Title: Aging-associated changes in microRNA expression profile of internal anal sphincter smooth muscle: Role of microRNA-133a. Rattus norvegicus

A comprehensive –omic, computational, and physiological approach was employed to examine the (previously unexplored) role of microRNAs (miRNAs) as regulators of IAS smooth muscle contractile phenotype and basal tone. MicroRNA 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 up-regulated in aging IAS. qRT-PCR confirmed the up-regulated expression of these miRNAs and down regulation of multiple, predicted targets (Eln, Col3a1, Col1a1, Zeb2, Myocd, SRF, Smad1, Smad2, RhoA/ROCK2, Fn1, Sm22-v2, Klf4, and Acta2) involved in regulation of SM 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 A2 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. Lastly, 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 miR-133a is feasible therapeutic target in aging-associated rectoanal incontinence. Overall design: Three rats (Fischer 344) provided by the National Institutes of Aging of three different age groups (6 months, 18 months, and 26 months of age) were used for these studies. The smooth muscle cells (SMCs) from the internal anal sphincter (IAS) from above different age groups were isolated using collagenase dispersion method. The cells were subjected to RNA collection; and RNA from these samples were used for mRNA microarray analysis. For another sets of studies, from another batch of Fischer 344 rats (of 6 months, 18 months, and 26 months of age), the IAS SMCs were isolated and their miRNA fractions using miRVana miRNA Isolation kit following the manufacturer’s protocol were collected for miRNA microarray analysis.

本研究采用多组学、计算生物学与生理学相结合的综合研究策略，探究此前尚未阐明的微小核糖核酸（microRNAs, miRNAs）对肛门内括约肌（internal anal sphincter, IAS）平滑肌收缩表型与基础张力的调控作用。研究通过miRNA表达谱分析、全基因组表达分析、验证分析与调控网络分析，评估年轻与衰老大鼠肛门内括约肌平滑肌中mRNA与miRNA的表达变化。结果发现，衰老大鼠肛门内括约肌中存在多种上调表达的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及rno-miR-206。实时荧光定量聚合酶链反应（qRT-PCR）验证了上述miRNA的上调表达，同时证实多个参与平滑肌收缩调控的预测靶基因表达下调，这些靶基因包括Eln、Col3a1、Col1a1、Zeb2、Myocd、SRF、Smad1、Smad2、RhoA/ROCK2、Fn1、Sm22-v2、Klf4及Acta2。后续研究显示，衰老过程伴随miR-133a表达上调，同时伴随RhoA、ROCK2、MYOCD、SRF及SM22α蛋白表达水平下降，RhoA信号通路活性降低，且肛门内括约肌的基础张力以及激动剂U-46619（血栓烷A2类似物）诱导的张力升高均出现减弱。此外，体外转染miR-133a可呈剂量依赖性增强大鼠肛门内括约肌肌条的张力，该效应可被抗miR-133a寡核苷酸逆转。最后，体内肛周注射抗miR-133a寡核苷酸可逆转衰老相关的肛门内括约肌张力丧失。本研究证实了miR-133a对肛门内括约肌基础张力及激动剂诱导张力的重要调控作用。此外，通过递送抗miR寡核苷酸逆转衰老相关张力丧失的实验结果，强烈提示miRNA表达失调是衰老导致肛门内括约肌张力降低的关键诱因，同时表明miR-133a可作为衰老相关性直肠肛门失禁的潜在治疗靶点。

实验设计：本研究使用美国国家衰老研究所提供的费希尔344大鼠，涵盖3个不同年龄组（6月龄、18月龄及26月龄），每组3只。采用胶原酶分散法分离不同年龄组大鼠肛门内括约肌平滑肌细胞（smooth muscle cells, SMCs），收集细胞总RNA并用于mRNA基因芯片分析。另一组实验中，从另一批同年龄组（6月龄、18月龄及26月龄）的费希尔344大鼠中分离肛门内括约肌平滑肌细胞，按照制造商说明书使用miRVana miRNA分离试剂盒提取miRNA组分，用于miRNA基因芯片分析。