱼肾脏细胞进行低温胁迫,分别在25、20、15、10℃进行细胞采集。随后,利用实时荧光定量PCR检测Gas2基因及P53基因mRNA表达变化,利用流式细胞仪检测细胞凋亡情况,并开展WST-1细胞增殖实验检测细胞的增殖情况。结果表明,低温胁迫下,阴性对照组及Gas2干扰组中Gas2和P53基因mRNA的表达在15℃和10℃时均显著升高;Gas2干扰组中Gas2和P53基因mRNA的表达水平在所有温度下均显著低于阴性对照组;随着温度降低,阴性对照组及Gas2干扰组的细胞凋亡率均明显升高,但Gas2干扰组的细胞凋亡率明显低于阴性对照组;低温胁迫下,对照组及Gas2干扰组的细胞增殖率与25℃时相比均显著下降;当温度降至15℃时,Gas2干扰组的细胞增殖率显著高于对照组。罗非鱼肾脏细胞在受低温胁迫时,抑制Gas2基因能够降低P53基因mRNA的表达水平以及细胞的凋亡率,并增加细胞的增殖。
The Gas2 short hairpin RNA(Gas2 shRNA) and negative control short hairpin RNA were transfected with lipofectin into tilapia kidney cells to investigate the effect of growth arrest specific 2 gene(Gas2) interference on proliferation and apoptosis of tilapia kidney cell line under low temperature stress, 24 hours after the transfection, Tilapia kidney cells were treated with decreasing temperature(5 ℃/d cooling rate, from 25 to 10 ℃), and sampled respectively at the temperature of 25, 20, 15 and 10 ℃ after 24 hours transfection. The mRNA expression of Gas2 gene and P53 gene was detected by real-time fluorescent quantitative PCR. Cell apoptosis was detected by flow cytometry. Cell proliferation was detected by WST-1 cell proliferation assay. In comparison with 25 ℃, the expression of Gas2 and P53 mRNA in the control group and Gas2 interference group at 15 ℃ and 10 ℃ increased significantly. Meanwhile, Gas2 interference tilapia kidney cell exhibited significantly decreased expression of Gas2 and P53 mRNA at all sampling times compared with the control group. With the decreasing temperature, apoptosis rates of the cells in both the control group and Gas2 interference group increased significantly. Simultaneously, the apoptosis rates of the Gas2 interference group were significantly lower than those of the control group at all the time points. The cell proliferation rates of the two groups both decreased significantly compared with 25 ℃. When the temperature dropped to 15 ℃, the cell proliferation rate of Gas2 interference group was significantly higher than that of the control group. The results indicated that inhibition of the Gas2 gene could reduce the expression of P53 mRNA, the apoptosis rate of the tilapia kidney cells and increase the proliferation of these cells under low temperature stress.
The Gas2 short hairpin RNA(Gas2 shRNA) and negative control short hairpin RNA were transfected with lipofectin into tilapia kidney cells to investigate the effect of growth arrest specific 2 gene(Gas2) interference on proliferation and apoptosis of tilapia kidney cell line under low temperature stress, 24 hours after the transfection, Tilapia kidney cells were treated with decreasing temperature(5 ℃/d cooling rate, from 25 to 10 ℃), and sampled respectively at the temperature of 25, 20, 15 and 10 ℃ after 24 hours transfection. The mRNA expression of Gas2 gene and P53 gene was detected by real-time fluorescent quantitative PCR. Cell apoptosis was detected by flow cytometry. Cell proliferation was detected by WST-1 cell proliferation assay. In comparison with 25 ℃, the expression of Gas2 and P53 mRNA in the control group and Gas2 interference group at 15 ℃ and 10 ℃ increased significantly. Meanwhile, Gas2 interference tilapia kidney cell exhibited significantly decreased expression of Gas2 and P53 mRNA at all sampling times compared with the control group. With the decreasing temperature, apoptosis rates of the cells in both the control group and Gas2 interference group increased significantly. Simultaneously, the apoptosis rates of the Gas2 interference group were significantly lower than those of the control group at all the time points. The cell proliferation rates of the two groups both decreased significantly compared with 25 ℃. When the temperature dropped to 15 ℃, the cell proliferation rate of Gas2 interference group was significantly higher than that of the control group. The results indicated that inhibition of the Gas2 gene could reduce the expression of P53 mRNA, the apoptosis rate of the tilapia kidney cells and increase the proliferation of these cells under low temperature stress.
引文
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[26] Sgorbissa A,Benetti R,Marzinotto S,et al.Caspase-3 and caspase-7 but not caspase-6 cleave Gas2 in vitro:implications for microfilament reorganization during apoptosis [J].J Cell Sci,1999,112(23):4475-4482.
[27] Benetti R,Sal G D,Monte M,et al.The death substrate Gas2 binds m-calpain and increases susceptibility to p53-dependent apoptosis [J].EMBO J,2014,20(11):2702-2714.
[2] Carginale V,Trinchella F,Capasso C,et al.Gene amplification and cold adaptation of pepsin in Antarctic fish.A possible strategy for food digestion at low temperature [J].Gene,2004,336(2):195-205.
