Wang Chenghe, Chen Zhong, Wu Jia, Zhang Yan, Hu Jia, Ge Qiangqiang, Yang Weimin, Xu Hua, Liu Jihong, Ye Zhangqun
Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, China.
Int Braz J Urol. 2015 Jul-Aug;41(4):764-72. doi: 10.1590/S1677-5538.IBJU.2014.0400.
RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double stranded RNAs (dsRNAs), also known as small activating RNAs (saRNAs). Myogenic regulatory factor MyoD is regarded as the master activator of myogenic differentiation cascade by binding to enhancer of muscle specific genes. Stress urinary incontinence (SUI) is a condition primarily resulted from urethral sphincter deficiency. It is thus expected that by promoting differentiation of adipose-derived stem cells (ADSCs) into myoblasts by activating MyoD gene through RNAa may offer benefits to SUI.
Rats ADSCs were isolated, proliferated in vitro, and identified by flow cytometry. Purified ADSCs were then transfected with a MyoD saRNA or control transfected. Real-time polymerase chain reaction (RT-PCR) and western blotting were used to detect MyoD mRNA and protein expression, respectively. Immunocytochemical staining was applied to determine the expression of desmin protein in transfected cells. Cell viability was measured by using CellTiter 96R AQueous One Solution Cell Proliferation Assay kit.
Transfection of a MyoD saRNA (dsMyoD) into ADSCs significantly induced the expression of MyoD at both the mRNA and protein levels, and inhibited cell proliferation. Desmin protein expression was detected in dsMyoD treated ADSCs 2 weeks later.
Our findings show that RNAa mediated overexpression of MyoD can promote transdifferentiation of ADSCs into myoblasts and may help treat stress urinary incontinence (SUI)-a condition primarily resulted from urethral sphincter deficiency.
RNA激活（RNAa）是一种由靶向启动子的小双链RNA（dsRNA）触发的基因激活机制，也被称为小激活RNA（saRNA）。成肌调节因子MyoD通过与肌肉特异性基因的增强子结合，被视为成肌分化级联反应的主要激活因子。压力性尿失禁（SUI）主要是由尿道括约肌缺陷引起的一种病症。因此，预期通过RNAa激活MyoD基因来促进脂肪来源干细胞（ADSCs）向成肌细胞分化可能对SUI有益。
分离大鼠ADSCs，在体外进行增殖，并通过流式细胞术进行鉴定。然后将纯化的ADSCs用MyoD saRNA转染或进行对照转染。分别使用实时聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测MyoD mRNA和蛋白质表达。应用免疫细胞化学染色来确定转染细胞中结蛋白的表达。使用CellTiter 96R AQueous One Solution Cell Proliferation Assay试剂盒测量细胞活力。
将MyoD saRNA（dsMyoD）转染到ADSCs中，在mRNA和蛋白质水平上均显著诱导了MyoD的表达，并抑制了细胞增殖。2周后在dsMyoD处理的ADSCs中检测到结蛋白表达。
我们的研究结果表明，RNAa介导的MyoD过表达可促进ADSCs向成肌细胞的转分化，并可能有助于治疗主要由尿道括约肌缺陷引起的压力性尿失禁（SUI）。
