SRPX2通过巨噬细胞促进人脐静脉内皮细胞血管生成能力的作用
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摘要
目的:探究含sushi重复蛋白X连锁2蛋白(sushi repeat-containing protein X-linked 2,SRPX2)是否可通过巨噬细胞对血管生成产生影响及其可能的机制。方法:用shRNA-SRPX2及shRNA空载体转染HCT116细胞,并收集以上两种细胞和空白HCT116的细胞培养基作条件培养基,用3种条件培养基作用于人源单核细胞(THP-1)诱导的巨噬细胞,而后巨噬细胞与人脐静脉内皮细胞(HUVEC)共培养,并以Transwell迁移实验检测HUVEC迁移能力,以Matrigel基质胶管腔形成实验检测管腔形成能力。用Western blot检测巨噬细胞中局部黏着斑激酶(FAK)的表达水平。结果:在Transwell迁移实验中,HCT116敲降SRPX2基因的条件培养基与巨噬细胞作用,并与HUVEC共培养后,HUVEC迁移细胞数明显减少(P <0. 01)。而在Matrigel管腔形成实验中,HCT116敲降组形成的管腔的交叉点数、成网数、节点数也有所减少(P <0. 05)。FAK蛋白检测发现,SRPX2重组蛋白作用后的巨噬细胞总FAK表达量和FAK蛋白磷酸化水平有所增高。结论:SRPX2可能通过FAK相关通路影响巨噬细胞,进而间接影响血管内皮细胞的血管生成能力。
AIM: To investigate whether sushi repeat-containing protein X-linked 2( SRPX2) can influence angiogenesis via macrophages,and its potential mechanism. METHODS: After transferring shRNA-SRPX2 and vacant shRNA vector into HCT116 cells,culture medium supernatant was extracted from three types of cells( HCT116-KD,HCT116-NC and HCT116-BC) as the conditioned medium. After culturing THP-1 induced macrophages in the conditioned medium,the macrophages were co-cultured with HUVECs for Transwell migration assay and Matrigel tube formation assay to verify the migration ability and tube formation ability of HUVECs. In addition, focal adhesion kinase( FAK) and phosphorylated FAK expression level in macrophages were measured by Western blot after activated by SRPX2 recombinant protein for 4 h.RESULTS: In migration assay,the number of HUVECs migrating to the lower chamber was significantly diminished in HCT116-KD group( P <0. 01). In tube formation assay,numbers of junctions, meshes and nodes also decreased in the HCT116-KD group( P < 0. 05). As for the FAK expression,the expression of FAK as well as the phosphorylated level of FAK in macrophages elevated after interacted with SRPX2 recombination protein.CONCLUSION: SRPX2 can affect angiogenesis via macrophages,which is probably depends on FAK related signal pathways.
AIM: To investigate whether sushi repeat-containing protein X-linked 2( SRPX2) can influence angiogenesis via macrophages,and its potential mechanism. METHODS: After transferring shRNA-SRPX2 and vacant shRNA vector into HCT116 cells,culture medium supernatant was extracted from three types of cells( HCT116-KD,HCT116-NC and HCT116-BC) as the conditioned medium. After culturing THP-1 induced macrophages in the conditioned medium,the macrophages were co-cultured with HUVECs for Transwell migration assay and Matrigel tube formation assay to verify the migration ability and tube formation ability of HUVECs. In addition, focal adhesion kinase( FAK) and phosphorylated FAK expression level in macrophages were measured by Western blot after activated by SRPX2 recombinant protein for 4 h.RESULTS: In migration assay,the number of HUVECs migrating to the lower chamber was significantly diminished in HCT116-KD group( P <0. 01). In tube formation assay,numbers of junctions, meshes and nodes also decreased in the HCT116-KD group( P < 0. 05). As for the FAK expression,the expression of FAK as well as the phosphorylated level of FAK in macrophages elevated after interacted with SRPX2 recombination protein.CONCLUSION: SRPX2 can affect angiogenesis via macrophages,which is probably depends on FAK related signal pathways.
引文
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[23]王丽,张勇. SRPX2在子宫内膜癌中的表达及对HEC-1A细胞增殖凋亡的影响[J].实用医学杂志,2017,33(4):529-532.
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[25] Walsh C,Tanjoni I,Uryu S,et al. Oral delivery of PND-1186 FAK inhibitor decreases tumor growth and spontaneous breast to lung metastasis in pre-clinical models[J]. Cancer Biol Ther,2010,9(10):778-790.
[26] Wendt MK,Schiemann WP. Therapeutic targeting of the focal adhesion complex prevents oncogenic TGF-βsignaling and metastasis[J]. Breast Cancer Res:BCR,2009,11(5):R68.
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[28] Simon D,Wei YL,Rao N,et al. Identification of a urokinase receptor-integrin interaction site. Promiscuous regulator of integrin function[J]. J Biol Chem,2000,275(14):10228-10234.
[2] Yamada T,Oshima T,Yoshihara K,et al. Impact of overexpression of Sushi repeat-containing protein Xlinked 2 gene on outcomes of gastric cancer[J]. J Surg Oncol,2014,109(8):836-840.
[3] Tanaka K,Arao T,Tamura D,et al. SRPX2 is a novel chondroitin sulfate proteoglycan that is overexpressed in gastrointestinal cancer[J]. Plos One,2012,7(1):e27922.
