Singh Jagmohan, Boopathi Ettickan, Addya Sankar, Phillips Benjamin, Rigoutsos Isidore, Penn Raymond B, Rattan Satish
Department of Medicine, Division of Gastroenterology & Hepatology, Sidney Kimmel Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania.
Center for Translational Medicine, Sidney Kimmel Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania.
Am J Physiol Gastrointest Liver Physiol. 2016 Nov 1;311(5):G964-G973. doi: 10.1152/ajpgi.00290.2016. Epub 2016 Sep 15.
A comprehensive genomic and proteomic, computational, and physiological approach was employed to examine the (previously unexplored) role of microRNAs (miRNAs) as regulators of internal anal sphincter (IAS) smooth muscle contractile phenotype and basal tone. miRNA profiling, genome-wide expression, validation, and network analyses were employed to assess changes in mRNA and miRNA expression in IAS smooth muscles from young vs. aging rats. Multiple miRNAs, including rno-miR-1, rno-miR-340-5p, rno-miR-185, rno-miR-199a-3p, rno-miR-200c, rno-miR-200b, rno-miR-31, rno-miR-133a, and rno-miR-206, were found to be upregulated in aging IAS. qPCR confirmed the upregulated expression of these miRNAs and downregulation of multiple, predicted targets (Eln, Col3a1, Col1a1, Zeb2, Myocd, Srf, Smad1, Smad2, Rhoa/Rock2, Fn1, Tagln v2, Klf4, and Acta2) involved in regulation of smooth muscle contractility. Subsequent studies demonstrated an aging-associated increase in the expression of miR-133a, corresponding decreases in RhoA, ROCK2, MYOCD, SRF, and SM22α protein expression, RhoA-signaling, and a decrease in basal and agonist [U-46619 (thromboxane A analog)]-induced increase in the IAS tone. Moreover, in vitro transfection of miR-133a caused a dose-dependent increase of IAS tone in strips, which was reversed by anti-miR-133a. Last, in vivo perianal injection of anti-miR-133a reversed the loss of IAS tone associated with age. This work establishes the important regulatory effect of miRNA-133a on basal and agonist-stimulated IAS tone. Moreover, reversal of age-associated loss of tone via anti-miR delivery strongly implicates miR dysregulation as a causal factor in the aging-associated decrease in IAS tone and suggests that miR-133a is a feasible therapeutic target in aging-associated rectoanal incontinence.
采用综合的基因组学、蛋白质组学、计算生物学和生理学方法，研究微小RNA（miRNA）作为内括约肌（IAS）平滑肌收缩表型和基础张力调节因子（此前未被探索）的作用。采用miRNA谱分析、全基因组表达分析、验证和网络分析，评估年轻大鼠与老年大鼠IAS平滑肌中mRNA和miRNA表达的变化。发现多种miRNA，包括rno-miR-1、rno-miR-340-5p、rno-miR-185、rno-miR-199a-3p、rno-miR-200c、rno-miR-200b、rno-miR-31、rno-miR-133a和rno-miR-206，在老年IAS中上调。qPCR证实了这些miRNA的表达上调以及多个预测靶点（Eln、Col3a1、Col1a1、Zeb2、Myocd、Srf、Smad1、Smad2、Rhoa/Rock2、Fn1、Tagln v2、Klf4和Acta2）的下调，这些靶点参与平滑肌收缩的调节。随后的研究表明，与衰老相关的miR-133a表达增加，RhoA、ROCK2、MYOCD、SRF和SM22α蛋白表达相应降低，RhoA信号传导减少，基础张力和激动剂[U-46619（血栓素A类似物）]诱导的IAS张力增加减少。此外，miR-133a的体外转染导致条带中IAS张力呈剂量依赖性增加，而抗miR-133a可逆转这种增加。最后，在体内经肛门注射抗miR-133a可逆转与年龄相关的IAS张力丧失。这项工作确立了miRNA-133a对基础和激动剂刺激的IAS张力的重要调节作用。此外，通过抗miR递送逆转与年龄相关的张力丧失强烈表明miR失调是IAS张力与衰老相关降低的因果因素，并表明miR-133a是衰老相关直肠肛门失禁的可行治疗靶点。
