丝甘蛋白聚糖促进非小细胞肺癌的侵袭与转移
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摘要
丝甘蛋白聚糖(serglycin,SRGN)在肿瘤细胞的侵袭与转移中具有广泛的研究前景。本研究报道SRGN与肺癌细胞侵袭与转移能力之间的相关性。首先,通过检测SRGN在正常肺上皮细胞株BEAS-2B及不同侵袭与转移能力的肺癌细胞株95C、95D中的表达差异。利用shRNA干扰技术,在侵袭与转移能力强的95D细胞中建立稳定干扰SRGN表达的95D/sh SRGN的细胞株,并通过RT-q PCR、Western印迹、酶联免疫吸附测定验证其敲除效率。结果显示:干扰SRGN可抑制侵袭与转移性强的95D细胞的侵袭与转移能力,减弱细胞迁移与侵袭等生物学特性,导致上皮标志物上皮细胞钙黏连蛋白(E-cadherin)表达上调,间质标志物纤维连接蛋白1(fibronectin1,FN1)及EMT(epithelial-mesenchymal transition,EMT)相关转录因子锌指E盒结合同源框1(zinc finger E-box binding homeobox 1,ZEB1)表达下调。进一步分析发现,SRGN与上皮细胞钙黏连蛋白表达成负相关(P=-0.25),而与FN1(P=0.12)及ZEB1(P=0.35)表达成正相关,并且SRGN高表达的患者总生存时间明显少于SRGN低表达组(P=0.0077),SRGN与ZEB1同时高表达的患者,总生存时间显著小于SRGN与ZEB1低表达患者(P=0.0005)。研究结果证实,SRGN促进上皮间质转化发生,增强非小细胞肺癌的侵袭与转移能力,为非小细胞肺癌预后提供参考。
Serglycin( SRGN) plays a significant role in tumor invasion and metastasis. This study reports that SRGN specifically regulates invasion and metastasis of lung cancer. Firstly,we determined the expression levels of SRGN in the normal lung epithelial cell line BEAS2 B and lung cancer cell lines95 C and 95 D with different invasive and metastatic abilities. Then,using the shRNA method,we established a 95 D/Sh SRGN cell line,which stably knocks down SRGN expression in 95 D cells. The knockdown efficiency was verified by RT-q PCR,Western blotting and ELISA. The results showed that interfering SRGN inhibits invasion and metastasis of 95 D cells, upregulates the epithelial markerepithelial cell cadherin( E-cadherin) and down-regulates the expression of fibronectin 1( FN1) and zinc finger E-box binding homeobox 1( ZEB1). Further analysis showed that SRGN negatively correlated with E-cadherin expression( P =-0. 25) and positively correlated with FN1( P = 0. 12) and ZEB1( P =0. 35). Clinically,the overall survival time of patients with SRGN overexpression was significantly lower( P = 0. 0077),and the overall survival of patients with high expression of SRGN and ZEB1 was significantly lower than that of patients with low expression of SRGN and ZEB1( P = 0. 0005). In this study,we found that SRGN glycoproteins promote the occurrence of EMT,enhance the invasion and metastasis of non-small cell lung cancer cell lines,and provide a potential prognostic molecular marker for non-small cell lung cancer.
Serglycin( SRGN) plays a significant role in tumor invasion and metastasis. This study reports that SRGN specifically regulates invasion and metastasis of lung cancer. Firstly,we determined the expression levels of SRGN in the normal lung epithelial cell line BEAS2 B and lung cancer cell lines95 C and 95 D with different invasive and metastatic abilities. Then,using the shRNA method,we established a 95 D/Sh SRGN cell line,which stably knocks down SRGN expression in 95 D cells. The knockdown efficiency was verified by RT-q PCR,Western blotting and ELISA. The results showed that interfering SRGN inhibits invasion and metastasis of 95 D cells, upregulates the epithelial markerepithelial cell cadherin( E-cadherin) and down-regulates the expression of fibronectin 1( FN1) and zinc finger E-box binding homeobox 1( ZEB1). Further analysis showed that SRGN negatively correlated with E-cadherin expression( P =-0. 25) and positively correlated with FN1( P = 0. 12) and ZEB1( P =0. 35). Clinically,the overall survival time of patients with SRGN overexpression was significantly lower( P = 0. 0077),and the overall survival of patients with high expression of SRGN and ZEB1 was significantly lower than that of patients with low expression of SRGN and ZEB1( P = 0. 0005). In this study,we found that SRGN glycoproteins promote the occurrence of EMT,enhance the invasion and metastasis of non-small cell lung cancer cell lines,and provide a potential prognostic molecular marker for non-small cell lung cancer.
