肿瘤抑制因子DACT2在神经胶质瘤细胞上皮间质转化中的作用机制
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摘要
目的探讨肿瘤抑制因子β-连环蛋白抑制基因2(DACT2)对神经胶质瘤细胞上皮间质转化的影响及潜在机制。方法以神经胶质瘤细胞U87、U251、A172和SHG44为研究对象,5-Aza处理后RT-PCR检测细胞中DACT2基因表达,甲基化特异PCR法(MSP)检测细胞中DACT2启动子甲基化状态,Western blot检测细胞中DACT2蛋白表达;构建过表达DACT2的U87细胞株,并用Western blot进行验证。Transwell实验检测细胞迁移侵袭能力,Western blot检测细胞中上皮间质标记物E-cadherin、Vimentin、MMP2及Wnt/β-cadherin通路蛋白active-β-cadherin、p-β-cadherin和totalβ-cadherin的表达变化。结果 5-Aza处理后4株细胞中均观察到DACT2基因表达,而未处理的U87和U251细胞中无DACT2基因表达,A172和SHG44细胞中DACT2有微弱表达。U87和U251细胞中DACT2启动子区完全甲基化,而A172和SHG44细胞中部分甲基化。U87和U251细胞中DACT2蛋白和mRNA表达低于A172和SHG44细胞(P<0.05)。转染pcDNA3.1-DACT2载体后,U87细胞中DACT2表达显著增加(P<0.05),过表达DACT2的U87细胞构建成功。过表达DACT2能抑制U87细胞上皮间质转化、侵袭迁移并阻断Wnt/β-catenin通路激活(P<0.05)。结论神经胶质瘤细胞中DACT2启动子呈高度甲基化状态,外源过表达DACT2能够促进胶质瘤细胞U87上皮间质转化和侵袭迁移,其机制可能与DACT2调控Wnt/β-catenin信号通路有关。
Objective To investigate the effects and potential mechanism of tumor suppressor dishevelled-binding antagonist of beta-catenin 2(DACT2) on epithelial-mesenchymal transition of glioma cells. Methods The expressions of DACT2 in glioma cells U87, U251, A172 and SHG44 were detected by RT-PCR after 5-Aza treatment. The methylation status of DACT2 promoter was detected by methylation specific PCR(MSP). Western blot was used to detect the expression of DACT2 protein. U87 cells overexpressing DACT2 were constructed and verified by Western blot. Transwell assay was used to detect cell migratory and invasive ability. The protein levels of E-cadherin, Vimentin, MMP2 and Wnt/β-cadherin pathway proteins, i.e., active-β-cadherin, p-β-cadherin and total β-cadherin, in cells were detected by Western blot. Results DACT2 expression was observed in all these cells after 5-Aza treatment; untreated U87 and U251 cells did not express DACT2 while A172 and SHG44 cells showed weak expression. The DACT2 promoter was completely methylated in U87 and U251 cells, and partially methylated in A172 and SHG44 cells. The level of DACT2 in U87 and U251 cells was lower than that in A172 and SHG44 cells(P<0.05). After transfection of pcDNA3.1-DACT2, the expression of DACT2 in U87 cells increased significantly(P<0.05), U87 cells overexpressing DACT2 were successfully constructed. Overexpression of DACT2 could significantly inhibit epithelial-mesenchymal transition, invasion and migration of U87 cells and block Wnt/β-catenin pathway activation(P<0.05).Conclusion The DACT2 promoter in glioma cells is highly methylated,and the exogenous overexpression of DACT2 can promote the epithelial mesenchymal transition,invasion and migration of U87 cells.The underling mechanism may be related to the regulation of Wnt/β-catenin pathway by DACT2.
