沉默Versican基因对鼠源性肝癌细胞株Hepa1-6体外增殖及迁移影响的研究
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摘要
目的:检测Versican基因在鼠源性肝癌细胞的表达情况,观察沉默Versican基因对肝癌细胞增殖及迁移能力的影响。方法:采用Western blot技术检测鼠源性肝癌细胞株Hepa1-6中Versican的表达。构建沉默Versican基因的shRNA质粒(shVCAN)稳定转染Hepa1-6,通过荧光显微镜观察shVCAN质粒转染效率,采用RT-PCR及Western blot技术检测Versican基因的沉默效率。CCK-8法检测稳定转染shVCAN质粒后Hepa1-6的增殖能力变化;划痕及Transwell实验检测稳定转染shVCAN质粒后Hepa1-6的迁移能力变化;免疫荧光及Western blot检测稳定转染shVCAN质粒后Hepa1-6上皮细胞-间质细胞转化(epithelial-mesenchymal transition,EMT)相关蛋白的表达情况。结果:鼠源性肝癌细胞株Hepa1-6中Versican蛋白呈阳性表达。荧光显微镜显示shVCAN质粒稳定转染Hepa1-6效率高;RT-PCR及Western blot显示,shVCAN质粒稳定转染Hepa1-6后,其内的Versican表达受到明显抑制。此外,shVCAN质粒稳定转染Hepa1-6后,其增殖及迁移能力降低,差异具有统计学意义(P<0.05)。同时,免疫荧光及Western blot显示,shVCAN质粒稳定转染的Hepa1-6内,EMT相关蛋白E-cadherin的表达上调,N-cadherin的表达下调。结论:沉默鼠源性肝癌细胞株Hepa1-6内Versican基因的表达,其增殖及迁移能力均受到抑制,其部分机理可能与沉默Versican基因引发Hepa1-6内E-cadherin上调及N-cadherin下调有关。
Objective: To detect the expression of Versican and study the impacts of silencing Versican on the proliferation and migration of hepatocellular carcinoma cells. Methods: The expression of Versican in mouse original hepatocellular carcinoma cells Hepa1-6 was detected by Western blot. Constructing plasmid silencing Versican stably transfected Hepa1-6 cells. The transfected effect of the plasmid in Hepa1-6 cells was observed under fluorescence microscope. The silenced effects of Versican in Hepa1-6 cells were detected by RT-PCR and Western blot. The CCK-8 assay was used to detect the proliferative ability of Hepa1-6/shVCAN. Migration ability of Hepa1-6/shVCAN was examined by Scratch and Transwell assays. The expression of EMT-related proteins was detected by immunofluorescence and Western blot. Results:The expression of Versican in Hepa1-6 cells was positive. The transfected effect of the shVCAN plasmid in Hepa1-6 cells was high under fluorescence microscope. The expression of Versican in Hepa1-6 cells was obviously inhibited after transfected shVCAN plasmid shown by RT-PCR and Western blot analysis. Moreover,the abilities of proliferation and migration of Hepa1-6 cells were also significantly suppressed after transfected shVCAN plasmid compared with those of Hepa1-6/sh C and Hepa1-6 cells( P < 0. 05). The immunofluorescence and Western blot analysis revealed that the expression level of Ecadherin was up-regulated while N-cadherin was downregulated in Hepa1-6/shVCAN cells compared with those in Hepa1-6/sh C and Hepa1-6 cells. Conclusion: Silencing the expression of Versican could inhibit the proliferative and migration abilities of mouse original hepatocellular carcinoma cells Hepal-6 and the partly mechanisms may be related to the up-regulation of E-cadherin and down-regulation of N-cadherin.
Objective: To detect the expression of Versican and study the impacts of silencing Versican on the proliferation and migration of hepatocellular carcinoma cells. Methods: The expression of Versican in mouse original hepatocellular carcinoma cells Hepa1-6 was detected by Western blot. Constructing plasmid silencing Versican stably transfected Hepa1-6 cells. The transfected effect of the plasmid in Hepa1-6 cells was observed under fluorescence microscope. The silenced effects of Versican in Hepa1-6 cells were detected by RT-PCR and Western blot. The CCK-8 assay was used to detect the proliferative ability of Hepa1-6/shVCAN. Migration ability of Hepa1-6/shVCAN was examined by Scratch and Transwell assays. The expression of EMT-related proteins was detected by immunofluorescence and Western blot. Results:The expression of Versican in Hepa1-6 cells was positive. The transfected effect of the shVCAN plasmid in Hepa1-6 cells was high under fluorescence microscope. The expression of Versican in Hepa1-6 cells was obviously inhibited after transfected shVCAN plasmid shown by RT-PCR and Western blot analysis. Moreover,the abilities of proliferation and migration of Hepa1-6 cells were also significantly suppressed after transfected shVCAN plasmid compared with those of Hepa1-6/sh C and Hepa1-6 cells( P < 0. 05). The immunofluorescence and Western blot analysis revealed that the expression level of Ecadherin was up-regulated while N-cadherin was downregulated in Hepa1-6/shVCAN cells compared with those in Hepa1-6/sh C and Hepa1-6 cells. Conclusion: Silencing the expression of Versican could inhibit the proliferative and migration abilities of mouse original hepatocellular carcinoma cells Hepal-6 and the partly mechanisms may be related to the up-regulation of E-cadherin and down-regulation of N-cadherin.
引文
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[10]Navarro AN,Kondo E,Kawamoto H,et al.Isolation and propagation of a human CD133(-)colon tumor-derived cell line with tumorigenic and angiogenic properties[J].Cell Transplant,2010,19(6):865-877.
