SLUG基因可调控性干扰载体的构建及其在鼻咽癌细胞中的表达
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摘要
目的探讨鼻咽癌细胞中基因表达,构建可调控性相关基因干扰载体,并验证其干扰作用。方法将高转移人鼻咽癌细胞株5-8F、5-8F-H3细胞进行培养,实时荧光定量RT-PCR检测5-8F及5-8F-H3细胞中SLUG、CBX3、MAD2L1、PLAU、HMGA1基因的表达水平。应用酶切、连接技术构建pSUPERIOR-SLUG干扰载体,采用脂质体转染方法转染5-8F-H3细胞,经嘌呤霉素筛选获得阳性克隆,实时荧光定量RT-PCR检测干扰效果。结果5-8F及5-8F-H3细胞中SLUG基因表达水平均明显高于CBX3、MAD2L1、PLAU、HMGA1基因(均P<0.05),相对倍数分别为(11.23±1.78)倍、(1.23±0.17)倍、(1.57±0.27)倍、(6.36±0.38)倍、(3.66±1.14)倍,以SLUG基因倍数最高。所构建的pSUPERIOR-SLUG重组质粒,经酶切鉴定及序列分析证实为所需的目标序列,并成功转染鼻咽癌5-8F-H3细胞。阳性克隆S2-18基因表达水平高于阳性克隆S1-7、S1-9、S2-22基因(P<0.05),干扰率分别为67.42%、72.45%、84.20%、75.61%。结论 SLUG基因在鼻咽癌5-8F-H3中高表达。成功构建了重组质粒——pSUPERIOR-SLUG,并成功转染鼻咽癌5-8F-H3细胞,为后续进一步研究SLUG在鼻咽癌的作用奠定了基础。
Objective To construct the inducible SLUG interference vector,and to investigate the changes in SLUG expression in nasopharyngeal carcinoma cells.Methods The highly metastatic human nasopharyngeal carcinoma cell lines 5-8 F and 5-8 F-H3 were cultured in this study.Real-time quantitative RT-PCR was performed to detect the expression of SLUG,CBX3,MAD2 L1,PLAU and HMGA1 genes in 5-8 F and 5-8 F-H3 cells.The pSUPERIOR-SLUG-shRNA interference plasmid was constructed using enzyme restriction and ligation technology,and then were transfected into 5-8 F-H3 cells by lipofectamine.The positive clones were selected with puromycin,and the interference efficiency was identified by real-time quantitative RT-PCR.ResultsThe expression levels of SLUG gene in 5-8 F and 5-8 F-H3 cells were significantly higher than those in CBX3,MAD2 L1,PLAU and HMGA1 gene(P<0.05),Relative multiples were(11.23±1.78) times,(1.23±0.17) times,(1.57±0.27) times,(6.36±0.38) times and(3.66±1.14) times,respectively.SLUG gene multiple was highest.The constructed pSUPERIOR-SLUG recombinant plasmid was confirmed as the desired target sequence by restriction enzyme digestion and sequence analysis.Successfully transferred into nasopharyngeal cancer 5-8 F-H3 cells.The interference level of S2-18 positive clones was higher than that of S1-7,S1-9 or S2-22 positive clones(P<0.05).The interference rates were 67.42%,72.45%,84.20% and 75.61%,respectively.Conclusion SLUG gene was highly expressed in 5-8 F-H3 cells.The pSUPERIOR-SLUG-shRNA plasmid was successfully transferred into nasopharyngeal cancer 5-8 F-H3 cells.Our results laid a foundation for further studying the role of SLUG in hepatic metastasis of nasopharyngeal carcinoma.
