利用慢病毒载体构建稳定干扰AMPKα1的HeLa细胞系
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摘要
目的建立稳定干扰AMPKα1的He La细胞系。方法利用AMPKα1慢病毒干扰质粒PLKO.1-puro-AMPKα1转染293T细胞制备重组慢病毒,然后用重组慢病毒感染He La细胞,荧光显微镜观察病毒感染效率,免疫印迹实验检测Sh-AMPKα1病毒感染组的AMPKα1表达。然后采用嘌呤霉素筛选出稳定干扰AMPKα1的He La细胞单克隆,免疫印迹实验和免疫荧光实验检测AMPKα1表达情况。最后用AMPK激活剂Metformin实验验证AMPKα1稳定干扰的Hela细胞中AMPKα1蛋白的表达以及AMPKα1的生物功能。结果荧光拍照结果显示慢病毒成功感染Hela细胞;免疫印迹实验显示特异性Sh-AMPKα1病毒感染组的AMPKα1表达[(0.58±0.02)DPI]比WT组[(1.00±0.00)DPI]低,免疫荧光实验结果也显示Sh-AMPKα1病毒感染组中AMPKα1的平均光密度[(0.09±0.01)IOD/area]比WT组[(1.00±0.00)IOD/area]要低,差异均具有显著统计学意义(P<0.01);经AMPK激活剂Metformin处理后,AMPKα1稳定干扰的Hela细胞中仍无AMPKα1蛋白表达,并且Sh-AMPKα1组中LC3-Ⅱ/Ⅰ/Actin[(1.00±0.00I)DPI]也显著低于HA-AMPKα1组[(1.62±0.02)DPI],表明病毒感染组细胞中AMPKα1不能发挥其生物功能,差异具有显著统计学意义(P<0.01)。结论利用Sh RNA-AMPKα1慢病毒筛选出了高效干扰AMPKα1表达的He La细胞系,为后续深入研究AMPKα1的生物功能奠定了基础。
Objective To establish adenosine monophosphate-activated protein kinase α1(AMPKα1) stably in-terfered Hela cell line. Methods Lentiviral vector PLKO.1-puro-AMPKα1 was transfected into 293 T cells to prepare re-combinant lentivirus. Then He La cells were infected with the recombinant lentivirus, and the efficiency of virus infectionwas detected by fluorescent photography. The monoclonal Hela cell stably interfered AMPKα1 was screened by puromycin,and Western blot and immunofluorescence were used to detecte the expression of AMPKα1 in specific Sh-AMPKα1 virusinfected group. Finally, the biological function of AMPKα1 in Hela cells stably interfered with AMPKα1 under the treat-ment of metformin, a AMPK activator, was detected. Results The results of fluorescent photography showed that the virusinfection was highly efficient. Immunoblotting results showed that the expression of AMPKα1 was significantly reduced inHela cells stably interfered with AMPKα1(0.58±0.02) DPI compared with the WT group(1.00±0.00) DPI. Immunofluores-cence results also showed that the mean optical density of AMPKα1 was(0.09±0.01) IOD/area in Sh-AMPKα1 virus infect-ed group versus(1.00±0.00) IOD/area in the WT group. All the above differences were statistically significant(P<0.01). Af-ter the treatment of metformin, the expression of AMPKα1 in the Sh-AMPKα1 virus infected group was still not expressed.Moreover, the relative ratio of LC3-Ⅱ/Ⅰ/Actinin in the Sh-AMPKα1 group was(1.00±0.00) DPI, which was significantlylower than(1.62±0.02) DPI in the HA-AMPKα1 group, indicating that AMPKα1 could not play its biological function(P<0.01). Conclusion The above results revealed that AMPKα1 effectively interfered Hela cell line was established by Sh R-NA-AMPKα1 lentivirus, which laid the foundation for the further research of AMPKα1 biological function.
