Wu Jing, Liu Shang, Yuan Zhi-Wei, Liu Jian-He, Li Jiong-Ming, Chen Tao, Fang Ke-Wei
Department of Biochemistry and Molecular Biology, the Basic Medical School of Kunming Medical University, Kunming, China.
Department of Urology, The Second Hospital of Kunming Medical University, Kunming, China.
Cell Physiol Biochem. 2018;45(4):1333-1349. doi: 10.1159/000487560. Epub 2018 Feb 15.
BACKGROUND/AIMS: We examined the effects of microRNA-27a (miR-27a) on detrusor fibrosis in streptozotocin (STZ)-induced diabetic rats.
Eighty healthy Sprague-Dawley (SD) rats were randomly allocated into control, diabetic, miR-27a mimics, mimics control, miR-27a inhibitors, inhibitors control, siRNA-PRKAA2 (siPRKAA2) and inhibitors + siPRKAA2 groups (the latter 7 groups were established as STZ-induced diabetic rat models and treated in different manners). Detrusor cell apoptosis in bladder tissues was determined through terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) staining. Detrusor cells were assigned to the blank, miR-27a mimics, mimics control, miR-27a inhibitors, inhibitors control, siPRKAA2 and inhibitors + siPRKAA2 groups. Flow cytometry determined the cell cycle stage and apoptosis. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting (WB) were used to assess the expression of miR-27a, PRKAA2, TGF-β1, Smad3, p-Smad3, fibronectin (FN), connective tissue growth (CTGF), and collagen-I (COL-I) in tissues and cells.
Compared with the control group, the diabetic, miR-27a mimics, and siPRKAA2 groups showed reduced weight and PRKAA2 expression, but elevated blood glucose, serum creatinine (sCr), blood urea nitrogen (BUN), cell apoptosis, and expression of TGF-β1, Smad3, FN, COL-I, CTGF, and p-Smad3. The opposite trend was observed in the miR-27a inhibitors group. PRKAA2 is a target gene of miR-27a. Compared to the blank group, the miR-27a mimics and siPRKAA2 groups indicated markedly increased TGF-β1, Smad3, FN, COL-I, CTGF and p-Smad3 expression; decreased PRKAA2 expression; and increased cell apoptosis. The miR-27a inhibitors group showed the opposite trend.
These results indicate that miR-27a may contribute to detrusor fibrosis in STZ-induced diabetic rats by targeting PRKAA2 via the TGF-β1/Smad3 signaling pathway.
背景/目的：我们研究了微小RNA-27a（miR-27a）对链脲佐菌素（STZ）诱导的糖尿病大鼠逼尿肌纤维化的影响。
将80只健康的Sprague-Dawley（SD）大鼠随机分为对照组、糖尿病组、miR-27a模拟物组、模拟物对照组、miR-27a抑制剂组、抑制剂对照组、siRNA-PRKAA2（siPRKAA2）组和抑制剂+siPRKAA2组（后7组建立为STZ诱导的糖尿病大鼠模型并采用不同方式处理）。通过末端脱氧核苷酸转移酶介导的dUTP-生物素缺口末端标记法（TUNEL）染色测定膀胱组织中逼尿肌细胞凋亡情况。将逼尿肌细胞分为空白组、miR-27a模拟物组、模拟物对照组、miR-27a抑制剂组、抑制剂对照组、siPRKAA2组和抑制剂+siPRKAA2组。流式细胞术测定细胞周期阶段和凋亡情况。采用定量逆转录聚合酶链反应（qRT-PCR）和蛋白质印迹法（WB）评估组织和细胞中miR-27a、PRKAA2、转化生长因子-β1（TGF-β1）、Smad3、磷酸化Smad3（p-Smad3）、纤连蛋白（FN）、结缔组织生长因子（CTGF）和I型胶原（COL-I）的表达。
与对照组相比，糖尿病组、miR-27a模拟物组和siPRKAA2组体重减轻、PRKAA2表达降低，但血糖、血清肌酐（sCr）、血尿素氮（BUN）、细胞凋亡以及TGF-β1、Smad3、FN、COL-I、CTGF和p-Smad3的表达升高。miR-27a抑制剂组观察到相反趋势。PRKAA2是miR-27a的靶基因。与空白组相比，miR-27a模拟物组和siPRKAA2组TGF-β1、Smad3、FN、COL-I、CTGF和p-Smad3表达明显增加；PRKAA2表达降低；细胞凋亡增加。miR-27a抑制剂组呈现相反趋势。
这些结果表明，miR-27a可能通过TGF-β1/Smad3信号通路靶向PRKAA2，从而导致STZ诱导的糖尿病大鼠逼尿肌纤维化。
