摘要
目的观察LncRNA HOX转录反向RNA(HOTAIR)对低氧条件下角质形成细胞低氧诱导因子-1α(HIF-1α)表达的影响,探讨LncRNA参与瘢痕疙瘩形成的可能机制。方法2018年1—12月,在中国医学科学院组织工程研究中心取传代培养的HaCat细胞分为常氧组、低氧组、sh-control组、sh-HOTAIR组。常氧组在常氧条件下培养,低氧组在低氧条件下培养,sh-control组转染sh-control后在低氧条件下培养,sh-HOTAIR组转染sh-HOTAIR后在低氧条件下培养。培养24 h后用双荧光素酶检测LncRNA HOTAIR与HIF-1α的作用关系,用蛋白印迹法检测HIF-1α蛋白表达。结果双荧光素酶检测显示常氧组、低氧组、sh-control组、sh-HOTAIR组荧光素酶相对活性分别为0.94±0.30、20.39±1.15、18.09±0.80、3.04±1.15,其中低氧组高于常氧组(t=29.03,P<0.05),而sh-HOTAIR组低于低氧组(t=18.57,P<0.05),差异均有统计学意义。蛋白印迹法显示低氧组、sh-control组HIF-1α蛋白表达高于常氧组、sh-HOTAIR组,分别为1.19±0.07、1.15±0.06、0.56±0.29、0.37±0.38。结论LncRNA HOTAIR参与低氧条件下角质形成细胞HIF-1α表达上调,LncRNA HOTAIR可能通过HIF-1α途径参与瘢痕疙瘩形成。
Objective To observe the effect of LncRNA HOTAIR on the expression of HIF-1αin HaCat cell under low oxygen condition,and to explore the role of LncRNA HOTAIR in the pathogenesis and development of keloid.Methods From Jan.2018 to Dec.2018 in Chinese Academy of Medical Science,recombinant plasmids were designed and constructed by specific shRNA-HOTAIR.After transfected HaCat cells with sh-LncRNA HOTAIR,RT-PCR was used to detect the expression level of HOTAIR.HaCat cells were cultured in different conditions and divided into four groups:group A cultured under normoxia condition,the other three groups cultured under hypoxia condition.Group C was transfected with sh-control before hypoxic culture,while group D were transfected with sh-LncRNA HOTAIR.After 24 hours′culture,dual luciferase reporter gene assay was used to verify the relationship of LncRNA HOTAIR and HIF-1α.Expression of HIF-1αin four groups of HaCat cells were determined by Western blot analysis.Results Recombinant plasmids of shRNA-HOTAIR were successfully constructed,and the HOTAIR expression was significantly decreased.The relative luciferase activity was 0.94±0.30 in group A,20.39±1.15 in group B,18.09±0.80 in group C and 3.04±1.15 in group D.The relative luciferase activity of group B was higher than those of group A(t=29.03,P<0.05),and the difference was statistically significant(P<0.05).According to the results of Western blotting,group B(1.19±0.07)and group D(1.15±0.06)had higher expression of HIF-1αthan group A(0.56±0.29)and group C(0.37±0.38).Down-regulation of HOTAIR significantly inhibited the protein level of HIF-1α.Conclusions LncRNA HOTAIR plays a positive role in upregulation of HIF-1αin HaCat cells under hypoxia condition.Thus LncRNA HOTAIR may take part in the pathogenesis and development of keloid through HIF-1αpathway.
作者
陈志雄
刘志飞
龙笑
王晓军
Chen Zhixiong;Liu Zhifei;Long Xiao;Wang Xiaojun(Department of Plastic Surgery,Peking Union Medical College Hospital of Chinese Academy of Medical Science and Peking Union Medical College,Beijing 100730,China)
出处
《中华医学美学美容杂志》
2022年第4期323-327,共5页
Chinese Journal of Medical Aesthetics and Cosmetology
基金
国家重点研发计划(2016YFA0101003)
北京市自然科学基金(7162156)。
