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Alterations in microRNA Expression in a Murine Model of Diet-Induced Vasculogenic Erectile Dysfunction Carlos E. Barbery, BS,* Frank A. Celigoj, MD,* Stephen D. Turner, PhD,† Ryan P. Smith, MD,* Parviz K. Kavoussi, MD,‡ Brian H. Annex, MD,§ and Jeffrey J. Lysiak, PhD* *Department of Urology, University of Virginia, Charlottesville, VA, USA; †Department of Public Health Sciences, University of Virginia, Charlottesville, VA, USA; ‡Department of Austin Center for Men’s Health Clinic, University of Virginia, Charlottesville, VA, USA; §Department of Cardiovascular Medicine, University of Virginia, Charlottesville, VA, USA DOI: 10.1111/jsm.12793

ABSTRACT

Introduction. MicroRNAs (miRs) are noncoding, endogenous RNA molecules that regulate gene expression and play roles in response to vascular injury. Aim. The aim of this study was to identify miRs expressed in corporal tissue (CT) and to determine whether miRs demonstrate differential expression in a mouse model of diet-induced erectile dysfunction (ED). Methods. RNA was isolated from the CT from control mice and mice with diet-induced ED. A quantifiable miR profiling technique (NanoString) was used to determine the expression of over 600 miRs. Main Outcome Measures. Differential expression analysis was performed using a negative binomial regression model for count-based data. Mean expression levels, fold change, and false discovery-corrected P values were determined. Candidate miRs were validated via quantitative polymerase chain reaction (Q-PCR). Results. In control mice, NanoString analysis revealed that 181 miRs were expressed above background levels and 5 miRs were expressed at high levels. Diet-induced ED resulted in the up-regulation of 6 miRs and the downregulation of 65 miRs in the CT compared with mice on control diet. Focusing on the upregulated miRs, we chose five for Q-PCR validation. Of these five, two (miR-151-5p and miR-1937c) demonstrated significance via Q-PCR, whereas the other three (miR-720, miR-1937a, miR-205) trended in the correct direction. Conclusions. MiRs may play a significant role in mRNA regulation in CT and specific miRs may be involved in diet-induced vasculogenic ED. Future studies are aimed at determining the mRNA targets of these miRs. Barbery CE, Celigoj FA, Turner SD, Smith RP, Kavoussi PK, Annex BH, and Lysiak JJ. Alterations in microRNA expression in a murine model of diet-induced vasculogenic erectile dysfunction. J Sex Med 2015;12:621–630. Key Words. Erectile Dysfunction; microRNA; NanoString; Diet-Induced ED

Introduction

O

ur understanding of the role and function of microRNA (miR) has grown exponentially in the past years. It is now estimated that miRs regulate the expression in over 60% of mRNA transcripts in the human genome [1]. The central dogma of protein expression and gene regulation has been overhauled by these small, noncoding

© 2014 International Society for Sexual Medicine

RNAs. miRs begin as long precursor molecules that undergo essential processing catalyzed by enzymes such as Drosha and Dicer before reaching their mature forms [2]. Once in the cytoplasm, they associate with the RNA-induced silencing complex that facilitates their interaction with target mRNA molecules. By binding to the 3′-untranslated region of target mRNAs, miRs can inhibit their translation through either causing J Sex Med 2015;12:621–630

622 degradation of the mRNA, repressing translation of the mRNA, or deadenylating the mRNA [2]. While some studies have shown that miRs can cause disease, they appear to have a greater role as a modifier of disease severity and thus miRs can serve as therapeutic agents [3]. A somewhat unique property of miRs lies in their ability to regulate the expression of several functionally related genes, thus possibly affecting an entire biologic pathway [4]. Additionally, some miRs have been shown to be stable in plasma when bound to specific carrier molecules, such as Argonaut or high-density lipoprotein, and have been suggested to be biomarkers for certain disease states [5]. The role of miRs in the context of benign urological disease, specifically erectile dysfunction (ED), remains limited [6]. Current estimates place the number of men with ED in the United States to be greater than 30 million [7]. When considering the entire population of men with ED, a sizeable fraction will have diet-induced ED. Obesity, hyperglycemia, hyperinsulinemia (i.e., insulin resistance), and type 2 diabetes mellitus (DM) have all been linked to a high-fat diet (HFD) and all are contributing factors to ED [8]. The overwhelming majority of preclinical models of DM-induced ED use approaches where hyperglycemia is present, but hyperglycemia is often not associated with obesity and is not associated with insulin resistance. For example, when hyperglycemia is induced by streptozotocin injection [9] or using the Akita genetic model [10], ED will be observed; however, obesity is not observed. Thus, most preclinical studies of DM-induced ED fail to incorporate some of the most essential aspects of the human condition of ED in adult onset DM, which includes not only hyperglycemia but obesity and insulin resistance. Studies from our group have shown that feeding C57BL/6 mice with an HFD where 45% of its daily calories come from fat caused obesity, hyperglycemia, and insulin resistance. By examining the corpus cavernosum in these mice, we found that a number of findings were consistent with ED including: (i) abnormalities in corporal endothelium-dependent and endotheliumindependent vasoreactivity; (ii) a decrease in the ratio of the smooth muscle to collagen content; (iii) a reduction in NADPH diaphorase staining (measure of bioavailable NO); and (iv) increases in apoptosis, as measured by TUNEL staining [11,12]. When these results were compared with J Sex Med 2015;12:621–630