[11]Feng LX,Sun P,Mi T,et al.Agglutinin isolated from Arisema heterophyllum blume induces apoptosis and autophagy in A549cells through inhibiting PI3K/Akt pathway and inducing ER stress[J].Chin J Nat Med,2016,14(11):856-864.
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[13]Zhu Y,Wei W,Ye T,et al.Small molecule TH-39 potentially targets Hec1/Nek2 interaction and exhibits antitumor efficacy in K562 cells via G0/G1 cell cycle arrest and apoptosis indcution[J].Cell Physiol Biochem,2016,40(1-2):297-308.
[14]Hernandez D,Miquel SL,Docampo MJ,et al.V3 versican isoform alters the behavior of human melanoma cells by interfering with CD44/Erb B-dependent signaling[J].J Biol Chem,2011,286(2):1475-1485.
[15]Yan S,Holderness BM,Li Z,et al.Epithelial-mesenchymal expression phenotype of primary melanoma and matched metastases and relationship with overall survival[J].Anticancer Res,2016,36(12):6449-6456.
[16]Feng M,Feng J,Chen W,et al.Lipocalin2 suppresses metastasis of colorectal cancer by attenuating nf-kb-dependent activation of snail and epithelial mesenchymal transition[J].Mol Cancer,2016,15(1):77.
[17]Wang YJ,Liu JZ,Lv P,et al.Long non-coding RNA CCAT2promotes gastric cancer proliferation and invasion by regulating the E-cadherin and LATS2[J].Am J Cancer Res,2016,6(11):2651-2660.
[18]Chen H,Chen Q,Luo Q.Expression of netrin-1 by hypoxia contributes to the invasion and migration of prostate carcinoma cells by regulating YAP activity[J].Exp Cell Res,2016,349(2):302-309.
[19]Gao D,Vahdat LT,Wong S,et al.Microenvironmental regulation of epithelial-mesenchymal transitions in cancer[J].Cancer Res,2012,72(19):4883-4889.
[2]Folkman J.Tumor angiogenesis:therapeutic implications[J].N Engl J Med,1971,285(21):1182-1186.
[3]Holleb AI,Folkman J.Tumor angiogenesis[J].CA Cancer J Clin,1972,22(4):226-229.
[4]Wu YJ,La Pierre DP,Wu J,et al.The interaction of Versican with its binding partners[J].Cell Research,2005,15(7):483–494.
[5]Aspberg A,Adam S,Kostka G,et al.Fibulin-1 is a ligand for the C-type lectin domains of aggrecan and Versican[J].J Biol Chem,1999,274(29):20444–20449.
[6]Wang Z,Li ZX,Wang YY,et al.Versican silencing improves the antitumor efficacy of endostatin by alleviating its induced inflammatory and immunosuppressive changes in the tumor microenvironment[J].Oncol Rep,2015,6(33):2981-2991.
[7]Inokawa Y,Inaoka K,Sonohara F,et al.Molecular alteration in the carcinogenesis and progression of hepatocellular carcinoma:Tumor factors and background liver factors[J].Oncol Lett,2016,12(5):3662-3668.
[8]Lu K,Liu G,Yang L,et al.Sustainable inflammation transforms hepatic cells by causing oxidative stress injury and potential epithelial-mesenchymal transition[J].Int J Oncol.2016,49(3):971-980.
[9]Matter MS,Marquardt JU,Andersen JB,et al.Oncogenic driver genes and the inflammatory microenvironment dictate liver tumor phenotype[J].Hepatology,2016,63(6):1888-1899.
[10]Navarro AN,Kondo E,Kawamoto H,et al.Isolation and propagation of a human CD133(-)colon tumor-derived cell line with tumorigenic and angiogenic properties[J].Cell Transplant,2010,19(6):865-877.
[11]Feng LX,Sun P,Mi T,et al.Agglutinin isolated from Arisema heterophyllum blume induces apoptosis and autophagy in A549cells through inhibiting PI3K/Akt pathway and inducing ER stress[J].Chin J Nat Med,2016,14(11):856-864.
[12]方胜涛,林采余,张全波,等.海芋粗提物对人肝癌细胞的细胞毒和凋亡诱导作用[J]。肿瘤预防与治疗,2011,24(2):69-73。
[13]Zhu Y,Wei W,Ye T,et al.Small molecule TH-39 potentially targets Hec1/Nek2 interaction and exhibits antitumor efficacy in K562 cells via G0/G1 cell cycle arrest and apoptosis indcution[J].Cell Physiol Biochem,2016,40(1-2):297-308.
[14]Hernandez D,Miquel SL,Docampo MJ,et al.V3 versican isoform alters the behavior of human melanoma cells by interfering with CD44/Erb B-dependent signaling[J].J Biol Chem,2011,286(2):1475-1485.
[15]Yan S,Holderness BM,Li Z,et al.Epithelial-mesenchymal expression phenotype of primary melanoma and matched metastases and relationship with overall survival[J].Anticancer Res,2016,36(12):6449-6456.
[16]Feng M,Feng J,Chen W,et al.Lipocalin2 suppresses metastasis of colorectal cancer by attenuating nf-kb-dependent activation of snail and epithelial mesenchymal transition[J].Mol Cancer,2016,15(1):77.
[17]Wang YJ,Liu JZ,Lv P,et al.Long non-coding RNA CCAT2promotes gastric cancer proliferation and invasion by regulating the E-cadherin and LATS2[J].Am J Cancer Res,2016,6(11):2651-2660.
[18]Chen H,Chen Q,Luo Q.Expression of netrin-1 by hypoxia contributes to the invasion and migration of prostate carcinoma cells by regulating YAP activity[J].Exp Cell Res,2016,349(2):302-309.
[19]Gao D,Vahdat LT,Wong S,et al.Microenvironmental regulation of epithelial-mesenchymal transitions in cancer[J].Cancer Res,2012,72(19):4883-4889.