过表达CXCR7介导AKT信号通路对人肝细胞癌细胞新血管生成的调控机制
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摘要
目的研究过表达CXCR7通过AKT信号通路诱导人肝细胞癌细胞新血管生成的作用机制。方法 (1)检测CXCR7基因在SMMC-7721,Huh-7,Hep G2,BEL-7402和MHCC-97H五种肝细胞癌细胞中的表达量,选取最适合癌细胞株进行转染实验;(2)转染Lenti-CXCR7-GFP慢病毒到BEL-7402细胞,观察转染后细胞形态,获得纯Lenti-CXCR7-GFP转染的BEL-7402细胞液;(3)检测CXCR7、p-AKT (phospho-AKT)、AKT、MMP2和MMP9表达量;(4) qRT-PCR法检测肝细胞癌中CXCR7与VEGF mRNA表达;(5)通过AKT通路抑制剂LY294002处理BEL-7402细胞,检测4组(Lenti-CXCR7-GFP-BEL-7402+DMEM处理组、Lenti-CXCR7-GFP-BEL-7402+LY294002处理组、Lenti-GFP-BEL-7402+DMEM处理组、Lenti-GFP-BEL-7402+LY294002处理组)细胞中CXCR7与VEGF蛋白表达;(6)采用鸡胚绒毛尿囊膜法检测BEL-7402肿瘤血管生成情况。结果 (1) Western blot实验结果表明正常肝细胞中CXCR7基因表达量很少,5株癌细胞中BEL-7402的CXCR7表达量相对较低,所以我们选择BEL-7402肝癌细胞株进行转染实验;(2)用倒置荧光显微镜观察细胞形态,可见大部分细胞均呈绿色荧光显色,表明Lenti-CXCR7-GFP转染到BEL-7402细胞后细胞活性较好,转染已经成功;(3) AKT通路相关蛋白表达量检测结果表明CXCR7、p-AKT,MMP2和MMP9蛋白表达量结果显示实验组显著高于对照组;(4)转染Lenti-CXCR7-GFP的BEL-7402肝癌细胞中CXCR7基因处于过表达状态,VEGF mRNA表达量显著高于对照组(P<0. 05);(5)转染Lenti-CXCR7-GFP的BEL-7402细胞+LY294002处理组中VEGF蛋白表达量比正常转染Lenti-CXCR7-GFP的BEL-7402细胞组明显下降(P<0. 05)。通过单纯Lenti-GFP转染的细胞(对照组)与Lenti-GFP转染+LY294002 (处理组)相比,VEGF表达水变化较小(P>0. 05);(6)接种BEL-7402细胞后,鸡胚绒毛尿囊膜中血管密度增加形成血管瘤,CAM增厚,结构不规则,血管加粗且呈放射状分布,Lenti-CXCR7-GFP转染BEL-7402肝细胞后,鸡胚绒毛尿囊膜中血管密度增加,增强了血管生成能力。结论肝细胞癌中CXCR7的过表达促进新血管生成的作用是经由激活了肝细胞癌中的AKT信号通路而诱导产生实现,CXCR7可能是AKT信号通路的一个重要激活剂。
Objective To investigate the mechanism of the overexpression of CXCR7 in inducing the neovascularization of human hepatocellular carcinoma cells through the AKT signaling pathway. Methods( 1) The expression levels of CXCR7 gene in SMMC-7721,Huh-7,Hep G2,BEL-7402 and MHCC-97 H liver cancer cells were detected,and the most suitable cell lines were selected for transfection experiment.( 2) Lenti-CXCR7-GFP lentivirus were transfected into BEL-7402 cells to observe the morphology of the transfected cells,and obtain BEL-7402 cell fluid transfected by pure Lenti-CXCR7-GFP.( 3) The expressions of CXCR7,p-AKT,AKT,MMP2,and MMP9 were detected.( 4) The expressions of CXCR7 and VEGF mRNA were detected through qRT-PCR.( 5) BEL-7402 cells were treated with AKT pathway inhibitor LY294002 to detect CXCR7 and VEGF protein expression in Lenti-CXCR7-GFP-BEL-7402+DMEM treatment group,Lenti-CXCR7-GFP-BEL-7402 + LY294002 treatment group,Lenti-GFP-BEL-7402+DMEM treatment group and Lenti-GFP-BEL-7402+LY294002 treatment group.( 6) The angiogenesis of BEL-7402 tumor was detected by chick embryo villoallantoic membrane method. Results( 1) Western blot results showed that the expression of CXCR7 gene in normal liver cells was very low,and the expression of CXCR7 in BEL-7402 cancer cells in 5 cancer cells was relatively low,so we chose BEL-7402 liver cancer cell line for transfection.( 2) The morphology of the cells was observed by inverted fluorescence microscope,and most of the cells were stained with green fluorescence. The results showed that BEL-7402 cells' activity was good after Lenti-CXCR7-GFP was transferred into BEL-7402 cells,and the transfection was successful.( 3) The detection results of AKT pathway related protein expression showed that CXCR7,p-AKT,MMP2 and MMP9 protein expression levels in the experimental group were significantly higher than those in the control group.( 4) CXCR7 gene was overexpressed in BEL-7402 liver cancer cells transfected with Lenti-CXCR7-GFP,and the expression level of VEGF was significantly higher than that of the control group( P <0. 05).( 5) The protein expression of VEGF in BEL-7402 cells transfected with Lenti-CXCR7-GFP +LY294002( treatment group)was significantly lower than that in BEL-7402 cells transfected with Lenti-CXCR7-GFP( P<0. 05).( 6) After inoculation of BEL-7402 cells,hemangioma was formed by increasing vascular density in the villi allantoic membrane of chick embryo. CAM was thickened with irregular structure,and the blood vessels were thickened and distributed radially. Conclusion The overexpression of CXCR7 in hepatocellular carcinoma promotes neovascularization by activating AKT signaling pathway in hepatocellular carcinoma. CXCR7 may be an important activator of AKT signaling pathway.
