环状RNA hsa_circUCK2参与调控二氧化硅致肺纤维化进程  

Circular RNA hsa_circUCK2 involved in regulation of pulmonary fibrosis induced by silicon dioxide

作　　者：张聪 罗薇 王莎 巢杰 ZHANG Cong;LUO Wei;WANG Sha;CHAO Jie(Key Laboratory of Environmental Medicine Engineering,Ministry of Education/School of Public Health,School of Medicine,Southeast University,Nanjing,Jiangsu 210009,China;Department of Physiology,School of Medicine,Southeast University,Nanjing,Jiangsu 210009,China)

机构地区：[1]东南大学公共卫生学院,环境医学工程教育部重点实验室,江苏南京210009 [2]东南大学医学院医学生理系,江苏南京210009

出　　处：《环境与职业医学》2024年第4期362-366,374,共6页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金项目(81972987)。

摘　　要：[背景]肺纤维化疾病目前早期缺少筛查诊断方法,后期缺乏特效治疗措施,因此急需探究其机制并开发针对性的治疗方法。[目的]筛选病理条件下差异表达的环状RNA(circRNA)hsa_circUCK2的表达情况,探究其对肺纤维化的影响。[方法]在细胞实验中,使用小干扰RNA(siRNA)敲除HPF-a细胞中hsa_circUCK2,TGF-β1刺激HPF-a细胞,设立si-NC组、si-circUCK2组、si-NC+TGF-β1处理组、si-circUCK2+TGF-β1处理组4组,Western blot法检测4组HPF-a细胞中纤连蛋白(FN1)表达情况,划痕实验检测HPF-a细胞迁移能力,CCK-8实验检测TGF-β1刺激的2组(si-NC+TGF-β1处理组和si-circUCK2+TGF-β1处理组)HPF-a细胞活性。在动物实验中,健康SPF级雄性C57BL/6小鼠48只,随机分为生理盐水+si-con组,生理盐水+si-circ_0000115组,SiO_(2)+si-con组,SiO2+si-circ_0000115组4组。mmu_circ_0000115是hsa_circUCK2的小鼠同源基因,气管滴注siRNA敲除小鼠肺circRNA mmu_circ_0000115,48 h后通过气管滴注SiO2悬液(0.2 g·kg^(-1),50 mg·mL^(-1))构建小鼠肺纤维化模型,实时荧光定量PCR检测小鼠各脏器中敲除效率,Western blot检测肺组织中I型胶原α2蛋白(COL1A2)表达情况,天狼星红检测肺组织中胶原合成情况。[结果]在细胞实验中,Western blot结果显示,敲除hsa_circUCK2可下调TGF-β1刺激的HPFa细胞中FN1的表达水平(P <0.05);CCK-8实验和细胞划痕实验结果显示,hsa_circUCK2基因的下调可以抑制HPF-a细胞的增殖和迁移(P <0.01)。在动物实验中,实时荧光定量PCR结果显示,在各脏器中只有肺组织中被显著敲除mmu_circ_0000115(P <0.000 1);Western blot结果表明,与SiO2+si-con组相比,敲低mmu_circ_0000115可以降低COL1A2蛋白表达水平(P <0.000 1);天狼星红结果显示,敲低mmu_circ_0000115后,模型组小鼠肺组织中胶原的产生和沉积减少。[结论]敲低hsa_circUCK2抑制成纤维细胞活化和减少肺纤维化模型小鼠中胶原沉积。提示hsa_circUCK2参与肺纤维化进程,可能是肺�[Background]Pulmonary fibrosis currently lacks screening and diagnostic methods in the early stages and effective treatments in the later stages,so there is an urgent need to explore the mechanisms and develop targeted treatments.[Objective]To screen the expression of differentially expressed circular RNA(circRNA)hsa_circUCK2 under pathological conditions and to explore its effect on pulmonary fibrosis.[Methods]In the cell-based experiments,hsa_circUCK2 was knocked down in HPF-a cells using small interfering RNA(siRNA),and HPF-a cells were stimulated by TGF-β1.Four groups were set up:si-NC group,si-circUCK2 group,si-NC+TGF-β1-treated group,and si-circUCK2+TGF-β1-treated group.Western blot assay was used to detect the expression of fibronectin(FN1)in HPF-a cells of the four groups,scratch assay was used to detect the migration ability of HPF-a cells,and CCK-8 assay was used to detect the proliferation ability of HPF-a cells in the two groups with TGF-β1 stimulation,the si-NC+TGF-β1-treated group and the si-circUCK2+TGF-β1-treated group.In the animal experiments,forty-eighty healthy SPF-grade male C57BL/6 mice were randomly divided into four groups:saline+si-con group,saline+si-circ_0000115 group,SiO2+si-con group,and SiO2+si-circ_0000115 group.Mouse lung circRNA mmu_circ_0000115(mouse homolog of hsa_circUCK2)was knocked down by tracheal drip injection of siRNA,and a mouse lung fibrosis model was constructed by tracheal drip injection of SiO_(2) suspension(0.2 g·kg^(−1),50 mg·mL^(−1))after 48 h.Real-time fluorescence quantitative PCR was used to detect the knockout efficiency in each organ of the mouse,Western blot was applied to detect the expression of type I collagenα2(COL1A2)in the lung tissues,and Sirius red was used to detect collagen synthesis in the lung tissues.[Results]In the cell-based experiments,after the knockdown of hsa_circUCK2,the Western blot results showed that the expression level of the FN1 protein in TGF-β1-stimulated HPF-a cells was significantly down-regulated(P<0.05);the CCK-8

关 键 词：环状RNA 转录组芯片 hsa_circUCK2 肺纤维化 人肺成纤维-成人细胞 

分 类 号：R114[医药卫生—卫生毒理学]