miR-214-5p通过DNMT1介导的AXIN2基因DNA甲基化修饰在皮肤基底细胞癌中的作用机制

Mechanism of miR-214-5p in skin basal cell carcinoma through DNMT1-mediated DNA methylation modification of AXIN2 gene

目的探讨皮肤基底细胞癌中轴抑制蛋白2(Axis inhibition protein 2,AXIN2)基因启动子甲基化对基因转录的影响及miR-214-5p通过靶向DNA甲基转移酶1(DNA methyltransferase1,DNMT1)对AXIN2甲基化率的调控机制。方法收集2022年1月-2023年6月在广州市皮肤病防治所就诊治疗的102例皮肤基底细胞癌(Cutaneous basal cell carcinoma,BCC)患者作为研究对象,提取癌组织和癌旁正常组织标本及基线资料。焦磷酸测序法检测AXIN2基因启动子区甲基化率。实时荧光定量PCR检测AXIN2、DNMT1基因mRNA和miR-214-5p的表达水平。将miR-214-5p模拟物(mimic)、抑制物(inhibitor)及其阴性对照(mimic NC和inhibitor NC)分别对基底细胞癌A431细胞进行转染,48 h后检测DNMT1基因mRNA表达水平和AXIN2基因甲基化率。结果BCC癌组织的AXIN2基因甲基化率显著高于癌旁正常组织(t=5.128,P<0.001),AXIN2基因mRNA相对表达水平显著低于癌旁正常组织(t=7.826,P<0.001),DNMT1基因mRNA表达水平显著高于癌旁正常组织(t=4.838,P<0.001),miR-214-5p表达水平显著低于癌旁正常组织(t=5.426,P<0.001)。BCC癌组织的AXIN2基因甲基化率与其mRNA表达水平呈负相关(r=-0.793,P<0.001),DNMT1基因mRNA水平与AXIN2基因甲基化率呈正相关(r=0.814,P<0.001),miR-214-5p表达水平与DNMT1基因mRNA水平呈负相关(r=-0.747,P<0.001)。双荧光素酶报告基因实验结果证实,DNMT1是miR-214-5p的靶基因。细胞转染后,与mimic NC、inhibitor和inhibitor NC比较,mimic的DNMT1基因mRNA水平、AXIN2基因甲基化率显著降低(P<0.001);而inhibitor的DNMT1基因mRNA水平和AXIN2基因甲基化率相较于其他三组明显上升(P<0.001)。结论miR-214-5p可通过调控下游靶蛋白DNMT1表达,影响AXIN2基因的DNA甲基化率,调控AXIN2基因的表达水平,参与皮肤基底细胞癌的发生机制。

Objective To investigate the effect of promoter methylation of axis inhibition protein 2(AXIN2)gene on gene transcription in cutaneous basal cell carcinoma(BCC)and the mechanismof miR-214-5p sregulation of Axis inhibition protein 2(AXIN2)methylation rate by targeting DNA methyltransferase1(DNMT1).Methods From January 2022 to June 2023,102 BCC patients'sample and baseline data who were treated in Guangzhou Institute of Dermatology were collected as the study objects.The methylation rate of AXIN2 gene promoter region was quantitatively determined by pyrosequencing.real-time quantitative(PCR)was used to detect AXIN2,DNMT1 mRNA and miR-214-5p expression levels.The basal cell carcinoma A431 cells were transfected with miR-214-5p mimic,inhibitor and negative control mimic NC,respectively.The mRNA expression level of DNMT1 gene and the methylation rate of AXIN2 gene were detected 48 h later.Results The methylation rate of AXIN2 gene in BCC was significantly higher than that in paracanceral normal tissues(t=5.128,P<0.001),and the mRNA relative expression level of AXIN2 gene was significantly lower than that in paracanceral normal tissues(t=7.826,P<0.001).The mRNA expression level of DNMT1 gene was significantly higher than that of paracancerous normal tissues(t=4.838,P<0.001),and the expression level of miR-214-5p was significantly lower than that of paracancerous normal tissues(t=5.426,P<0.001).The methylation rate of AXIN2 gene in BCC cancer tissue was negatively correlated with its mRNA expression level(r=-0.793,P<0.001),and the mRNA level of DNMT1 gene was positively correlated with the methylation rate of AXIN2 gene(r=0.814,P<0.001).The expression level of miR-214-5p was negatively correlated with the mRNA level of DNMT1 gene(r=-0.747,P<0.001).The results of double luciferase reporter gene experiment confirmed that DNMT1 was the target gene of miR-214-5p.After transfection,the mRNA levels of DNMT1 gene and the methylation rate of AXIN2 gene in mimic were significantly decreased compared with those of mimic NC,inhibitor and inhibitor NC(P<0.001).The mRNA level of DNMT1 gene and the methylation rate of AXIN2 gene in inhibitor were significantly increased compared with the other three(P<0.001).Conclusion miR-214-5p can affect the DNA methylation rate of AXIN2 gene by regulating the expression of downstream target protein DNMT1,regulate the expression level of AXIN2 gene and participate in the pathogenesis of skin basal cell carcinoma.