[4] Miljkoviclicina M,Hammel P,Garridourbani S,et al.Sushi repeat protein X-linked 2,a novel mediator of angiogenesis[J]. FASEB J,2009,23(12):4105-4116.
[5] Liu K,Fan J,Wu J. Sushi repeat-containing protein X-linked 2 promotes angiogenesis through the urokinase-type plasminogen activator receptor dependent integrinαvβ3/focal adhesion kinase pathways[J]. Drug Discov Ther,2017,11(4):212-217.
[6] Bingle L,Lewis CE,Corke KP,et al. Macrophages promote angiogenesis in human breast tumour spheroids in vivo[J]. Br J Cancer,2006,94(1):101-107.
[7] Lu P,Weaver VM,Werb Z. The extracellular matrix:A dynamic niche in cancer progression[J]. J Cell Biol,2012,196(4):395-406.
[8]樊江浩,刘揆亮,周跃,等.细胞外基质蛋白SRPX2促进HUVECs血管生成能力[J].基础医学与临床,2015,35(10):1336-1340.
[9] Tanaka K,Arao T,Maegawa M,et al. SRPX2 is overexpressed in gastric cancer and promotes cellular migration and adhesion[J]. Int J Cancer,2009,124(5):1072-1080.
[10] Kurosawa H,Goi K,Inukai T,et al. Two candidate downstream target genes for E2A-HLF[J]. Blood,1999,93(1):321-332.
[11] Royer B,Soares DC,Barlow PN,et al. Molecular evolution of the human SRPX2 gene that causes brain disorders of the Rolandic and Sylvian speech areas[J]. BMC Genet,2007,8(1):72.
[12] Royerzemmour B,Ponsolelenfant M,Gara H,et al. Epileptic and developmental disorders of the speech cortex:ligand/receptor interaction of wild-type and mutant SRPX2 with the plasminogen activator receptor u PAR[J]. Hum Mol Genet,2008,17(23):3617-3630.
[13] Liu KL,Wu J,Zhou Y,et al. Increased Sushi repeatcontaining protein X-linked 2 is associated with progression of colorectal cancer[J]. Med Oncol,2015,32(4):99.
[14] Yamada T,Oshima T,Yoshihara K,et al. Impact of overexpression of Sushi repeat-containing protein Xlinked 2 gene on outcomes of gastric cancer[J]. J Surg Oncol,2014,109(8):836-840.
[15] Lin X,Chang W,Wang Y,et al. SRPX2,an independent prognostic marker, promotes cell migration and invasion in hepatocellular carcinoma[J]. Biomed Pharmacother,2017,93:398-405.
[16] Gao Z,Zhang J,Bi M,et al. SRPX2 promotes cell migration and invasion via FAK dependent pathway in pancreatic cancer[J]. Int J Clin Exp Pathol,2015,8(5):4791-4798.
[17] Tang H,Zhao J,Zhang L,et al. SRPX2 enhances the epithelial-mesenchymal transition and temozolomide resistance in glioblastoma cells[J]. Cell Mol Neuro biol,2016,36(7):1067-1076.
[18] Birbrair A,Zhang T,Wang ZM,et al. Type-2 pericytes participate in normal and tumoral angiogenesis[J]. Am J Physiol Cell Physiol,2014,307(1):25-38.
[19]陈伟,周赟,龚建平.肿瘤相关巨噬细胞在原发性肝癌发生发展中的作用[J].临床肝胆病杂志,2016,32(7):1422-1424.
[20] Bingle L,Brown NJ,Lewis CE. The role of tumour‐associated macrophages in tumour progression:implications for new anticancer therapies[J]. J Pathol,2002,196(3):254-265.
[21] Wu H,Xu J,He Y,et al. Tumor‐associated macrophages promote angiogenesis and lymph angiogenesis of gastric cancer[J]. J Surg Oncol,2012,106(4):462-468.
[22]李树裕,王志钢.粘附斑激酶(FAK)及其信号通路研究进展[J].生物技术通报,2009,10(12):6-10.
[23]王丽,张勇. SRPX2在子宫内膜癌中的表达及对HEC-1A细胞增殖凋亡的影响[J].实用医学杂志,2017,33(4):529-532.
[24] Sulzmaier FJ,Jean C,Schlaepfer DD. FAK in cancer:mechanistic findings and clinical applications[J].Nat Rev Cancer,2014,14(9):598-610.
[25] Walsh C,Tanjoni I,Uryu S,et al. Oral delivery of PND-1186 FAK inhibitor decreases tumor growth and spontaneous breast to lung metastasis in pre-clinical models[J]. Cancer Biol Ther,2010,9(10):778-790.
[26] Wendt MK,Schiemann WP. Therapeutic targeting of the focal adhesion complex prevents oncogenic TGF-βsignaling and metastasis[J]. Breast Cancer Res:BCR,2009,11(5):R68.
[27] Kroon ME,Koolwijk P,Goor HV,et al. Role and localization of urokinase receptor in the formation of new microvascular structures in fibrin matrices[J]. Am J Pathol,1999,154(6):1731-1742.
[28] Simon D,Wei YL,Rao N,et al. Identification of a urokinase receptor-integrin interaction site. Promiscuous regulator of integrin function[J]. J Biol Chem,2000,275(14):10228-10234.
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