Objective To establish adenosine monophosphate-activated protein kinase α1(AMPKα1) stably in-terfered Hela cell line. Methods Lentiviral vector PLKO.1-puro-AMPKα1 was transfected into 293 T cells to prepare re-combinant lentivirus. Then He La cells were infected with the recombinant lentivirus, and the efficiency of virus infectionwas detected by fluorescent photography. The monoclonal Hela cell stably interfered AMPKα1 was screened by puromycin,and Western blot and immunofluorescence were used to detecte the expression of AMPKα1 in specific Sh-AMPKα1 virusinfected group. Finally, the biological function of AMPKα1 in Hela cells stably interfered with AMPKα1 under the treat-ment of metformin, a AMPK activator, was detected. Results The results of fluorescent photography showed that the virusinfection was highly efficient. Immunoblotting results showed that the expression of AMPKα1 was significantly reduced inHela cells stably interfered with AMPKα1(0.58±0.02) DPI compared with the WT group(1.00±0.00) DPI. Immunofluores-cence results also showed that the mean optical density of AMPKα1 was(0.09±0.01) IOD/area in Sh-AMPKα1 virus infect-ed group versus(1.00±0.00) IOD/area in the WT group. All the above differences were statistically significant(P<0.01). Af-ter the treatment of metformin, the expression of AMPKα1 in the Sh-AMPKα1 virus infected group was still not expressed.Moreover, the relative ratio of LC3-Ⅱ/Ⅰ/Actinin in the Sh-AMPKα1 group was(1.00±0.00) DPI, which was significantlylower than(1.62±0.02) DPI in the HA-AMPKα1 group, indicating that AMPKα1 could not play its biological function(P<0.01). Conclusion The above results revealed that AMPKα1 effectively interfered Hela cell line was established by Sh R-NA-AMPKα1 lentivirus, which laid the foundation for the further research of AMPKα1 biological function.
引文
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[18]Ducommun S,Deak M,Sumpton D,et al.Motif affinity and mass spec-trometry proteomic approach for the discovery of cellular AMPK tar-gets:identification of mitochondrial fission factor as a new AMPK sub-strate[J].Cell Signal,2015,27(5):978-988.
[19]Matic I,Strobbe D,Di Guglielmo F,et al.Molecular biology digest ofcell mitophagy[J].Int Rev Cell Mol Biol,2017,332:233-258.
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[24]Charbgoo F,Behmanesh M,Nikkhah M,et al.RNAi mediated gene si-lencing of ITPA using a targeted nanocarrier:apoptosis induction inSKBR3 cancer cells[J].Clin Exp Pharmacol Physiol,2017,44(8):888-894.
[2]Kurumbail RG,Calabrese MF.Structure and Regulation of AMPK[J].EXS,2016,107:3-22.
[3]Morales-Alamo D,Ponce-Gonzalez JG,Guadalupe-Grau A,et al.Crit-ical role for free radicals on sprint exercise-induced Ca MKII andAMPKalpha phosphorylation in human skeletal muscle[J].J ApplPhysiol(1985),2013,114(5):566-577.
[4]Zhao B,Qiang L,Joseph J,et al.Mitochondrial dysfunction activatesthe AMPK signaling and autophagy to promote cell survival[J].GenesDis,2016,3(1):82-87.
[5]Zhang CS,Lin SC.AMPK promotes autophagy by facilitating mitochon-drial fission[J].Cell Metab,2016,23(3):399-401.
[6]Bremer K,Kocha KM,Snider T,et al.Sensing and responding to ener-getic stress:The role of the AMPK-PGC1alpha-NRF1 axis in controlof mitochondrial biogenesis in fish[J].Comp Biochem Physiol B Bio-chem Mol Biol,2016,199:4-12.
[7]Li J,Wang Y,Wang Y,et al.Pharmacological activation of AMPK pre-vents Drp1-mediated mitochondrial fission and alleviates endoplasmicreticulum stress-associated endothelial dysfunction[J].J Mol Cell Car-diol,2015,86:62-74.