Objective To investigate the mechanism of the overexpression of CXCR7 in inducing the neovascularization of human hepatocellular carcinoma cells through the AKT signaling pathway. Methods( 1) The expression levels of CXCR7 gene in SMMC-7721,Huh-7,Hep G2,BEL-7402 and MHCC-97 H liver cancer cells were detected,and the most suitable cell lines were selected for transfection experiment.( 2) Lenti-CXCR7-GFP lentivirus were transfected into BEL-7402 cells to observe the morphology of the transfected cells,and obtain BEL-7402 cell fluid transfected by pure Lenti-CXCR7-GFP.( 3) The expressions of CXCR7,p-AKT,AKT,MMP2,and MMP9 were detected.( 4) The expressions of CXCR7 and VEGF mRNA were detected through qRT-PCR.( 5) BEL-7402 cells were treated with AKT pathway inhibitor LY294002 to detect CXCR7 and VEGF protein expression in Lenti-CXCR7-GFP-BEL-7402+DMEM treatment group,Lenti-CXCR7-GFP-BEL-7402 + LY294002 treatment group,Lenti-GFP-BEL-7402+DMEM treatment group and Lenti-GFP-BEL-7402+LY294002 treatment group.( 6) The angiogenesis of BEL-7402 tumor was detected by chick embryo villoallantoic membrane method. Results( 1) Western blot results showed that the expression of CXCR7 gene in normal liver cells was very low,and the expression of CXCR7 in BEL-7402 cancer cells in 5 cancer cells was relatively low,so we chose BEL-7402 liver cancer cell line for transfection.( 2) The morphology of the cells was observed by inverted fluorescence microscope,and most of the cells were stained with green fluorescence. The results showed that BEL-7402 cells' activity was good after Lenti-CXCR7-GFP was transferred into BEL-7402 cells,and the transfection was successful.( 3) The detection results of AKT pathway related protein expression showed that CXCR7,p-AKT,MMP2 and MMP9 protein expression levels in the experimental group were significantly higher than those in the control group.( 4) CXCR7 gene was overexpressed in BEL-7402 liver cancer cells transfected with Lenti-CXCR7-GFP,and the expression level of VEGF was significantly higher than that of the control group( P <0. 05).( 5) The protein expression of VEGF in BEL-7402 cells transfected with Lenti-CXCR7-GFP +LY294002( treatment group)was significantly lower than that in BEL-7402 cells transfected with Lenti-CXCR7-GFP( P<0. 05).( 6) After inoculation of BEL-7402 cells,hemangioma was formed by increasing vascular density in the villi allantoic membrane of chick embryo. CAM was thickened with irregular structure,and the blood vessels were thickened and distributed radially. Conclusion The overexpression of CXCR7 in hepatocellular carcinoma promotes neovascularization by activating AKT signaling pathway in hepatocellular carcinoma. CXCR7 may be an important activator of AKT signaling pathway.
引文
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[2] Zheng X,Wang X,Zhou J,et al. Diagnosis and treatment of community-associated methicillin-resistant Staphylococcus aureus prostatic abscess involving the seminal vesicle:A case report[J]. Exp Ther Med,2015,9(3):835-838.
[3] Movahedi K,Laoui D,Gysemans C,et al. Different Tumor Microenvironments Contain Functionally Distinct Subsets of Macrophages Derived from Ly6C(high)Monocytes[J]. Cancer Res,2010,70(14):5728-5739.