引文
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[4]Jin X,Zhu Z,Shi Y.Metastasis mechanism and gene/protein expression in gastric cancer with distant organs metastasis[J].Bull Cancer,2014,101(1):E1-12
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[8]Li CY,Tan L,Zhang GJ,et al.Transcriptional regulation of urokinase receptor in high-(95D)and low-metastatic(95C)human lung cancer cells[J].Acta Biochim Biophys Sin(Shanghai),2004,36(6):405-411
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[12]Greenburg G,Hay ED.Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells[J].J Cell Biol,1982,95(1):333-339
[13]Nieto MA.The snail superfamily of zinc-finger transcription factors[J].Nat Rev Mol Cell Biol,2002,3(3):155-166
[14]Cano A,Pérez-Moreno MA,Rodrigo I,et al.The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression[J].Nat Cell Biol,2000,2(2):76-83
[15]Peinado H,Olmeda D,Cano A.Snail,Zeb and b HLH factors in tumour progression:an alliance against the epithelial phenotype[J].Nat Rev Cancer,2007,7(6):415-428
[16]Kang Y,MassaguéJ.Epithelial-mesenchymal transitions:twist in development and metastasis[J].Cell,2004,118(3):277-279
[17]Katsuno Y,Lamouille S,Derynck R.TGF-βsignaling and epithelial-mesenchymal transition in cancer progression[J].Curr Opin Oncol,2013,25(1):76-84
[18]Huber MA,Kraut N,Beug H.Molecular requirements for epithelial-mesenchymal transition during tumor progression[J].Curr Opin Cell Biol,2005,17(5):548-558
[19]Onder TT,Gupta PB,Mani SA,et al.Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways[J].Cancer Res,2008,68(10):3645-3654
[20]Shen Y,Liu S,Yuan H,et al.A long non-coding RNA lncRNA-PE promotes invasion and epithelial-mesenchymal transition in hepatocellular carcinoma through the miR-200a/bZEB1 pathway[J].Tumour Biol,2017,39(5):1010428317705756
[21]Krebs AM,Mitschke J,Lasierra Losada M,et al.The EMTactivator Zeb1 is a key factor for cell plasticity and promotes metastasis in pancreatic cancer[J].Nat Cell Biol,2017,19(5):518-529
[22]Ma J,Zhan Y,Xu Z,et al.ZEB1 induced miR-99b/let-7e/miR-125a cluster promotes invasion and metastasis in esophageal squamous cell carcinoma[J].Cancer Lett,2017,398:37-45
[23]Humtsoe JO,Koya E,Pham E,et al.Transcriptional profiling identifies upregulated genes following induction of epithelialmesenchymal transition in squamous carcinoma cells[J].Exp Cell Res,2012,318(4):379-390
[24]Ma D,Rudd E,Edner J,et al.Sequence analysis of the SRGN,AP3B1,ARF6,and SH2D1A genes in familial hemophagocytic lymphohistiocytosis[J].Pediatr Blood Cancer,2008,50(5):1067-1069
[25]Zhang W,Chen M,Zhang W,et al.Use of electrophysiological monitoring in selective rhizotomy treating glossopharyngeal neuralgia[J].J Craniomaxillofac Surg,2014,42(5):e182-185
[26]Gianazza E,Chinello C,Mainini V,et al.Alterations of the serum peptidome in renal cell carcinoma discriminating benign and malignant kidney tumors[J].J Proteomics,2012,76 Spect No.:125-140
[2]Reckamp KL.Advances in immunotherapy for non-small cell lung cancer[J].Clin Adv Hematol Oncol,2015,13(12):847-853
[3]Yu H,Han Z,Wang Y,et al.The clonal evolution and therapeutic approaches of lung cancer[J].Cell Biochem Biophys,2014,70(1):63-71
[4]Jin X,Zhu Z,Shi Y.Metastasis mechanism and gene/protein expression in gastric cancer with distant organs metastasis[J].Bull Cancer,2014,101(1):E1-12
[5]Kolset SO,Tveit H.Serglycin-structure and biology[J].Cell Mol Life Sci,2008,65(7-8):1073-1085
