去泛素化酶USP10 RNAi慢病毒载体的构建及其对PC12细胞增殖的影响
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摘要
目的:构建大鼠去泛素化酶USP10 siRNA重组慢病毒载体,为探讨USP10对神经元的调控作用提供体外模型。方法:设计并合成3对特异性大鼠USP10基因的RNAi靶序列,聚合酶链反应(PCR)扩增后,用AgeⅠ及EcoRⅠ双酶切及连接酶构建线性化的GV208慢病毒载体;转化感受态细胞,经Amp抗性筛选阳性克隆,PCR鉴定阳性克隆载体并测序;病毒包装并转染至293T细胞,观察绿色荧光蛋白(GFP)表达,荧光法检测滴度。用重组慢病毒感染PC12细胞,Western Blot检测USP10蛋白的表达,CCK-8法检测PC12细胞增殖。结果:经测序鉴定成功构建了3对USP10 RNAi慢病毒载体,感染PC12细胞后,USP10蛋白的表达显著被抑制,PC12细胞的增殖能力明显减弱。结论:成功构建能高效、稳定抑制USP10表达的PC12细胞株,并明显抑制PC12细胞增殖能力,为探讨USP10对神经元的调控作用提供基础。
Objective: To construct a lentiviral vector of RNA interference(RNAi) of rat ubiquitination enzyme10(USP10), and then provide a cell model for exploring the regulation of USP10 on neurons.Methods: Three interfering sequences targeting USP10 were designed, amplification of USP10 gene fragment was performed by polymerase chain reaction(PCR). The GV208 lenti-virus vector was digested with Age Ⅰ and EcoR Ⅰ to linearize, then linked with the USP10 gene fragment by a ligase.The ligation products were transformed to competent cells, the positive clones were screened, and then identified by PCR follwed by RNA sequencing. Next, the lentiviral vectors carrying USP10 RNAi were packaged and transfected in 293 cells by using the GV208 Lentiviral Packaging Kit, and then the virus titer was measured by fluorescence method. Furthermore, the lentiviral particles were collected to infect PC12 cells, and the transfection efficiency was observed under fluorescence microscope.PC12 cells with stable USP10 down-regulation were screened with puromycin, and the expres-sion level of USP10 was detected by Western blotting. The proliferation of PC12 cells after the infection was assessed using CCK-8 assay. Results: RNA sequencing demonstrated successful construction of three USP10 RNAi lentivirus vectors. After infected PC12 cell, the stable cell line with USP10 knockdown was successfully screened and the proliferation of PC12 cells was also significantly decreased.Conclusion: PC12 cells line transfected by USP10 RNAi lentiviral vector is successfully constructed,which can effectively and stably inhibit the expression of USP10, and decrease proliferation of PC12 cells significantly, thus laying a foundation for further study on the regulation of USP10 on neurons
Objective: To construct a lentiviral vector of RNA interference(RNAi) of rat ubiquitination enzyme10(USP10), and then provide a cell model for exploring the regulation of USP10 on neurons.Methods: Three interfering sequences targeting USP10 were designed, amplification of USP10 gene fragment was performed by polymerase chain reaction(PCR). The GV208 lenti-virus vector was digested with Age Ⅰ and EcoR Ⅰ to linearize, then linked with the USP10 gene fragment by a ligase.The ligation products were transformed to competent cells, the positive clones were screened, and then identified by PCR follwed by RNA sequencing. Next, the lentiviral vectors carrying USP10 RNAi were packaged and transfected in 293 cells by using the GV208 Lentiviral Packaging Kit, and then the virus titer was measured by fluorescence method. Furthermore, the lentiviral particles were collected to infect PC12 cells, and the transfection efficiency was observed under fluorescence microscope.PC12 cells with stable USP10 down-regulation were screened with puromycin, and the expres-sion level of USP10 was detected by Western blotting. The proliferation of PC12 cells after the infection was assessed using CCK-8 assay. Results: RNA sequencing demonstrated successful construction of three USP10 RNAi lentivirus vectors. After infected PC12 cell, the stable cell line with USP10 knockdown was successfully screened and the proliferation of PC12 cells was also significantly decreased.Conclusion: PC12 cells line transfected by USP10 RNAi lentiviral vector is successfully constructed,which can effectively and stably inhibit the expression of USP10, and decrease proliferation of PC12 cells significantly, thus laying a foundation for further study on the regulation of USP10 on neurons
引文
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[17]廖晓红,尹蔚兰,王芳,等.shRNA-PAX6慢病毒载体构建及其对胶质瘤U251细胞增殖的影响[J].南方医科大学学报,2017,37(12):1 603-1 608.Liao XH,Yin WL,Wang F,et al.Construction of shRNA-PAX6 lentiviral vector and its effect on proliferation of glioma U251 cells[J].Journal of Southern Medical University,2017,37(12):1 603-1 608.