Objective To construct the inducible SLUG interference vector,and to investigate the changes in SLUG expression in nasopharyngeal carcinoma cells.Methods The highly metastatic human nasopharyngeal carcinoma cell lines 5-8 F and 5-8 F-H3 were cultured in this study.Real-time quantitative RT-PCR was performed to detect the expression of SLUG,CBX3,MAD2 L1,PLAU and HMGA1 genes in 5-8 F and 5-8 F-H3 cells.The pSUPERIOR-SLUG-shRNA interference plasmid was constructed using enzyme restriction and ligation technology,and then were transfected into 5-8 F-H3 cells by lipofectamine.The positive clones were selected with puromycin,and the interference efficiency was identified by real-time quantitative RT-PCR.ResultsThe expression levels of SLUG gene in 5-8 F and 5-8 F-H3 cells were significantly higher than those in CBX3,MAD2 L1,PLAU and HMGA1 gene(P<0.05),Relative multiples were(11.23±1.78) times,(1.23±0.17) times,(1.57±0.27) times,(6.36±0.38) times and(3.66±1.14) times,respectively.SLUG gene multiple was highest.The constructed pSUPERIOR-SLUG recombinant plasmid was confirmed as the desired target sequence by restriction enzyme digestion and sequence analysis.Successfully transferred into nasopharyngeal cancer 5-8 F-H3 cells.The interference level of S2-18 positive clones was higher than that of S1-7,S1-9 or S2-22 positive clones(P<0.05).The interference rates were 67.42%,72.45%,84.20% and 75.61%,respectively.Conclusion SLUG gene was highly expressed in 5-8 F-H3 cells.The pSUPERIOR-SLUG-shRNA plasmid was successfully transferred into nasopharyngeal cancer 5-8 F-H3 cells.Our results laid a foundation for further studying the role of SLUG in hepatic metastasis of nasopharyngeal carcinoma.
引文
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[13] EMADI BAYGI M,SOHEILI Z S,ESSMANN F,et al.Slug/SNAI2 regulates cell proliferation and invasiveness of metastatic prostate cancer cell lines[J].Tumour Biol,2010,31(4):297-307.
[14] TOIYAMA Y,YASUDA H,SAIGUSA S,et al.Increased expression of Slug and Vimentin as novel predictive biomarkers for lymph node metastasis and poor prognosis in colorectal cancer[J].Carcinogenesis,2013,34(11):2548-2557.
[15] HASLEHURST A M,KOTI M,DHARSEE M,et al.EMT transcription factors snail and slug directly contribute to cisplatin resistance in ovarian cancer[J].BMC Cancer,2012,12:91.
[16] FERRARI AMOROTTI G,CHIODONI C,SHEN F,et al.Suppression of invasion and metastasis of triple-negative breast cancer lines by pharmacological or genetic inhibition of slug activity[J].Neoplasia,2014,16(12):1047-1058.
[17] WU K,ZENG J,ZHOU J,et al.Slug contributes to cadherin switch and malignant progression in muscle-invasive bladder cancer development[J].Urol Oncol,2013,31(8):1751-1760.
[18] GOSSEN M,FREUNDLIEB S,BENDER G,et al.Transcriptional activation by tetracyclines in mammalian cells[J].Science,1995,268(5218):1766-1769.
[19] GOSSEN M,BUJARD H.Tight control of gene expression in mammalian cells by tetracycline-responsive promoters[J].Proc Natl Acad Sci U S A,1992,89(12):5547-5551.
[20] CHEN D L,WANG Z Q,ZENG Z L,et al.Identification of microRNA-214 as a negative regulator of colorectal cancer liver metastasis by way of regulation of fibroblast growth factor receptor 1 expression[J].Hepatology,2014,60(2):598-609.
[2] CHUA M L K,WEE J T S,HUI E P,et al.Nasopharyngeal carcinoma[J].Lancet,2016,387(10022):1012-1024.
[3] CHEN Q,TANG L,LIU N,et al.Famitinib in combination with concurrent chemoradiotherapy in patients with locoregionally advanced nasopharyngeal carcinoma:a phase 1,open-label,dose-escalation study[J].Cancer Commun(Lond),2018,38(1):66.
