西达苯胺抑制人胃癌SGC-7901细胞侵袭机制探讨

Chidamide inhibits invasion of human gastric carcinoma SGC-7901 cells via up-regulating SFRP2

目的西达苯胺(chidamide,CDM)是一种苯甲酰胺类组蛋白去乙酰化酶抑制剂,通过表观遗传调控机制发挥抗肿瘤作用。本研究旨在探讨CDM对人胃癌SGC-7901细胞侵袭能力的影响及其作用机制。方法用不同浓度的CDM(0、0. 2,0.4.0. 8、1. 6.3. 2、6. 4 μmol/L)处理SGC-7901细胞48 h后,CCK-8法检测细胞活力,脂质体介导法将人分泌型卷曲相关蛋白2 (secreted frizzled-related protein 2 ,SFRP2)真核表达质粒转染SGC-7901细胞,Transwell法检测细胞侵袭,反转录-聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)法检测 SFRP2 的 mRNA 表达水平,蛋白质印迹法检测SFRP2、锌指E盒结合同源盒1( zinc finger E-box binding homeobox 1, ZEB1)、E-钙粘蛋白(E-cadherin)、基质金属蛋白酶2 (matrix metalloproteinase 2, MMP2)和MMP9的蛋白表达水平。结果不同浓度的CDM均可抑制SGC-7901细胞活力,组间比较差异有统计学意义,F=58. 407,P<0. 001;低剂量CDM(0. 2~0. 8 μmol/L)可抑制SGC-7901细胞侵袭(F=334. 816,P<0. 001),下调ZEB1、MMP2和 MMP9蛋白表达(F值分别为186. 422、237. 054 和 171. 358,均 P<0. 001),上调 SFRP2 和E-cadherin 蛋白表达(F 值分别为 419. 462 和 215. 368,均 P<0. 001),上调SFRP2的mRNA表达水平(F=178.905 ,P<0. 001),并呈剂量依赖性;SFRP2高表达可抑制SGC-7901细胞侵袭(t=56. 385,P<0.001),上调E-cadherin 蛋白表达(t=13. 074, P<0. 001),下调 ZEB1、MMP2 和 MMP9 蛋白表达(t值分别为13. 583、11.232和13. 911,均P<0.001);沉默SFRP2基因可削弱CDM对SGC-7901细胞侵袭的抑制作用,F=548.043,P<0.001。结论 CDM可通过上调SFRP2基因表达抑制人胃癌SGC-7901细胞侵袭。

OBJECTIVE Chidamide( CDM) is a histone deacetylase inhibitor( HDACi) of benzamides and has antineoplastic activity through epigenetic regulation. This study aimed to investigate the effect of CMD on invasion of human gastric carcinoma SGC-7901 cells and its possible mechanism. METHODS SGC-7901 cells were cultured in vitro and treated with CMD (0,0. 2,0. 4,0. 8,1. 6,3. 2,6. 4 ptmol/L) for 48 h and then the cell viability were detected by CCK-8 assay. The secreted frizzled-related protein 2 (SFRP2) high-expression SGC-7901 cells were stably established by transfecting with eukaryotic expression vector (pCMV6-SFRP2). Transwell assay was used to determine the ability of invasion. The mRNA expression level of SFRP2 were measured by RT-PCR. Western blot was performed to analyze the protein expression of SFRP2, zinc finger E-box binding homeobox 1 ( ZEB1 ), E-cadherin, matrix metalloproteinase 2 ( MMP2 ) and MMP9. RESULTS CCK-8 assay showed that CMD remarkably reduced the viability of SGC-7901 cells in a dose-dependent manner (F=58.407,P<0. 001). Low-level CMD suppressed migration and invasion of SGC-7901 cells (F=334. 816, P<0. 001) and down-regulated the protein levels of ZEB1, MMP2 and MMP9 (F= 186. 422,237. 054,171. 358,all P<0. 001),up-regulated the protein levels of SFRP2 and E-cadherin (F=419. 462,215. 368,all P<0. 001) and up-regulated the mRNA expression of SFRP2 in a dose-dependent manner (F=178.905,P<0. 001). High expression of SFRP2 also inhibited invasion of SGC-7901 cells (t=56. 385,P<0. 001),up-regulated the protein level of E-cadherin (Z= 13. 074,P< 0. 001) and down-regulated the protein levels of ZEB1, MMP2 and MMP9(t= 13. 583,11. 232,13. 911, all P<0. 001).Knock-down of SFRP2 gene expression reversed CMD-induced suppression of invasion. CONCLUSION CMD may inhibit invasion of SGC-7901 cells most likely via up-regulating the expression of SFRP2, and then down-regulating the protein levels of ZEB1, MMP2 and MMP9 ,and up-regulating the protein level of E-cadherin.