[4] Subramaniam P,Whye D,Efimenko E,et al. Targeting Nonclassical Oncogenes for Therapy in T-ALL[J]. Cancer Cell,2012,21(4):459-472.
[5] Thompson AA,Liu W,Chun E,et al. Structure of the Nociceptin/Orphanin FQ Receptor in Complex with a Peptide Mimetic[J]. Nature,2012,485(7398):395-399.
[6] Szpakowska M,Fievez V,Arumugan K,et al. Function,diversity and therapeutic potential of the N-terminal domain of human chemokine receptors[J]. Biochem Pharmacol,2012,84(10):1366-1380.
[7] Cho JS,Kang JH,Park IH,et al. Steroids inhibit vascular endothelial growth factor expression via TLR4/Akt/NF-κB pathway in chronic rhinosinusitis with nasal polyp[J]. Exp Biol Med(Maywood),2014,239(8):912-913.
[8] Yu DI,Zhang Y,Nie Q,et al. CCN1/Cyr61-PI3K/AKT signaling promotes retinal neovascularization in oxygen-induced retinopathy[J]. Int J Mol Med,2015,36(6):1507-1518.
[9] Gavalas NG,Michalis L,Sofia-Paraskevi T,et al. Angiogenesis-Related Pathways in the Pathogenesis of Ovarian Cancer[J]. Int J Mol Sci,2013,14(8):15885-15909.
[10] Xue T,Chen R,Han D,et al. Down-regulation of CXCR7inhibits the growth and lung metastasis of human hepatocellular carcinoma cells with highly metastatic potential[J]. Exp Ther Med,2012,3(1):117-123.
[11] Wang J,Hu W,Wang K,et al. Repertaxin,an inhibitor of the chemokine receptors CXCR1 and CXCR2,inhibits malignant behavior of human gastric cancer MKN45 cells in vitro and in vivo and enhances efficacy of 5-fluorouracil[J]. Int J Oncol,2016,48(4):1341-1352.
[12] Hartmann TN,Grabovsky V,Pasvolsky R,et al. A crosstalk between intracellular CXCR7 and CXCR4 involved in rapid CXCL12-triggered integrin activation but not in chemokine-triggered motility of human T lymphocytes and CD34+cells[J]. J Leukoc Biol,2008,84(4):1130-1140.
[13] Luker KE,Gupta M,Steele JM,et al. Imaging ligand-dependent activation of CXCR7[J]. Neoplasia, 2009, 11(10):1022-1035.
[14] Starr AE,Bellac CL,Dufour A,et al. Overall CM. Biochemical characterization and N-terminomics analysis of leukolysin,the membrane-type 6 matrix metalloprotease(MMP25):chemokine and vimentin cleavages enhance cell migration and macrophage phagocytic activities[J]. J Biol Chem,2012,287(16):13382-13395.
[15] Meng FD,Li Y,Tian X,et al. Synergistic effects of snail and quercetin on renal cell carcinoma Caki-2 by altering AKT/m TOR/ERK1/2 signaling pathways[J]. Int J Clin Exp Pathol,2015,8(6):6157-6168.
[16] Katsura M,Shoji F,Okamoto T,et al. Correlation between CXCR4/CXCR7/CXCL12 chemokine axis expression and prognosis in lymph-node-positive lung cancer patients[J]. Cancer Sci,2017:10. 1111/cas. 13422.
[17] Li X,Ma Q,Xu Q,et al. SDF-1/CXCR4 signaling induces pancreatic cancer cell invasion and epithelial-mesenchymal transition in vitro,through non-canonical activation of Hedgehog pathway[J]. Cancer Lett,2012,322(2):169-176.
[18] Wang Y,Han G,Wang K,et al. Tumor-derived GM-CSF promotes inflammatory colon carcinogenesis via stimulating epithelial release of VEGF[J]. Cancer Res,2014,74(3):716-726.
[19] Chen JS,Wang Q,Fu XH,et al. Involvement of PI3K/PTEN/AKT/m TOR pathway in invasion and metastasis in hepatocellular carcinoma:Association with MMP‐9[J]. Hepatol Res,2009,39(2):177-186.
[20] Hickman JA,Graeser R,De Hoogt R,et al. Three‐dimensional models of cancer for pharmacology and cancer cell biology:Capturing tumor complexity in vitro/ex vivo[J]. Biotechnol J,2014,9(9):1115-1128.
[21] Chen D,Tian W,Li Y,et al. Osteoblast-specific transcription factor Osterix(Osx)and HIF-1 cooperatively regulate gene expression of vascular endothelial growth factor(VEGF)[J]. Biochemical&Biophysical Research Communications,2012,424(1):176-181.