摘要
背景微小RNA-107(miRNA-107)在多种肿瘤(如膀胱癌、乳腺癌)的发生发展过程中具有重要作用,但其在过敏性哮喘发生发展中的作用尚不完全清楚。目的探讨miRNA-107对气道平滑肌细胞(ASMCs)增殖和迁移的影响。方法本研究时间为2019年9月—2020年12月。原代培养SPF级雌性BABL/c小鼠的ASMCs,运用免疫荧光鉴定ASMCs。将细胞随机分为miRNA-107模拟物组(miRNA-107 mimic组)、模拟物对照组(mimic NC组)和miRNA-107抑制剂组(miRNA-107 inhibitor组)、抑制剂对照组(inhibitor NC组)。miRNA-107 mimic组和mimic NC组的细胞分别转染5μl 20μmol/L的miRNA-107 mimic或5μl 20μmol/L的mimic NC+7.5μl viafect+200μl Opti-MEM无血清培养基;miRNA-107 inhibitor组和inhibitor NC组的细胞分别转染10μl 20μmol/L的miRNA-107 inhibitor或10μl 20μmol/L的inhibitor NC+15μl viafect+200μl Opti-MEM无血清培养基。采用荧光定量试剂盒检测各组ASMCs中miRNA-107相对表达水平,BrdU细胞增殖检测试剂盒(比色分析法)检测各组细胞增殖能力(吸光度差值),穿梭小室检测各组细胞迁移能力(迁移细胞数)。分别比较miRNA-107 mimic组与mimic NC组、miRNA-107 inhibitor组与inhibitor NC组ASMCs中miRNA-107相对表达水平、细胞增殖能力和细胞迁移能力。结果ASMCs从组织块周围呈放射状萌出,7 d后逐渐长满培养瓶,呈谷峰状;首次传代后,ASMCs呈梭形,多有突起,生长致密且平行排列;培养2代后的ASMCs经免疫荧光鉴定后有绿色荧光;经鉴定,99%以上的细胞均是ASMCs。miRNA-107 mimic组ASMCs中miRNA-107相对表达水平高于mimic NC组,吸光度差值、迁移细胞数低于mimic NC组(P<0.05)。miRNA-107 inhibitor组ASMCs中miRNA-107相对表达水平低于inhibitor NC组,吸光度差值、迁移细胞数高于inhibitor NC组(P<0.05)。结论miRNA-107可以抑制ASMCs的增殖和迁移。抑制miRNA-107的表达可以促进ASMCs的增殖和迁移,这可能是哮喘患者发生气道重塑的机制之一。因此,miRNA-107有望作为抑制气道重塑的一个靶点,为哮喘的治疗提供新的方法。
Background It is confirmed that microRNA-107(miRNA-107)plays an important role in the occurrence and development of kinds of tumors(such as bladder cancer and breast cancer),however its role in the occurrence and development of allergic asthma is not completely clear so far.Objective To investigate the effect of miRNA-107 on the proliferation and migration of airway smooth muscle cells(ASMCs).Methods The study time was from September 2019 to December 2020.The primary ASMCs of SPF female BABL/c mice were cultured,and the ASMCs were identified by immunofluorescence.The cells were randomly divided into miRNA-107 mimic group(miRNA-107 mimic group),mimic control group(mimic NC group)and miRNA-107 inhibitor group(miRNA-107 inhibitor group),inhibitor control group(inhibitor NC group),respectively.Cells in miRNA-107 mimic group and mimic NC group were transfected with 5μl 20μmol/L miRNA-107 mimic or 5μl 20μmol/L mimic NC+7.5μl viafect+200μl Opti-MEM serum-free medium;cells in the miRNA-107 inhibitor group and inhibitor NC group were transfected with 10μl 20μmol/L miRNA-107 inhibitor or 10μl 20μmol/L inhibitor NC+15μl viafect+200μl Opti-MEM serum-free medium.Fluorescence quantitative kit was used to detect the relative expression level of miRNA-107 in each group of ASMCs,BrdU cell proliferation detection kit(colorimetric analysis method)was used to detect the cell proliferation ability(absorbance difference)of each group,and the transwell chamber was used to detect the cell migration ability(number of migrating cells)of each group.The relative expression levels of miRNA-107 in ASMCs,cell proliferation and cell migration of miRNA-107 mimic group and mimic NC group,miRNA-107 inhibitor group and inhibitor NC group were compared,respectively.Results ASMCs sprouted radially from around the tissue mass,and gradually overgrown the culture flask after 7 days,showing a valley peak shape;after the first passage,ASMCs were fusiform,with many protrusions,dense growth and parallel arrangement;ASMCs cultured for 2 generations showed green fluorescence after being identified by immunofluorescence.It had been identified that more than 99%of the cells were ASMCs.The relative expression level of miRNA-107 in ASMCs in the miRNA-107 mimic group was higher than that in the mimic NC group,and the absorbance difference and number of migrating cells were lower than those in the mimic NC group(P<0.05).The relative expression level of miRNA-107 in the ASMCs of the miRNA-107 inhibitor group was lower than that of the inhibitor NC group,and the absorbance difference and numben of migrating cells were higher than those of the inhibitor NC group(P<0.05).Conclusion miRNA-107 can inhibit the proliferation and migration of ASMCs.Inhibiting the expression of miRNA-107 can promote the proliferation and migration of ASMCs,which may be one of the mechanisms of airway remodeling in asthma patients.Therefore,miRNA-107 is expected to serve as a target to inhibit airway remodeling and provide a new method for the treatment of asthma.
作者
宇文婷
罗雅妮
黎满慧
史菲
熊轶
YUWEN Ting;LUO Yani;LIManhui;SHI Fei;XIONG Yi(Biomedical Research Institute,Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center,Shenzhen 518036,China;Shenzhen People's Hospital/Shenzhen Institute of Respiratory Diseases,Shenzhen 518020,China)
出处
《实用心脑肺血管病杂志》
2021年第6期68-72,共5页
Practical Journal of Cardiac Cerebral Pneumal and Vascular Disease
基金
广东省自然科学基金资助项目(2020A1515011040)。
