5⁃杂氮-2’-脱氧胞苷对肺癌A549细胞系中SFRP2基因表达以及细胞迁移侵袭的影响

Effects of5-aza-2’-deoxycytidine on SFRP2gene expression andd ecellularized migration and invasion in lung cancer A549cell line

目的研究肺癌A549细胞系中5-杂氮-2’-脱氧胞苷(5-Aza-CdR)对分泌型卷曲相关蛋白2基因(SFRP2)甲基和基因表达的影响,以及探究5-Aza-CdR对A549细胞迁移和侵袭的影响。方法收集2018年至2019年于南阳市中心医院切除的肺癌样本以及相应的癌旁组织,分为肺癌和癌旁组,通过实时定量PCR(qPCR)和蛋白质印迹法(Western blot)检测SFRP2基因mRNA和蛋白表达水平。甲基化特异性PCR(MSP)法分别检测60例肺癌组和癌旁组中SFRP2基因甲基化水平;采用对肺癌A549细胞系进行去甲基化处理,得到SFRP2基因去甲基化组。未处理肺癌A549细胞系为对照组。对照组和SFRP2基因去甲基化组通过MSP法检测SFRP2基因基因的甲基化状态;采用划痕实验和侵袭迁移小室法(Transwell小室)分别检测肺癌A549细胞系的迁移和侵袭能力;通过qPCR和Western blot检测SFRP2、GSK3β、p-GSK3β、β-catenin和p-β-catenin的mRNA和蛋白表达水平。结果qPCR和Western blot结果显示,与癌旁组比较,肺癌组中的SFRP2基因的mRNA和蛋白表达量显著降低(P<0.05);MSP结果显示,与癌旁组比较,48例肺癌组的SFRP2基因发生甲基化。MSP结果显示,与对照组比较,SFRP2基因去甲基化组的SFRP2基因甲基化水平显著降低(P<0.05)。划痕实验和Transwell小室结果显示,与对照组比较,SFRP2基因去甲基化组的迁移和侵袭能力显著降低(P<0.05)。qPCR和Western blot结果显示,与对照组比较,SFRP2基因去甲基化组的p-GSK3β、β-catenin的mRNA和蛋白表达显著减少(P<0.05),SFRP2、GSK3β和p-β-catenin的mRNA和蛋白表达显著增多(P<0.05)。结论SFRP2基因基因去甲基化可以恢复SFRP基因转录表达,阻碍Wnt/β-catenin信号通路,从而抑制肺癌A549细胞系迁移和侵袭能力。

Objective To study the effect of 5-Aza-2’-deoxycytidine(5-Aza-CdR)on the methyl and gene ex⁃pression of secreted frizzled-related protein two gene(SFRP2)in lung cancer A549 cell line,and explore the effect of 5-Aza-CdR on A549 cell migration and invasion.Methods Collected lung cancer samples and corresponding para⁃cancerous tissues resected at the Nanyang Central Hospital from 2018 to 2019,and divided them into lung cancer and pa⁃racancerous groups.We used real-time quantitative PCR(qPCR)and Western blot to detect mRNA and protein ex⁃pression levels of SFRP2 gene.Methylation-specific PCR(MSP)method was used to detect the methylation level of SFRP2 gene in 60 cases of lung cancer group and paracancerous group;The demethylation treatment of lung cancer A549 cell line was used to obtain the SFRP2 gene demethylation group.The untreated lung cancer A549 cell line served as the control group.The control group and the SFRP2 gene demethylation group were used to detect the methylation status of the SFRP2 gene by the MSP method;The scratch test and the invasion and migration chamber method(Transwell cham⁃ber)were used to detect the migration and invasion ability of the lung cancer A549 cell line;qPCR And Western blot was used to detect the mRNA and protein expression levels of SFRP2,GSK3β,p-GSK3β,β-catenin and p-β-catenin.Results Compared with the paracancerous group,the results of qPCR and Western blot showed that the mRNA and pro⁃tein expression of the SFRP2 gene in the lung cancer group were significantly reduced(P<0.05);The MSP results showed that compared with the paracancerous group,48 cases of SFRP2 gene in the lung cancer group were methylated.MSP results showed that compared with the control group,the SFRP2 gene methylation level of the SFRP2 gene demethy⁃lation group was significantly reduced(P<0.05).The scratch test and Transwell chamber results showed that compared with the control group,the migration and invasion ability of the SFRP2 gene demethylation group were significantly re⁃duced(P<0.05).The results of qPCR and Western blot showed that compared with the control group,themRNA and protein expressions of p-GSK3βandβ-catenin in the SFRP2 gene demethylation group were significantly reduced(P<0.05),and the levels mRNA and protein expression of of SFRP2,GSK3βand p-β-catenin increased significantly(P<0.05).Conclusion Demethylation of SFRP2 gene could restore the transcriptional expression of SFRP gene,hinder the Wnt/β-catenin signaling pathway,and inhibit the migration and invasion of lung cancer cell line A549.