[4] SETTON J,WOLDEN S,CARIA N,et al.Definitive treatment of metastatic nasopharyngeal carcinoma:report of 5 cases with review of literature[J].Head Neck,2012,34(5):753-757.
[5] 付向春,祝冬香,曾晓琦,等.牛磺酸对人肺癌细胞株A549细胞凋亡的影响[J].南昌大学学报(医学版),2018,58(1):43-47,52.
[6] VILLAREJO A,CORTEsCABRERA A,MOLINA ORTíZ P,et al.Differential role of Snail1 and Snail2 zinc fingers in E-cadherin repression and epithelial to mesenchymal transition[J].J Biol Chem,2014,289(2):930-941.
[7] GRZEGRZOLKA J,BIALA M,WOJTYRA P,et al.Expression of EMT markers SLUG and TWIST in breast cancer[J].Anticancer Res,2015,35(7):3961-3968.
[8] TIAN X,ZHOU D,CHEN L,et al.Polo-like kinase 4 mediates epithelial-mesenchymal transition in neuroblastoma via PI3K/Akt signaling pathway[J].Cell Death Dis,2018,9(2):54.
[9] LUO W R,CHEN X Y,LI S Y,et al.Neoplastic spindle cells in nasopharyngeal carcinoma show features of epithelial-mesenchymal transition[J].Histopathology,2012,61(1):113-122.
[10] WANG W1,LI X2,ZHANG W,et al.Oxidored-nitro domain containing protein 1(NOR1)expression suppresses slug/vimentin but not snail in nasopharyngeal carcinoma:Inhibition of EMT in vitro and in vivo in mice[J].Cancer Lett,2014,348(1/2):109-118.
[11] XU T,FAN B,LV C,et al.Slug mediates nasopharyngeal carcinoma radioresistance via downregulation of PUMA in a p53-dependent and -independent manner[J].Oncol Rep,2015,33(5):2631-2638.
[12] UYGUR B,WU W S.SLUG promotes prostate cancer cell migration and invasion via CXCR4/CXCL12 axis[J].Mol Cancer,2011,10:139.
[13] EMADI BAYGI M,SOHEILI Z S,ESSMANN F,et al.Slug/SNAI2 regulates cell proliferation and invasiveness of metastatic prostate cancer cell lines[J].Tumour Biol,2010,31(4):297-307.
[14] TOIYAMA Y,YASUDA H,SAIGUSA S,et al.Increased expression of Slug and Vimentin as novel predictive biomarkers for lymph node metastasis and poor prognosis in colorectal cancer[J].Carcinogenesis,2013,34(11):2548-2557.
[15] HASLEHURST A M,KOTI M,DHARSEE M,et al.EMT transcription factors snail and slug directly contribute to cisplatin resistance in ovarian cancer[J].BMC Cancer,2012,12:91.
[16] FERRARI AMOROTTI G,CHIODONI C,SHEN F,et al.Suppression of invasion and metastasis of triple-negative breast cancer lines by pharmacological or genetic inhibition of slug activity[J].Neoplasia,2014,16(12):1047-1058.
[17] WU K,ZENG J,ZHOU J,et al.Slug contributes to cadherin switch and malignant progression in muscle-invasive bladder cancer development[J].Urol Oncol,2013,31(8):1751-1760.
[18] GOSSEN M,FREUNDLIEB S,BENDER G,et al.Transcriptional activation by tetracyclines in mammalian cells[J].Science,1995,268(5218):1766-1769.
[19] GOSSEN M,BUJARD H.Tight control of gene expression in mammalian cells by tetracycline-responsive promoters[J].Proc Natl Acad Sci U S A,1992,89(12):5547-5551.
[20] CHEN D L,WANG Z Q,ZENG Z L,et al.Identification of microRNA-214 as a negative regulator of colorectal cancer liver metastasis by way of regulation of fibroblast growth factor receptor 1 expression[J].Hepatology,2014,60(2):598-609.