circIGF2BP3调控光老化皮肤成纤维细胞自噬水平的研究  

Regulatory role of circIGF2BP3 in autophagy in photoaged dermal fibroblasts

作　　者：曲莹莹 方嘉琦 欧阳梦婷 王梦瑶 黄羡殷 郑跃[1] 赖维[1] 许庆芳[1] Qu Yingying;Fang Jiaqi;Ouyang Mengting;Wang Mengyao;Huang Xianyin;Zheng Yue;Lai Wei;Xu Qingfang(Department of Dermatology,The Third Affiliated Hospital of Sun Yat-sen University,Guangzhou 510630,China)

机构地区：[1]中山大学附属第三医院皮肤科,广州510630

出　　处：《中华皮肤科杂志》2022年第1期40-46,共7页Chinese Journal of Dermatology

基　　金：国家自然科学基金(81773340);广东省自然科学基金(2021A1515012622)。

摘　　要：目的初步探究circIGF2BP3对光老化皮肤成纤维细胞自噬水平的影响。方法取中山大学附属第三医院泌尿外科6例儿童包皮环切术后包皮组织,分离培养成纤维细胞,以每日10 J/cm^(2)长波紫外线(UVA)连续照射14 d,建立UVA诱导的光老化成纤维细胞模型(UVA处理组),未经处理的正常成纤维细胞作为对照组,β半乳糖苷酶染色、Western印迹法检测P21蛋白表达,CCK8法检测细胞活力验证建模是否成功。Western印迹法检测光老化成纤维细胞中自噬相关蛋白P62、LC3-Ⅱ、LC3-Ⅰ表达,qRT-PCR验证光老化与正常成纤维细胞间circIGF2BP3表达差异,并对其进行生物学注释。将原代成纤维细胞分为4组:空载组、UVA+空载组,circIGF2BP3过表达组、UVA+circIGF2BP3过表达组,Western印迹法检测各组细胞中自噬相关蛋白表达水平。两独立样本均数的比较采用t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果UVA处理组β半乳糖苷酶染色阳性率(61.33%±5.78%)、P21蛋白表达(1.25±0.03)均显著高于对照组(6.37%±0.32%、1.00±0.05,t=9.49、4.26,P<0.01、<0.05),而细胞活力(74.33%±3.48%)显著低于对照组(100%,t=7.38,P<0.01)。UVA处理组P62蛋白表达及LC3-Ⅱ/Ⅰ比值均显著高于对照组(均P<0.05)。光老化成纤维细胞中circIGF2BP3的相对表达量为0.72±0.04,显著低于对照组(1.00±0.03),t=5.46,P<0.01。circIGF2BP3过表达组P62蛋白表达(0.60±0.01)及LC3-Ⅱ/Ⅰ比值(0.71±0.01)均显著低于空载组(1.00±0.02、1.00±0.01;t=16.25、2.75,P<0.01、P<0.05);UVA+circIGF2BP3过表达组P62蛋白表达(1.05±0.02)及LC3-Ⅱ/Ⅰ比值(2.04±0.05)亦均显著低于UVA+空载组(1.31±0.02、2.72±0.14;t=10.49、6.47,均P<0.01)。结论circIGF2BP3可调控UVA诱导的光老化皮肤成纤维细胞自噬水平,为防治光老化提供了新的潜在治疗靶点。Objective To preliminarily investigate the effect of circIGF2BP3 on autophagy in photoaged dermal fibroblasts.Methods Human dermal fibroblasts were isolated from circumcised foreskin tissues from 6 children in the Department of Urological Surgery,Third Affiliated Hospital of Sun Yat-sen University.An ultraviolet A(UVA)-induced photoaged human dermal fibroblast model(UVA radiation group)was established by repeated UVA radiation at a dose of 10 J/cm^(2) for 14 consecutive days,and human dermal fibroblasts receiving no treatment served as control group.The photoaged cell model was verified byβ-galactosidase staining,Western blot analysis for determining P21 protein expression,and cell counting kit-8(CCK8)assay for evaluating cell viability.Moreover,Western blot analysis was performed to determine the protein expression of autophagy-related proteins P62,microtubule-associated protein 1 light chain 3(LC3)-Ⅰand LC3-Ⅱin photoaged human dermal fibroblasts,and real-time quantitative RCR(qRT-PCR)to verify the differential expression of circIGF2BP3 between photoaged and normal human dermal fibroblasts.Furthermore,circIGF2BP3 was biologically annotated.Some cultured primary human dermal fibroblasts were divided into 4 groups:empty vector group transfected with an empty vector,UVA+empty vector group transfected with an empty vector followed by repeated UVA radiation,circIGF2BP3 group transfected with a circIGF2BP3-overexpressing lentiviral vector,UVA+circIGF2BP3 group transfected with a circIGF2BP3-overexpressing lentiviral vector followed by repeated UVA radiation.Western blot analysis was performed to determine the expression of autophagy-related proteins.Statistical analysis was carried out by using t test,one-way analysis of variance and least significant difference-t test.Results Compared with the control group,the UVA radiation group showed significantly increased proportions ofβ-galactosidase-positive cells(61.33%±5.78%vs.6.37%±0.32%,t=9.49,P<0.01)and P21 expression(1.25±0.03 vs.1.00±0.05,t=4.26,P<0.05),but s

关 键 词：皮肤衰老 细胞衰老 自噬 成纤维细胞 环状RNA circIGF2BP3 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]