FTO诱导TSG-6 mRNA去甲基化对瘢痕疙瘩的影响

Effect of TSG-6 mRNA demethylation induced by FTO on keloid

目的探讨去甲基化酶肥胖相关蛋白(FTO)调控肿瘤坏死因子-α刺激基因-6(TSG-6)mRNA去甲基化在瘢痕疙瘩组织中的表达及其在瘢痕形成中的作用。方法选取新鲜瘢痕疙瘩组织(实验组)和正常皮肤组织(对照组),应用细胞免疫荧光方法检测TSG-6在实验组和对照组中的表达水平;构建表达TSG-6 mRNA正义和反义链探针用于RNA pulldown,随后用N^(6)-甲基腺嘌呤(m^(6)A)抗体对各组样本进行Western blot检测;应用实时荧光定量多聚核苷酸链式反应(qPCR)和Western blot检测实验组和对照组中TSG-6 mRNA去甲基化酶和甲基化酶的表达;应用细胞免疫荧光方法检测FTO在实验组和对照组的表达水平。结果细胞免疫荧光结果显示,实验组中TSG-6表达水平低于对照组(P<0.01);生物素标记TSG-6 RNA pulldown实验结果显示,实验组中TSG-6 mRNA的m^(6)A表达水平低于对照组(P<0.01);qPCR实验结果显示实验组中FTO mRNA表达水平高于对照组(P<0.01);Western blot实验结果显示实验组中FTO蛋白表达水平高于对照组(P<0.001);细胞免疫荧光结果显示实验组FTO表达水平高于对照组(P<0.001)。结论去甲基化酶FTO催化TSG-6 mRNA去甲基化,进而促进瘢痕疙瘩的形成,这可能为瘢痕疙瘩的临床治疗提供新的思路和方向。

Objective To investigate the effect of demethylase obesity-associated protein(FTO)on the expression of tumor necrosis factor-α-stimulating gene-6(TSG-6)mRNA demethylation in keloid tissue and its role in scar formation.Methods Fresh keloid tissue(experimental group)and normal skin tissue(control group)were randomlyselected,and the expression level of TSG-6 in the experimental group and control group was detected by immunofluorescence method.A probe expressing TSG-6 mRNA sense and antisense chain were constructed for RNA pulldown,followed by Western blot detection of samples in each group with N^(6)-methyladenine(m^(6)A)antibody.Real-time fluorescence quantitative polynucleotide chain reaction(qPCR)and Western blot were used to detect the expression of TSG-6 mRNA demethylase and methylase in the experimental group and control group.The expression levels of FTO in experimental group and control group were detected by immunofluorescence assay.Results Immunofluorescence results showed that the expression level of TSG-6 in the experimental group was lower than that in the control group(P<0.01).Pulldown test results of biotin-labeled TSG-6 RNA showed that the m^(6)A expression level of TSG-6 mRNA in the experimental group was lower than that in the control group(P<0.01).The results of qPCR showed that the expression level of FTO mRNA in the experimental group was higher than that in the control group(P<0.01).Western blot analysis showed that FTO protein expression in the experimental group was higher than that in the control group(P<0.001).Immunofluorescence results showed that the expression level of FTO in experimental group was higher than that in control group(P<0.001).Conclusion The demethylase FTO catalyzes the demethylation of TSG-6 mRNA and promotes the formation of keloid,which may provide a new idea and direction for clinical treatment of keloid.