抑癌基因DKK2过表达对人膀胱癌细胞增殖和迁移的抑制作用及其机制探讨
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摘要
目的 :分析dickkopf 2(DKK2)基因在人膀胱癌细胞系及膀胱癌组织中的表达水平,以及DKK2过表达对人膀胱癌细胞增殖与迁移的影响,并初步探讨其作用机制。方法:通过RT-PCR、实时荧光定量PCR和蛋白质印迹法检测人膀胱癌细胞系及组织中DKK 2基因的相对表达水平。选用DKK2低表达的膀胱癌细胞系T24,进行DKK2过表达质粒转染后,采用RT-PCR、实时荧光定量PCR和蛋白质印迹法验证DKK2过表达效果;然后采用CCK-8法、平板克隆形成实验、划痕愈合实验、Transwell小室法和FCM法分别检测DKK2过表达对细胞增殖、克隆形成、迁移、侵袭及细胞周期的影响;进一步采用蛋白质印迹法和实时荧光定量PCR法检测DKK2过表达后膀胱癌细胞中增殖和迁移相关分子表达水平的变化。结果:人膀胱癌细胞系5637、T24、SW780、J82、HT-1197和HT-1376中DKK2 mRNA及蛋白表达水平均明显低于正常膀胱上皮细胞SVHUC-1(P值均<0.01);同时与正常膀胱组织及配对的癌旁组织相比,膀胱癌组织中DKK2 mRNA及蛋白的表达均明显下调(P值均<0.001)。DKK2过表达质粒转染后,膀胱癌T24细胞中DKK2 mRNA及蛋白的表达水平明显上调,而细胞的增殖、克隆形成、迁移和侵袭能力均受到明显抑制(P值均<0.05),同时细胞周期被阻滞于G0/G1期(P<0.001)。而且DKK2过表达的膀胱癌T24细胞中,p21和E-cadherin表达水平明显升高(P值均<0.001),cyclin D1、vimentin和N-cadherin表达水平明显降低(P值均<0.001)。结论:DKK2在人膀胱癌细胞及组织中低表达。DKK 2可能作为一个潜在的抑癌基因,参与膀胱癌进展。
Objective: To investigate the expression level of dickkopf 2(DKK 2) gene in human bladder cancer cell lines and tissues, and the effects of DKK2 overexpression on proliferation and migration of bladder cancer cells, and to explore the possible mechanism.Methods: The relative expression levels of DKK2 mRNA and protein in human bladder cancer cell lines and tissues were detected by RT-PCR, real-time fluorescent quantitative PCR and Western blotting, respectively. The bladder cancer T24 cells with low expression of endogenous DKK 2 gene were transfected with DKK2 overexpression plasmids, the expression levels of DKK2 mRNA and protein were measured by RT-PCR, real-time fluorescent quantitative PCR and Western blotting, respectively. Then the proliferation, colony formation,migration, invasion and cell cycle were detected by CCK-8, plate clone formation assay,scratch wound healing assay, Transwell assay and FCM assay, respectively. Furthermore,the expressions of proliferation-and migration-related molecules in T24 cells after DKK2 overexpression were detected by real-time fluorescent quantitative PCR and Western blotting,respectively.Results: The expressions of DKK2 mRNA and protein were downregulated in human bladder cancer 5637, T24, SW780, J82, HT-1197 and HT-1376 cells as compared with the normal bladder SV-HUC-1 cells(all P < 0.01), and those in human bladder cancer tissues were also downregulated as compared with the normal bladder tissues and paired para-cancerous tissues(all P < 0.001). After transfection with DKK2 overexpression plasmids, the expression levels of DKK2 mRNA and protein were increased, the proliferation, colony formation,migration and invasion of bladder cancer T24 cells were inhibited(all P < 0.05), and the cell cycle was arrested in G0/G1 phase(P < 0.001). Furthermore, the mRNA and protein expressions of p21 and E-cadherin were significantly upregulated(both P < 0.001), and the expressions of cyclin D1, vimentin and N-cadherin were downregulated in T24 cells with DKK2 overexpression(all P < 0.001).Conclusion: DKK 2 gene is low-expressed in human bladder cancer cells and tissues, and it may be a potential tumor suppressor gene involved in the progression of human bladder cancer.