[6]Zhang Z,Deng Y,Zheng G,et al.SRGN-TGFβ2 regulatory loop confers invasion and metastasis in triple-negative breast cancer[J].Oncogenesis,2017,6(7):e360
[7]Guo JY,Hsu HS,Tyan SW,et al.Serglycin in tumor microenvironment promotes nonsmall cell lung cancer aggressiveness in a CD44-dependent manner[J].Oncogene,2017,36(17):2457-2471
[8]Li CY,Tan L,Zhang GJ,et al.Transcriptional regulation of urokinase receptor in high-(95D)and low-metastatic(95C)human lung cancer cells[J].Acta Biochim Biophys Sin(Shanghai),2004,36(6):405-411
[9]Winberg JO,Kolset SO,Berg E,et al.Macrophages secrete matrix metalloproteinase 9 covalently linked to the core protein of chondroitin sulphate proteoglycans[J]J Mol Biol,2000,304(4):669-680
[10]Li XJ,Ong CK,Cao Y,et al.Serglycin is a theranostic target in nasopharyngeal carcinoma that promotes metastasis[J].Cancer Res,2011,71(8):3162-3172
[11]He L,Zhou X,Qu C,et al.Serglycin(SRGN)overexpression predicts poor prognosis in hepatocellular carcinoma patients[J].Med Oncol,2013,30(4):707
[12]Greenburg G,Hay ED.Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells[J].J Cell Biol,1982,95(1):333-339
[13]Nieto MA.The snail superfamily of zinc-finger transcription factors[J].Nat Rev Mol Cell Biol,2002,3(3):155-166
[14]Cano A,Pérez-Moreno MA,Rodrigo I,et al.The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression[J].Nat Cell Biol,2000,2(2):76-83
[15]Peinado H,Olmeda D,Cano A.Snail,Zeb and b HLH factors in tumour progression:an alliance against the epithelial phenotype[J].Nat Rev Cancer,2007,7(6):415-428
[16]Kang Y,MassaguéJ.Epithelial-mesenchymal transitions:twist in development and metastasis[J].Cell,2004,118(3):277-279
[17]Katsuno Y,Lamouille S,Derynck R.TGF-βsignaling and epithelial-mesenchymal transition in cancer progression[J].Curr Opin Oncol,2013,25(1):76-84
[18]Huber MA,Kraut N,Beug H.Molecular requirements for epithelial-mesenchymal transition during tumor progression[J].Curr Opin Cell Biol,2005,17(5):548-558
[19]Onder TT,Gupta PB,Mani SA,et al.Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways[J].Cancer Res,2008,68(10):3645-3654
[20]Shen Y,Liu S,Yuan H,et al.A long non-coding RNA lncRNA-PE promotes invasion and epithelial-mesenchymal transition in hepatocellular carcinoma through the miR-200a/bZEB1 pathway[J].Tumour Biol,2017,39(5):1010428317705756
[21]Krebs AM,Mitschke J,Lasierra Losada M,et al.The EMTactivator Zeb1 is a key factor for cell plasticity and promotes metastasis in pancreatic cancer[J].Nat Cell Biol,2017,19(5):518-529
[22]Ma J,Zhan Y,Xu Z,et al.ZEB1 induced miR-99b/let-7e/miR-125a cluster promotes invasion and metastasis in esophageal squamous cell carcinoma[J].Cancer Lett,2017,398:37-45
[23]Humtsoe JO,Koya E,Pham E,et al.Transcriptional profiling identifies upregulated genes following induction of epithelialmesenchymal transition in squamous carcinoma cells[J].Exp Cell Res,2012,318(4):379-390
[24]Ma D,Rudd E,Edner J,et al.Sequence analysis of the SRGN,AP3B1,ARF6,and SH2D1A genes in familial hemophagocytic lymphohistiocytosis[J].Pediatr Blood Cancer,2008,50(5):1067-1069
[25]Zhang W,Chen M,Zhang W,et al.Use of electrophysiological monitoring in selective rhizotomy treating glossopharyngeal neuralgia[J].J Craniomaxillofac Surg,2014,42(5):e182-185
[26]Gianazza E,Chinello C,Mainini V,et al.Alterations of the serum peptidome in renal cell carcinoma discriminating benign and malignant kidney tumors[J].J Proteomics,2012,76 Spect No.:125-140