[8]Toyama EQ,Herzig S,Courchet J,et al.Metabolism.AMP-activatedprotein kinase mediates mitochondrial fission in response to energystress[J].Science,2016,351(6270):275-281.
[9]Tian W,Li W,Chen Y,et al.Phosphorylation of ULK1 by AMPK regu-lates translocation of ULK1 to mitochondria and mitophagy[J].FEBSLett,2015,589(15):1847-1854.
[10]Cai Z,Yan L J,Li K,et al.Roles of AMP-activated protein kinase inAlzheimer's disease[J].Neuromolecular Med,2012,14(1):1-14.
[11]Vingtdeux V,Chandakkar P,Zhao H,et al.Novel synthetic small-mol-ecule activators of AMPK as enhancers of autophagy and amyloid-betapeptide degradation[J].FASEB J,2011,25(1):219-231.
[12]Jin J,Gu H,Anders NM,et al.Metformin protects cells from mutanthuntingtin toxicity through activation of AMPK and modulation of mito-chondrial dynamics[J].Neuromolecular Med,2016,18(4):581-592.
[13]Hang L,Thundyil J,Lim KL.Mitochondrial dysfunction and Parkinsondisease:a Parkin-AMPK alliance in neuroprotection[M].Ann N YAcad Sci,2015,1350:37-47.
[14]Mohr SE,Smith JA,Shamu CE,et al.RNAi screening comes of age:im-proved techniques and complementary approaches[J].Nat Rev MolCell Biol,2014,15(9):591-600.
[15]Segura MM,Garnier A,Durocher Y,et al.New protocol for lentiviralvector mass production[J].Methods Mol Biol,2010,614:39-52.
[16]Song YM,Lee YH,Kim JW,et al.Metformin alleviates hepatosteatosisby restoring SIRT1-mediated autophagy induction via an AMP-activat-ed protein kinase-independent pathway[J].Autophagy,2015,11(1):46-59.
[17]Wikstrom JD,Israeli T,Bachar-Wikstrom E,et al.AMPK regulates ERmorphology and function in stressed pancreatic beta-cells via phosphor-ylation of DRP1[J].Mol Endocrinol,2013,27(10):1706-1723.
[18]Ducommun S,Deak M,Sumpton D,et al.Motif affinity and mass spec-trometry proteomic approach for the discovery of cellular AMPK tar-gets:identification of mitochondrial fission factor as a new AMPK sub-strate[J].Cell Signal,2015,27(5):978-988.
[19]Matic I,Strobbe D,Di Guglielmo F,et al.Molecular biology digest ofcell mitophagy[J].Int Rev Cell Mol Biol,2017,332:233-258.
[20]Mouton-Liger F,Jacoupy M,Corvol JC,et al.PINK1/Parkin-depen-dent mitochondrial surveillance:from pleiotropy to Parkinson's dis-ease[J].Front Mol Neurosci,2017,10:120.
[21]Kerr JS,Adriaanse BA,Greig NH,et al.Mitophagy and Alzheimer'sdisease:cellular and molecular mechanisms[J].Trends Neurosci,2017,40(3):151-166.
[22]Khalil B,El FN,Aouane A,et al.PINK1-induced mitophagy promotesneuroprotection in Huntington's disease[J].Cell Death Dis,2015,6:e1617.
[23]Subramanya S,Kim SS,Manjunath N,et al.RNA interference-basedtherapeutics for human immunodeficiency virus HIV-1 treatment:syn-thetic si RNA or vector-based sh RNA?[J].Expert Opin Biol Ther,2010,10(2):201-213.
[24]Charbgoo F,Behmanesh M,Nikkhah M,et al.RNAi mediated gene si-lencing of ITPA using a targeted nanocarrier:apoptosis induction inSKBR3 cancer cells[J].Clin Exp Pharmacol Physiol,2017,44(8):888-894.