[3] Guderley H.Metabolic responses to low temperature in fish muscle [J].Biol Rev,2010,79(2):409-427.
[4]王金凤,胡鹏,牛虹博,等.低温胁迫对鱼类PI3K/AKT/GSK-3β信号通路的影响[J].生物学杂志,2016,33(6):24-28.
[5] Zhao H Y,Han D,Xie S Q,et al.Effect of water temperature on the growth performance and digestive enzyme activities of Chinese longsnout catfish (Leiocassis longirostris Günther) [J].Aquacult Res,2010,40(16):1864-1872.
[6]李进荣.对池养罗非鱼耐寒能力的观察[J].水产科学,1985,(1):37.
[7]李思发.吉富品系尼罗罗非鱼引进史[J].中国水产,2001,(10):52-53.
[8] Ndong D,Chen Y Y,Lin Y H,et al.The immune response of tilapia Oreochromis mossambicus and its susceptibility to Streptococcus iniae under stress in low and high temperatures [J].Fish Shellfish Immunol,2007,22(6):686-694.
[9] Chebaani N,Guardiola F A,Sihem M,et al.Innate humoral immune parameters in Tilapia zillii under acute stress by low temperature and crowding [J].Fish Physiol Biochem,2014,40(3):797-804.
[10] Yang C,Jiang M,Wen H,et al.Analysis of differential gene expression under low-temperature stress in Nile tilapia (Oreochromis niloticus) using digital gene expression [J].Gene,2015,564(2):134-140.
[11] Brancolini C,Bottega S,Schneider C.Gas2,a growth arrest-specific protein,is a component of the microfilament network system [J].J Cell Biol,1992,117(6):1251-1261.
[12] Lee K K,Tang M K,Yew D T,et al.Gas2 is a multifunctional gene involved in the regulation of apoptosis and chondrogenesis in the developing mouse limb [J].Dev Biol,1999,207(1):14.
[13] Yao W,Gu L,Sun D,et al.Wild type p53 gene causes reorganization of cytoskeleton and,therefore,the impaired deformability and difficult migration of murine erythroleukemia cells [J].Cytoskeleton,2010,56(1):1-12.
[14] Araki K,Ebata T,Guo A K,et al.p53 regulates cytoskeleton remodeling to suppress tumor progression [J].Cell Mol Life Sci,2015,72(21):4077-4094.
[15] Yang C G,Wu F,Lu X,et al.Growth arrest specific gene 2 in tilapia (Oreochromis niloticus):molecular characterization and functional analysis under low-temperature stress [J].BMC Mol Biol,2017,18(1):18.
[16] K Nagasawa,N Matsuura,Y Takeshita,et al.Attenuation of cold stress-induced exacerbation of cardiac and adipose tissue pathology and metabolic disorders in a rat model of metabolic syndrome by the glucocorticoid receptor antagonist RU486 [J].Nutr Diabetes,2016,6(4):e207.
[17] Wen B,Jin S R,Chen Z Z,et al.Plasticity of energy reserves and metabolic performance of discus fish (Symphysodon aequifasciatus) exposed to low-temperature stress [J],Aquaculture,2017,481:169-171.
[18] Yao C L,Somero G N.The impact of acute temperature stress on hemocytes of invasive and native mussels (Mytilus galloprovincialis and Mytilus californianus):DNA damage,membrane integrity,apoptosis and signaling pathways [J].J Exp Biol,2012,215(24):4267-77.
[19] Hochachka P W.Defense strategies against hypoxia and hypothermia [J].Science,1986,231(4735):234-241.
[20] Nagle W A,Soloff B L,Jr A J M,et al.Cultured Chinese hamster cells undergo apoptosis after exposure to cold but nonfreezing temperatures [J].Cryobiology,1990,27(4):439-451.
[21] Sonna L A,Fujita J,Gaffin S L,et al.Invited review:Effects of heat and cold stress on mammalian gene expression [J].J Appl Physiol,2002,92(4):1725-1742.
[22] Los D A,Murata N.Membrane fluidity and its roles in the perception of environmental signals [J].Biochim Biophys Acta,2004,1666(1):142-157.
[23] Duan Z G,Wu J Y,Ge Y Y,et al.The functional significance of the plasma membrane in cold acclimation of two fish species:the tilapia(oreochromis niloticus) and the paradise fish(macropodus opercularis) [J].Acta Hydrobiol Sinica,2015.
[24]严文静,陈汉民,赵小立,等.低温诱导淡水白鲳尾鳍细胞系早期凋亡[J].实验生物学报,2005,38(2):105-110.
[25] Tong Z,Bama D,Isabelle R,et al.Growth-arrest-specific protein 2 inhibits cell division in xenopus embryos [J].Plos One,2011,6(9):e24698.
[26] Sgorbissa A,Benetti R,Marzinotto S,et al.Caspase-3 and caspase-7 but not caspase-6 cleave Gas2 in vitro:implications for microfilament reorganization during apoptosis [J].J Cell Sci,1999,112(23):4475-4482.
[27] Benetti R,Sal G D,Monte M,et al.The death substrate Gas2 binds m-calpain and increases susceptibility to p53-dependent apoptosis [J].EMBO J,2014,20(11):2702-2714.