ST8Sia1基因过表达载体构建与鉴定及对人黑色素瘤细胞增殖的影响
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摘要
目的构建ST8Sia1基因过表达载体,建立稳定过表达ST8Sia1的细胞株,检测ST8Sia1的表达及对细胞增殖的影响。方法通过PCR反应扩增ST8Sia1基因片段,双酶切目的基因片段和pEGFP-C1载体以及pHBLV-CMV-MCS-3flag-EF1-ZsGreen-T2A-Puro载体,分别将目的基因片段和不同载体连接,得到ST8Sia1-pEGFP-C1重组载体和重组慢病毒载体。采用PCR方法和DNA序列进行鉴定。分别用不同载体转染黑色素瘤细胞WM451,筛选建立稳定过表达ST8Sia1的细胞株,Real-time PCR检测稳转细胞ST8Sia1 mRNA水平;Western blot法检测稳转细胞中ST8Sia1蛋白表达水平;CCK-8法检测稳转细胞的生长增殖活性;软琼脂克隆形成实验分析稳转细胞的克隆形成能力。结果成功构建ST8Sia1-pEGFP-C1重组质粒及ST8Sia1过表达慢病毒载体。发现ST8Sia1过表达慢病毒载体转染效率较高,建立稳定过表达ST8Sia1的WM451细胞株,发现ST8Sia1过表达促进肿瘤细胞增殖和克隆形成。结论成功构建了ST8Sia1过表达载体,并发现ST8Sia1过表达能够促进黑色素瘤细胞WM451的增殖、克隆形成能力。
Aim To construct different over-expression vectors of ST8 Sia1 gene and establish a cell line with stable ST8 Sia1 over-expression, and to check the proliferation of cells with ST8 Sia1 over-expression. Methods Polymerase chain reaction(PCR) was used to amplify the code region of ST8 Sia1. The amplified ST8 Sia1 fragment and pEGFP-C1 vector, pHBLV-CMV-MCS-3 flag-EF1-ZsGreen-T2 A-Puro vector were digested with restriction enzymes. The target gene fragment and the different vectors were ligated to obtain the ST8 Sia1-pEGFP-C1 recombinant vector and the recombinant lentiviral vector, respectively. PCR and DNA sequencing were used to identify recombinant vectors. Melanoma cells WM451 were transfected with different vectors. Cell line with stable ST8 Sia1 over-expressing was established.ST8 Sia1 mRNA level was checked by real-time PCR. ST8 Sia1 protein expression level was checked by Western blot. Proliferation of the stable cells was assayed by CCK-8 method. The clony formation of stable cells was also performed. Results Both ST8 Sia1-pEGFP-C1 recombinant plasmid and ST8 Sia1 over-expression lentiviral vectors were successfully constructed. The transfection efficiency of ST8 Sia1 over-expression lentiviral vector was much higher than that of ST8 Sia1-pEGFP-C1 recombinant plasmid. A WM451 cell line with stable ST8 Sia1 over-expression lentiviral vectors was established. Results showed that the over-expression of ST8 Sia1 promoted the proliferation and colony formation of cells. Conclusions ST8 Sia1 over-expression vectors are successfully constructed. The over-expression of ST8 Sia1 promotes the proliferation and colony formation of WM451 cells.
Aim To construct different over-expression vectors of ST8 Sia1 gene and establish a cell line with stable ST8 Sia1 over-expression, and to check the proliferation of cells with ST8 Sia1 over-expression. Methods Polymerase chain reaction(PCR) was used to amplify the code region of ST8 Sia1. The amplified ST8 Sia1 fragment and pEGFP-C1 vector, pHBLV-CMV-MCS-3 flag-EF1-ZsGreen-T2 A-Puro vector were digested with restriction enzymes. The target gene fragment and the different vectors were ligated to obtain the ST8 Sia1-pEGFP-C1 recombinant vector and the recombinant lentiviral vector, respectively. PCR and DNA sequencing were used to identify recombinant vectors. Melanoma cells WM451 were transfected with different vectors. Cell line with stable ST8 Sia1 over-expressing was established.ST8 Sia1 mRNA level was checked by real-time PCR. ST8 Sia1 protein expression level was checked by Western blot. Proliferation of the stable cells was assayed by CCK-8 method. The clony formation of stable cells was also performed. Results Both ST8 Sia1-pEGFP-C1 recombinant plasmid and ST8 Sia1 over-expression lentiviral vectors were successfully constructed. The transfection efficiency of ST8 Sia1 over-expression lentiviral vector was much higher than that of ST8 Sia1-pEGFP-C1 recombinant plasmid. A WM451 cell line with stable ST8 Sia1 over-expression lentiviral vectors was established. Results showed that the over-expression of ST8 Sia1 promoted the proliferation and colony formation of cells. Conclusions ST8 Sia1 over-expression vectors are successfully constructed. The over-expression of ST8 Sia1 promotes the proliferation and colony formation of WM451 cells.
