SND1通过识别TINCR的甲基化位点促进瘢痕疙瘩成纤维细胞的生长  

SND1 recognize the methylation sites of TINCR to promote growth of keloid fibroblasts

作　　者：秦高平[1] 孙要文[1] 郭亚东[1] 李建武 程亮 韩峰 宋勇[2] Qin Gao-Ping;Sun Yao-Wen;Guo Ya-Dong;Li Jian-Wu;Cheng Liang;Han Feng;Song Yong(Department of Burn and Plastic Surgery,Shaanxi Provincial People’s Hospital,Xi’an 710068,China;Department of Hepatobiliary Surgery,Shaanxi Provincial People’s Hospital,Xi’an 710068,China)

机构地区：[1]陕西省人民医院烧伤整形医学美容外科,西安710068 [2]陕西省人民医院肝胆外科,西安710068

出　　处：《解放军医学杂志》2021年第11期1068-1076,共9页Medical Journal of Chinese People's Liberation Army

摘　　要：目的探讨葡萄球菌核酸酶结构域蛋白1(SND1)与组织分化诱导非编码RNA(TINCR)的相互作用及其对TINCR表达水平和瘢痕疙瘩生长的影响。方法收集2016年6月-2018年5月在陕西省人民医院接受治疗的Ⅱ、Ⅲ度烧伤患者的瘢痕疙瘩组织样本(n=27)。运用生物信息学方法预测TINCR序列中的RNA甲基化位点;培养人原代正常皮肤成纤维细胞(HFs)和瘢痕疙瘩成纤维细胞(KFs),设置HFs组和KFs组。取对数生长期KFs,设置:(1)对照组、空载组及甲基转移酶样蛋白3(METTL3)过表达组;(2)对照组和3-脱氮基腺苷(DAA)组;(3)对照组、SND1重组蛋白组及SND1重组蛋白+DAA组。采用实时定量PCR(RT-qPCR)检测HFs和KFs中TINCR的相对表达水平,甲基化RNA免疫沉淀技术(MeRIP)联合RT-qPCR检测TINCR的甲基化水平,Western blotting检测SND1和METTL3蛋白的相对表达水平,放线菌素D处理联合RT-qPCR检测TINCR残留水平,CCK-8法和克隆形成实验检测细胞增殖能力。取18只BALB/c裸鼠,构建瘢痕疙瘩异种移植模型,随机分为瘢痕疙瘩组、瘢痕疙瘩+SND1重组蛋白组及瘢痕疙瘩+SND1重组蛋白+DAA组,每组6只,采用HE染色观察瘢痕疙瘩组织生长情况,免疫组化染色和Western blotting检测SND1在瘢痕疙瘩组织中的表达水平。结果TINCR第三外显子含有7个潜在的RNA甲基化位点。与HFs相比,KFs中TINCR相对表达水平(5.43±0.35 vs.1.00±0.11,P<0.01)和甲基化水平(19.73%±1.56%vs.10.25%±1.13%,P<0.01)均明显升高,SND1和METTL3蛋白相对表达水平明显增高(P<0.01),甲基化的TINCR与SND1和METTL3均有结合。与空载组相比,METTL3过表达可促进METTL3 mRNA和蛋白的表达(mRNA:6.03±0.55 vs.1.09±0.09,P<0.01;蛋白:4.33±0.35 vs.0.96±0.08,P<0.01)、增高TINCR甲基化水平(32.89%±2.88%vs.19.04%±1.72%,P<0.01)和相对表达水平(4.65±0.32 vs.1.00±0.10,P<0.01)。与对照组相比,DAA处理可降低TINCR的稳定性[(8.50±1.13)h vs.(12.90±1.41)h,P<0.001]和甲基化水平(7.43%±0.55%vs.1Objective To investigate the interaction between staphylococcal nuclease domain containing protein 1(SND1)and tissue differentiation-inducing non-coding RNA(TINCR),and the effect of SND1 on TINCR expression level and keloid growth.Methods Keloid tissue samples(n=27)were collected from patients withⅡ-orⅢ-degree burns who received treatment in Shaanxi Provincial People's Hospital from June 2016 to May 2018.Bioinformatics methods were used to predict RNA methylation sites in TINCR sequences;human primary normal skin fibroblasts(HFs)and keloid fibroblasts(KFs)were cultured and divided into HFs group and KFs group.KFs in logarithmic growth phase were taken,and divided into:(1)control group,empty vector group and methyltransferase-like protein 3(METTL3)overexpression group,(2)control group and 3-deazaadenosine(DAA)group,and(3)control group,SND1 recombinant proteome group and SND1 proteome+DAA group.The relative expression level of TINCR in HFs and KFs was determined by real-time quantitative PCR(RT-qPCR);methylated RNA immunoprecipitation(MeRIP)followed by RT-qPCR was performed to analyze the methylation levels of TINCR;Western blotting was used to detect the relative expression levels of SND1 and METTL3 protein;the half-life of TINCR was determined by actinomycin D treatment combined with RT-qPCR,and the cell proliferation capacity was assessed with CCK-8 and clone assays.A keloid xenograft model was generated with 18 BALB/c nude mice,and then randomly divided into three groups(6 mice in each group):keloid group,keloid+SND1 recombinant protein group,and keloid+SND1 recombinant protein+DAA group.Keloid tissue growth was observed by HE staining;the expression level of SND1 in keloid tissue was detected by immunohistochemistry staining and Western blotting.Results The third exon of TINCR contains seven potential RNA methylation sites.The relative expression level and methylation level of TINCR elevated obviously in KFs than in HFs(5.43±0.35 vs.1.00±0.11,P<0.01;19.73%±1.56%vs.10.25%±1.13%,P<0.01),and the protein l

关 键 词：组织分化诱导非编码RNA 葡萄球菌核酸酶结构域蛋白1 RNA甲基化 RNA稳定性 细胞增殖 成纤维细胞 瘢痕疙瘩 

分 类 号：R394.3[医药卫生—医学遗传学]