Objective To investigate the effects and potential mechanism of tumor suppressor dishevelled-binding antagonist of beta-catenin 2(DACT2) on epithelial-mesenchymal transition of glioma cells. Methods The expressions of DACT2 in glioma cells U87, U251, A172 and SHG44 were detected by RT-PCR after 5-Aza treatment. The methylation status of DACT2 promoter was detected by methylation specific PCR(MSP). Western blot was used to detect the expression of DACT2 protein. U87 cells overexpressing DACT2 were constructed and verified by Western blot. Transwell assay was used to detect cell migratory and invasive ability. The protein levels of E-cadherin, Vimentin, MMP2 and Wnt/β-cadherin pathway proteins, i.e., active-β-cadherin, p-β-cadherin and total β-cadherin, in cells were detected by Western blot. Results DACT2 expression was observed in all these cells after 5-Aza treatment; untreated U87 and U251 cells did not express DACT2 while A172 and SHG44 cells showed weak expression. The DACT2 promoter was completely methylated in U87 and U251 cells, and partially methylated in A172 and SHG44 cells. The level of DACT2 in U87 and U251 cells was lower than that in A172 and SHG44 cells(P<0.05). After transfection of pcDNA3.1-DACT2, the expression of DACT2 in U87 cells increased significantly(P<0.05), U87 cells overexpressing DACT2 were successfully constructed. Overexpression of DACT2 could significantly inhibit epithelial-mesenchymal transition, invasion and migration of U87 cells and block Wnt/β-catenin pathway activation(P<0.05).Conclusion The DACT2 promoter in glioma cells is highly methylated,and the exogenous overexpression of DACT2 can promote the epithelial mesenchymal transition,invasion and migration of U87 cells.The underling mechanism may be related to the regulation of Wnt/β-catenin pathway by DACT2.
引文
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[22] KIM EY,KIM A,KIM SK,et al.Inhibition of mTORC1 induces loss of E-cadherin through AKT/GSK-3β signaling-mediated upregulation of E-cadherin repressor complexes in non-small cell lung cancer cells[J].Respir Res,2014,15(1):26.
[23] XIANG T,FAN Y,LI C,et al.DACT2 silencing by promoter CpG methylation disrupts its regulation of epithelial-to-mesenchymal transition and cytoskeleton reorganization in breast cancer cells[J].Oncotarget,2016,7(43):70924-70935.
[24] XIANG T,LI L,YIN X,et al.Epigenetic silencing of the WNT antagonist Dickkopf 3 disrupts normal Wnt/β-catenin signalling and apoptosis regulation in breast cancer cells[J].J Cell Mol Med,2013,17(10):1236-1246.
[25] WANG S,DONG YJ,XU L,et al.DACT2 is a functional tumor suppressor through inhibiting Wnt/β-Catenin pathway and associated with poor survival in colon cancer[J].Oncogene,2015,34(20):2575-2585.
[26] YU Y,YAN W,LIU X,et al.DACT2 is frequently methylated in human gastric cancer and methylation of DACT2 activated Wnt signaling[J].Am J Cancer Res,2014,4(6):710-724.
[27] ZHAO Z,HERMAN JG,BROCK MV,et al.Methylation of DACT2 promotes papillary thyroid cancer metastasis by activating Wnt signaling[J].PLoS One,2014,9(11):e112336.
[2] SIEGEL R,MA J,ZOU Z,et al.Cancer statistics,2014[J].CA Cancer J Clin,2014,64(1):9-29.
[3] 李云涛,陈谦学,吴庭枫,等.Rho激酶在胶质瘤侵袭调控中作用的研究进展[J].浙江临床医学,2015,(8):1418-1420.
[4] 李冠璋,张伟,江涛.视黄酸信号通路与神经胶质瘤的研究进展[J].中华神经外科杂志,2016,32(8):862-864.
[5] 张超超,于凤波,黄海燕,等.新诊断胶质母细胞瘤的治疗进展[J].牡丹江医学院学报,2016,37(1):111-113.
[6] 陈亮,李青松.Wnt信号通路在胶质瘤中的研究进展[J].国际神经病学神经外科学杂志,2016,43(2):184-188.
[7] GE C,WU S,WANG W,et al.miR-942 promotes cancer stem cell-like traits in esophageal squamous cell carcinoma through activation of Wnt/β-catenin signalling pathway[J].Oncotarget,2015,6(13):10964-10977.
[8] CHEYETTE BN,WAXMAN JS,MILLER JR,et al.Dapper,a dishevelled-associated antagonist of beta-catenin and JNK signaling,is required for notochord formation[J].Dev Cell,2002,2(4):449-461.
