长链非编码RNA Hotair在CKD小鼠肾脏纤维化中的作用及机制研究  

Effect of long non-coding RNA Hotair in renal fibrosis of CKD mice and its mechanism

作　　者：刘琳[1] 任燕[1] 陈茂盛[1] 王敏敏[1] LIU Lin;REN Yan;CHEN Maosheng;WANG Minmin(Department of Nephrology,Zhejiang Provincial People's Hospital(People's Hospital of Hangzhou Medical College),Hangzhou 310014,China)

机构地区：[1]浙江省人民医院(杭州医学院附属人民医院)肾脏病科,杭州310014

出　　处：《浙江医学》2023年第22期2357-2362,I0003,共7页Zhejiang Medical Journal

基　　金：浙江省自然科学基金项目(LQ19H050003)。

摘　　要：目的探讨长链非编码RNA(lncRNA)Hotair在慢性肾脏病(CKD)小鼠模型肾脏纤维化中的作用及其机制。方法采用随机数字表法将36只小鼠分为对照组、CKD组、CKD+短发夹RNA-对照(sh-Ctrl)组、CKD+短发夹RNA-Hotair(shHotair)组,每组9只。对照组喂食正常饲料,其余3组均予0.2%腺嘌呤饲料喂养造模。造模6周后,CKD+sh-Ctrl组和CKD+shHotair组分别予尾静脉注射100μL(10^(11)基因拷贝)包装了对照shRNA和Hotair-shRNA的腺相关病毒,对照组和CKD组予尾静脉注射100μL 0.9%氯化钠注射液。观察8周后处死,检测血清肌酐、尿素氮水平,取肾组织行HE染色和Masson染色观察肾脏纤维化情况。采用qRT-PCR法检测肾皮质Hotair、多梳抑制复合体2(PRC2)复合物[Zeste增强子同源物2(EZH2)、Zeste12同源物抑制因子(SUZ12)、胚胎外胚层发育蛋白(EED)]和赖氨酸特异性去甲基化酶1(LSD1)mRNA表达水平,Western blot法检测肾皮质纤维化相关蛋白胶原蛋白1A1(COL1A1)、胶原蛋白4A1(COL4A1)、α-平滑肌肌动蛋白(α-SMA)和E-钙黏蛋白(E-cadherin)的表达水平,并检测PRC2复合物和LSD1的蛋白表达水平。结果与对照组比较,CKD组小鼠体重下降、肾功能恶化,出现肾脏纤维化,肾皮质Hotair表达升高;与CKD+sh-Ctrl组比较,CKD+sh-Hotair组小鼠血清肌酐、尿素氮水平均降低(均P<0.01),病理检查显示肾脏纤维化程度减轻,肾皮质Hotair表达下降(P<0.01)。与对照组比较,CKD组小鼠COL1A1、COL4A1和α-SMA蛋白表达水平均增加,E-cadherin表达水平减少,PRC2复合物中EZH2、EED和LSD1 m RNA和蛋白表达水平均升高(均P<0.01)。与CKD+sh-Ctrl组比较,CKD+sh-Hotair组小鼠COL1A1、COL4A1和α-SMA蛋白表达水平均降低,E-cadherin蛋白表达水平恢复,PRC2复合物中EZH2、EED和LSD1 mRNA和蛋白表达水平均下降(均P<0.01)。结论lncRNA Hotair在CKD小鼠模型中表达升高,敲低Hotair可缓解肾脏纤维化,可能与Hotair作为PRC2和LSD1的分子支架介导的表观遗传修�Objective To explore the effect of long non-coding RNA(lncRNA)Hotair in renal fibrosis of mice with chronic kidney disease(CKD)and its mechanism.Methods A total of 36 mice were randomly divided into control group,CKD group,CKD+sh-Ctrl group,CKD+sh-Hotair group,with 9 mice in each group.The control group was fed with normal diet,and the other groups were fed with 0.2%adenine diet.After 6 weeks of modeling,the CKD+sh-Ctrl group and CKD+sh-Hotair group were injected with 100μL of adeno-associated virus(10^(11) genome copies/mice)packaged with control shRNA and Hotair-shRNA through the tail vein,respectively.The control group and CKD group were injected with 100μL of 0.9%sodium chloride through the tail vein.After 8 weeks of observation,mice were sacrificed.Serum creatinine and urea nitrogen were tested.HE and Masson staining were uesd to test renal fibrosis.qRT-PCR was conducted to detect the mRNA expression of Hotair,polycomb repressive complex 2(PRC2)[enhancer of Zeste homolog 2(EZH2),suppressor of Zeste 12 homolog(SUZ12),and embryonic ectoderm development(EED)],and histone lysine-specific demethylase 1(LSD1).Western blot was used to detect the protein levels of renal cortex fibrosis-related proteins collagen Iα1(COL1A1),collagenⅣα1(COL4A1),α-smooth muscle actin(α-SMA),and E-cadherin,as well as the protein levels of PRC2 complex and LSD1.Results Compared with the control group,mice in CKD group showed weight loss,deterioration of kidney function,renal fibrosis and elevation of Hotair in renal cortex.Compared with the CKD+sh-Ctrl group,the serum creatinine and urea nitrogen of mice in the CKD+sh-Hotair group were significantly reduced(both P<0.01),and the degree of renal fibrosis was significantly alleviated with decreased Hotair expression in renal cortex according to pathological examination(P<0.01).Compared with the control group,increased levels of fibrosis-related proteins COL1A1,COL4A1 andα-SMA,and decreased levels of E-cadherin presented in renal cotex of mice in CKD group,while the mRNA expression

关 键 词：长链非编码RNA Hotair 慢性肾脏病 纤维化 PRC2复合物 

分 类 号：R692[医药卫生—泌尿科学]