引文
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[15] Kang N Y,Kim C H,Kim K S,et al.Expression of the human CMP-NeuAc:GM3 alpha2,8-sialyltransferase (GD3 synthase) gene through the NF-kappaB activation in human melanoma SK-MEL-2 cells[J].Biochim Biophys Acta,2007,1769(11-12):622-30.
[2] Weinberg R A.How cancer arises[J].Sci Am,1996,275(3):62-70.
[3] 纪玉婷,杨燕,郑荣生,等.慢病毒介导稳定高表达Cx32的Huh7细胞系建立及其对细胞增殖的影响[J].中国药理学通报,2018,34(2):284-9.[3] Ji Y T,Yang Y,Zheng R S,et al.Construction of lentiviral vector-mediated Cx32 stably over-expressed Huh7 cell line and its effect on cell proliferation[J].Chin Pharmacol Bull,2018,34(2):284-9.
[4] Azoury S C,Lange J R.Epidemiology,risk factors,prevention,and early detection of melanoma[J].Surg Clin North Am,2014,94(5):945-62.
[5] Takashima S,Matsumoto T,Tsujimoto M,et al.Effects of amino acid substitutions in the sialylmotifs on molecular expression and enzymatic activities of alpha2,8-sialyltransferases ST8Sia-I and ST8Sia-VI[J].Glycobiology,2013,23:603-12.
[6] Rimoldi S,Papis E,Bernardini G,et al.Molecular cloning and expression of alpha2,8-sialyltransferase (ST8Sia I,GD3 synthase) in Xenopus[J].Mol Cell Biochem,2007,301(1-2):143-53.
[7] Yamashiro S,Okada M,Haraguchi M,et al.Expression of alpha 2,8-sialyltransferase (GD3 synthase) gene in human cancer cell lines:high level expression in melanomas and up-regulation in activated T lymphocytes[J].Glycoconj J,1995,12(6):894-900.
[8] Naldini L,Trono D,Verma I.Lentiviral vectors,two decades later[J].Science,2016,353(6304):1101-2.
[9] de Bruyns A,Geiling B,Dankort D.Construction of modular lentiviral vectors for effective gene expression and knockdown[J].Methods Mol Biol,2016,1448:3-21.
[10] Tiscornia G,Singer O,Verma I.Design and cloning of lentiviral vectors expressing small interfering RNAs[J].Nat Protoc,2006,1(1):234-40.
[11] Sarkar T,Battula V,Werden S,et al.GD3 synthase regulates epithelial-mesenchymal transition and metastasis in breast cancer[J].Oncogene,2015,34(23):2958-67.
[12] Battula V L,Shi Y,Evans K W,et al.Ganglioside GD2 identifies breast cancer stem cells and promotes tumorigenesis[J].J Clin Invest,2012,122(6):2066-78.
[13] Cazet A,Lefebvre J,Adriaenssens E,et al.GD3 synthase expression enhances proliferation and tumor growth of MDA-MB-231 breast cancer cells through c-Met activation[J].Mol Cancer Res,2010,8(11):1526-35.
[14] Birkle S,Gao L,Zeng G,et al.Down-regulation of GD3 ganglioside and its O-acetylated derivative by stable transfection with antisense vector against GD3-synthase gene expression in hamster melanoma cells:effects on cellular growth,melanogenesis,and dendricity[J].J Neurochem,2000,74(2):547-54.
[15] Kang N Y,Kim C H,Kim K S,et al.Expression of the human CMP-NeuAc:GM3 alpha2,8-sialyltransferase (GD3 synthase) gene through the NF-kappaB activation in human melanoma SK-MEL-2 cells[J].Biochim Biophys Acta,2007,1769(11-12):622-30.