[9] ZHANG M,LINGHU E,ZHAN Q,et al.Methylation of DACT2 accelerates esophageal cancer development by activating Wnt signaling[J].Oncotarget,2016,7(14):17957-17969.
[10] 樊博,齐盼,张爱莉,等.肾细胞癌中DACT2基因启动子区甲基化状态与mRNA表达的研究[J].重庆医学,2017,46(21):2895-2897.
[11] 郭艳丽,周珍,郭炜,等.β-环连蛋白抑制基因2甲基化状态对贲门腺癌发生发展的影响及机制[J].中国肿瘤,2016,25(8):653-658.
[12] 刘磊,周珍,邝刚,等.食管鳞癌中DACT2基因表达及甲基化状态研究[J].中国肿瘤,2017,26(4):302-307.
[13] YING T,LI QM,NING H,et al.Upregulation of DACT2 suppresses proliferation and enhances apoptosis of glioma cell via inactivation of YAP signaling pathway[J].Cell Death Dis,2017,8(8):e2981.
[14] 潘冬.表观遗传调控参与肿瘤细胞辐射应答的机制研究[D].北京:中国科学院大学(中国科学院近代物理研究所),2017.
[15] 刘馨,周鹏,卢奕,等.DNA甲基化在常见眼部肿瘤中的研究进展[J].中华眼科杂志,2015,51(12):950-954.
[16] YAN J,YANG Y,BROCK MV,et al.Epigenetic regulation of DACT2,a key component of the Wnt signalling pathway in human lung cancer[J].J Pathol,2013,230(2):194-204.
[17] GAO S,YANG Z,ZHENG ZY,et al.Reduced expression of DACT2 promotes hepatocellular carcinoma progression:Involvement of methylation-mediated gene silencing[J].World J Surg Oncol,2013,11(1):57.
[18] ZHANG X,YANG Y,LIU X,et al.Epigenetic regulation of the Wnt signaling inhibitor DACT2 in human hepatocellular carcinoma[J].Epigenetics,2013,8(4):373-382.
[19] HOU J,LIAO LD,XIE YM,et al.DACT2 is a candidate tumor suppressor and prognostic marker in esophageal squamous cell carcinoma[J].Cancer Prev Res,2013,6(8):791-800.
[20] SHI C,REN L,SUN C,et al.miR-29a/b/c function as invasion suppressors for gliomas by targeting CDC42 and predict the prognosis of patients[J].Br J Cancer,2017,117(7):1036-1047.
[21] 陈欢,王淼,伍会健.上皮–间质转化在肿瘤侵袭、转移及化疗耐药中的研究进展[J].中国细胞生物学学报,2017,39(7):978-983.
[22] KIM EY,KIM A,KIM SK,et al.Inhibition of mTORC1 induces loss of E-cadherin through AKT/GSK-3β signaling-mediated upregulation of E-cadherin repressor complexes in non-small cell lung cancer cells[J].Respir Res,2014,15(1):26.
[23] XIANG T,FAN Y,LI C,et al.DACT2 silencing by promoter CpG methylation disrupts its regulation of epithelial-to-mesenchymal transition and cytoskeleton reorganization in breast cancer cells[J].Oncotarget,2016,7(43):70924-70935.
[24] XIANG T,LI L,YIN X,et al.Epigenetic silencing of the WNT antagonist Dickkopf 3 disrupts normal Wnt/β-catenin signalling and apoptosis regulation in breast cancer cells[J].J Cell Mol Med,2013,17(10):1236-1246.
[25] WANG S,DONG YJ,XU L,et al.DACT2 is a functional tumor suppressor through inhibiting Wnt/β-Catenin pathway and associated with poor survival in colon cancer[J].Oncogene,2015,34(20):2575-2585.
[26] YU Y,YAN W,LIU X,et al.DACT2 is frequently methylated in human gastric cancer and methylation of DACT2 activated Wnt signaling[J].Am J Cancer Res,2014,4(6):710-724.
[27] ZHAO Z,HERMAN JG,BROCK MV,et al.Methylation of DACT2 promotes papillary thyroid cancer metastasis by activating Wnt signaling[J].PLoS One,2014,9(11):e112336.