CXXC型锌指蛋白4基因甲基化上调Wnt/β-连环蛋白信号通路调控胃癌细胞增殖和凋亡的研究

CXXC-type zinc finger protein 4 gene methylation up-regulates Wingless integrated/β-catenin signaling pathway to regulate gastric cancer proliferation and apoptosis

目的探讨CXXC型锌指蛋白4基因(CXXC4)对胃癌细胞增殖和凋亡的影响及其机制。方法采用甲基化特异性聚合酶链反应(PCR)检测江苏省苏北人民医院2015年1月至2019年12月诊治的100例胃癌患者(胃癌组)和50例胃良性疾病患者(良性组)CXXC4基因甲基化并分析其与临床病理因素关系。设计合成靶向CXXC4基因的甲基化寡核苷酸转染至SGC7901胃癌细胞(转染甲基化寡核苷酸组,MON组),选择未转染甲基化寡核苷酸细胞为对照(未转染寡核苷酸,CON组),单因素方差分析比较两组细胞CXXC4基因甲基化及信使核糖核酸(mRNA)表达、吸光度(A)值、细胞周期及凋亡、果蝇无翅基因(Wnt)、β-连环蛋白(β-catenin)、细胞周期蛋白D1(Cyclin D1)和c-Myc基因mRNA表达。两组间比较采用t或χ2检验,多组间比较采用单因素方差分析,采用SNK-q法分别比较组间差异。结果胃癌组患者CXXC4基因甲基化率显著高于CON组(67.0%比14.0%,χ2=35.371,P<0.01),差异有统计学意义。SGC7901胃癌细胞中CXXC4为未甲基化状态。胃癌组患者CXXC4基因甲基化率在分化程度、肿瘤T分期、淋巴结转移及肿瘤分期方面的差异有统计学意义(χ2=4.626、4.075、5.294、5.619,P值均<0.05)。MON组SGC7901胃癌细胞第1~6天A值、S期、G2/M期、增殖指数、Wnt、β-catenin、Cyclin D1和c-Myc基因mRNA相对表达量显著高于CON组(t=7.324、9.238、8.789、11.233、8.018、10.383、27.899、34.461、21.432、20.137、31.654、27.439、36.872,P<0.01),差异有统计学意义,CXXC4基因mRNA表达、G0/G1期和凋亡率显著低于CON组(t=55.637、45.256、32.009,P<0.01),差异有统计学意义。结论CXXC4基因甲基化与胃癌发病机制及临床病理因素有相关。CXXC4基因甲基化上调Wnt/β-catenin信号通路促进胃癌细胞增殖并抑制凋亡。

Objective To study the effects of CXXC zinc finger protein 4(CXXC4)gene methylation on proliferation and apoptosis of gastric cancer cells and the mechanism.Methods Methylation specific polymerase chain reaction(PCR)was used to detect the CXXC4 gene methylation in patients with gastric cancer and those with benign gastric diseases.The SGC7901 gastric cancer cells were divided into MON group(treated with methylated oligonucleotide)and CON group(treated with complete medium).The CXXC4 gene methylation and mRNA expression,absorbance,cell proliferation and apoptosis,Wingless integrated(Wnt),β-catenin,Cyclin D1 and c-Myc gene mRNA expression levels were detected and compared between two group by one-way analysis of variance.Results The CXXC4 gene methylation rate in gastric cancer group was significantly higher than that in benign gastric disease group(67.0%vs.14.0%,χ2=35.371,P<0.01).The methylated oligonucleotide could successfully induce the methylation of CXXC4 gene in PANC-1 pancreatic cancer cells.The d1-d6 absorbance,proportion of S phase and G2/M phase,proliferation index,Wnt,β-catenin,Cyclin D1 and c-Myc gene mRNA relative expression in the MON group were significantly higher than those in the CON group(t=7.324,9.238,8.789,11.233,8.018,10.383,27.899,34.461,21.432,20.137,31.654,27.439,36.872,P<0.01),while CXXC4 gene mRNA expression,proportion of G0/G1 phase and apoptosis rate were significantly lower in the MON group than in the CON group(t=55.637,45.256,32.009,P<0.01).Conclusion CXXC4 gene methylation is associated with gastric cancer pathogenesis and clinicopathological factors.CXXC4 gene methylation up-regulates Wnt/β-catenin signaling pathway to promote gastric cancer cell proliferation and inhibit apoptosis.