Objective: To investigate the expression level of dickkopf 2(DKK 2) gene in human bladder cancer cell lines and tissues, and the effects of DKK2 overexpression on proliferation and migration of bladder cancer cells, and to explore the possible mechanism.Methods: The relative expression levels of DKK2 mRNA and protein in human bladder cancer cell lines and tissues were detected by RT-PCR, real-time fluorescent quantitative PCR and Western blotting, respectively. The bladder cancer T24 cells with low expression of endogenous DKK 2 gene were transfected with DKK2 overexpression plasmids, the expression levels of DKK2 mRNA and protein were measured by RT-PCR, real-time fluorescent quantitative PCR and Western blotting, respectively. Then the proliferation, colony formation,migration, invasion and cell cycle were detected by CCK-8, plate clone formation assay,scratch wound healing assay, Transwell assay and FCM assay, respectively. Furthermore,the expressions of proliferation-and migration-related molecules in T24 cells after DKK2 overexpression were detected by real-time fluorescent quantitative PCR and Western blotting,respectively.Results: The expressions of DKK2 mRNA and protein were downregulated in human bladder cancer 5637, T24, SW780, J82, HT-1197 and HT-1376 cells as compared with the normal bladder SV-HUC-1 cells(all P < 0.01), and those in human bladder cancer tissues were also downregulated as compared with the normal bladder tissues and paired para-cancerous tissues(all P < 0.001). After transfection with DKK2 overexpression plasmids, the expression levels of DKK2 mRNA and protein were increased, the proliferation, colony formation,migration and invasion of bladder cancer T24 cells were inhibited(all P < 0.05), and the cell cycle was arrested in G0/G1 phase(P < 0.001). Furthermore, the mRNA and protein expressions of p21 and E-cadherin were significantly upregulated(both P < 0.001), and the expressions of cyclin D1, vimentin and N-cadherin were downregulated in T24 cells with DKK2 overexpression(all P < 0.001).Conclusion: DKK 2 gene is low-expressed in human bladder cancer cells and tissues, and it may be a potential tumor suppressor gene involved in the progression of human bladder cancer.
引文
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[15]Zhang N,Hong B,Lian W,et al.Vascular endothelial growth inhibitor174 and its functional domains inhibit epithelial-mesenchymal transition in renal cell carcinoma cells in vitro[J].Int J Mol Med,2017,40(2):569-575.
[2]Ojerholm E,Smith A,Hwang WT,et al.Reply to Neutrophil-to-lymphocyte ratio as a bladder cancer biomarker:Assessing prognostic and predictive value in SWOG 8710[J].Cancer,2017,123(19):3856.
[3]Niehrs C.Function and biological roles of the Dickkopf family of Wnt modulators[J].Oncogene,2006,25(57):7469-7481.
[4]Yuan H,Yu S,Cui Y,et al.Knockdown of mediator subunit Medl 9 suppresses bladder cancer cell proliferation and migration by downregulating Wnt/β-catenin signalling pathway[J/OL].J Cell Mol Med,2017(2017-06-19)[2017-09-05].http://onlinelibrary.wiley.com/doi/10.1111/jcmm.13229/full.
[5]Sato H,Suzuki H,Toyota M,et al.Frequent epigenetic inactivation of DICKKOPF family genes in human gastrointestinal tumors[J].Carcinogenesis,2007,28(12):2459-2466.
[6]Hirata H,Hinoda Y,Nakajima K,et al.Wnt antagonist gene DKK2is epigenetically silenced and inhibits renal cancer progressionthrough apoptotic and cell cycle pathways[J].Clinic Cancer Res,2009,15(18):5678-5687.
[7]Zhu J,Zhang S,Gu L,et al.Epigenetic silencing of DKK2 and Wnt signal pathway components in human ovarian carcinoma[J].Carcinogenesis,2012,33(12):2334-2343.
[8]Sun Y,Jin SD,Zhu Q,et al.Long non-coding RNA LUCAT1 is associated with poor prognosis in human non-small lung cancer and regulates cell proliferation via epigenetically repressing p21 and p57 expression[J].Oncotarget,2017,8(17):28297-28311.
[9]Li C,Lyu J,Meng QH.MiR-93 promotes tumorigenesis and metastasis of non-small cell lung cancer cells by activating the PI3K/Akt pathway via inhibition of LKB1/PTEN/CDKN1A[J].J Cancer,2017,8(5):870-879.
[10]Chikara S,Lindsey K,Dhillon H,et al.Enterolactone induces G,-phase cell cycle arrest in non small cell lung cancer cells by downregulating cyclins and cyclin-dependent kinases[J].Nutr Cancer,2017,69(4):652-662.
[11]Wu BQ,Jiang Y,Zhu F,et al.Long noncoding RNA PVT1 promotes EMT and cell proliferation and migrationthrough downregulating p21 in pancreatic cancer cells[J/OL].Technol Cancer Res Treat,2017(2017-03-30)[2017-07-03].http://journals.sagepub.com/doi/pdf/10.1177/1533034617700559.
[12]Antony J,Huang RY.AXL-driven EMT state as a targetable conduit in cancer[J].Cancer Res,2017,77(14):3725-3732.
[13]Pothoven KL,Schleimer RP.The barrier hypothesis and Oncostatin M:Restoration of epithelial barrier function as a novel therapeutic strategy for the treatment of type2 inflammatory disease[J].Tissue Barriers,2017,5(3):e1341367.
[14]Ying Y,Qingwu L,Mingming X,et al.Emodin:one main ingredient of Shufeng Jiedu Capsule reverses chemoresistance of lung cancer cells through inhibition of EMT[J].Cell Physiol Biochem,2017,42(3):1063-1072.
[15]Zhang N,Hong B,Lian W,et al.Vascular endothelial growth inhibitor174 and its functional domains inhibit epithelial-mesenchymal transition in renal cell carcinoma cells in vitro[J].Int J Mol Med,2017,40(2):569-575.