[2]Li Y,Kong Y,Zhou Z,et al.The HECTD3 E3 ubiquitin ligase facilitates cancer cell survival by promoting K63-linked polyubiquitination of caspase-8[J].Cell Death Dis,2013,4:e935.
[3]Luo P,Qin C,Zhu L,et al.Ubiquitin-specific peptidase10(USP10)inhibits hepatic steatosis,insulin resistance and inflammation via Sirt6[J].Hepatology,2018:doi:10.1002/hep.30062.[Epub ahead of print]
[4]Liu F,Walters KJ.Multitasking with ubiquitin through multivalent interactions[J].Trends Biochem Sci,2010,35(6):352-360.
[5]Reyes-Turcu FE,Ventii KH,Wilkinson KD.Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes[J].Annu Rev Biochem,2009,78:363-397.10
[6]Weisberg EL,Schauer NJ,Yang J,et al.Inhibition of USP10 induces degradation of oncogenic FLT3[J].Nat Chem Biol,2017,13(12):1 207-1 215.
[7]Pan L,Chen Z,Wang L,et al.Deubiquitination and stabilization of T-bet by USP10[J].Biochem Biophys Res Commun,2014,449(3):289-294.
[8]Takahashi M,Higuchi M,Makokha GN,et al.HTLV-1 Tax oncoprotein stimulates ROS production and apoptosis in T cells by interacting with USP10[J].Blood,2013,122(5):715-725.
[9]Deng M,Yang X,Qin B,et al.Deubiquitination and activation of AMPK by USP10[J].Mol Cell,2016,61(4):614-624.
[10]Yuan J,Luo K,Zhang L,et al.USP10 regulates p53localization and stability by deubiquitinating p53[J].Cell,2010,140(3):384-396.
[11]Liu J,Xia H,Kim M,et al.Beclin1 controls the levels of p53 by regulating the deubiquitination activity of USP10 and USP13[J].Cell,2011,147(1):223-234.
[12]Sowa ME,Bennett EJ,Gygi SP,et al.Defining the human deubiquitinating enzyme interaction landscape[J].Cell,2009,138(2):389-403.
[13]Lu C,Ning Z,Wang A,et al.USP10 suppresses tumor progression by inhibiting mTOR activation in hepatocellular carcinoma[J].Cancer Lett,2018,436:139-148.
[14]Sacco JJ,Coulson JM,Clague MJ,et al.Emerging roles of deubiquitinases in cancer-associated pathways[J].IUBMB Life,2010,62(2):140-157.
[15]刘联斌,张相民,曾汶,等.构建COX2基因慢病毒载体对肺腺癌A549细胞增殖与凋亡的影响[J].临床肿瘤学杂志,2018,23(1):7-12.Liu LB,Zhang XM,Zeng W,et al.Effects of constructing COX2 gene lentiviral vector on proliferation and apoptosis of lung adenocarcinoma A549[J].Journal of Clinical Oncology,2018,23(1):7-12.
[16]Das PC,Mcelroy WK,Cooper RL.Potential mechanisms responsible for chlorotriazine-induced alterations in catecholamines in pheochromocytoma(PC12)cells[J].Life Sci,2003,73(24):3 123-3 138.
[17]廖晓红,尹蔚兰,王芳,等.shRNA-PAX6慢病毒载体构建及其对胶质瘤U251细胞增殖的影响[J].南方医科大学学报,2017,37(12):1 603-1 608.Liao XH,Yin WL,Wang F,et al.Construction of shRNA-PAX6 lentiviral vector and its effect on proliferation of glioma U251 cells[J].Journal of Southern Medical University,2017,37(12):1 603-1 608.