Barbery et al. information in other modes of ED (type 1 DM [13] and hypercholesterolemia [14]), it appeared that some of the mechanisms for the HFDinduced vascular ED were different from findings in these other models of ED [13,14]. Aims

By employing a quantifiable miR profiling technique termed nCounter® miR Expression Assay (NanoString Technology, Inc., Seattle, WA, USA), the current study aims to identify miRs that are expressed in the corporal tissue (CT) as well as miRs that are differentially expressed in the CT of mice with diet-induced ED compared with control normal mice. Through the identification of differentially expressed miRs and their respective targets, we will gain a better understanding of the pathophysiologic mechanisms underlying dietinduced ED as well as develop new therapeutic targets. Methods

Murine Model of Diet-Induced Vasculogenic ED Animal studies were approved by the Institutional Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health. Mice were fed freely and maintained on a 12-hour dark–light cycle. Our laboratory has previously reported that mice fed with a diet consisting of 60% fat will develop ED compared with mice on normal chow diet [12,15]. C57Bl/6J mice were put onto a 60% HFD (Jackson Laboratories, Bar Harbor, ME, USA) at 6 weeks of age and remained on the HFD for 22 weeks. Age-matched control mice were placed on normal chow diet. Validations of the Murine Model System At 28 weeks of age, the mice were fasted for ∼16 hours and placed on Sani-Chip bedding. They were weighed and injected intraperitoneally with D-glucose (1 g/kg body weight; Sigma, St. Louis, MO, USA); for example, for a 25-g mouse, 0.1 mL was injected and for a 40-g mouse, 0.16 mL was injected. Blood samples were then taken at various time points (0–120 minutes) from the tail vein. Glucose measures were made using a glucometer (One Touch Profile meter; LifeScan, Milpitas, CA, USA). Endothelium-dependent relaxation was assessed using acetylcholine (Ach) on corporal

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miRs in Diet-Induced ED strips, as previously described by our group [12]. In brief, penises (n = 6 per group) were excised from deeply anesthetized mice, transferred to a Petri dish with ice-cold Krebs solution then fixed on slices of Modern Materials Modern Pink NO.3 Wax from Bayer (Whippany, NJ, USA). The penises were fixed by needle tips from Precision Glide Needle 30G1/2 then microscopically dissected to remove the tunica albuginea. The corpora spongiosa and the dorsal penile vessels and nerves were dissected from the corpora cavernosa. The corpora were then dissected longitudinally in the middle to get two segments and two sutures of 4.0 Sofsilk were fixed at either end. The segments were then transferred to a 20-mL organ bath attached to Radnoti Channel Tissue Bath System (Monrovia, CA, USA) with a 2-L reservoir and Radnoti Heater Circulating Pump (17001A). Changes in contractions were transferred through a transducer (model no. MP100A; Biopac System, Goleta, CA, USA) and recorded using AcqKnowledge 3.9.1 software (Biopac System). The bath was filled with Krebs solution (prepared as 10× from NaCl, KCl, MgSO4, KH2PO4, and CaCl2 and diluted to 1× just before usage by adding NaHCO3 25 mM, glucose 11 mM, and EDTA 0.03 mM) at 37°C bubbled with 95% O2 and 5% CO2, and stretched at a 1-mN tension. The tension was increased gradually to 2.5 mN for 60 minutes then released back to 1 mN and set to equilibrate for 15 minutes. Endothelial-dependent relaxation was tested by cumulatively adding ACh [10−9] M to [3 × 10−6] M to the medium of the precontracted segments with phenylephrine [10−5] M for 10 minutes. Intracorporal pressures (ICPs) were obtained before and during cavernosal nerve electrical stimulation (CNES) in both HFD-fed and control mice (n = 7 per group). Baseline ICPs and CNES ICPs were performed as previously described [15]. Briefly, under anesthesia, the cavernous nerves were exposed. A heparinized (100 U/mL) 25-gauge needle attached to PE-30 tubing, connected to a BD™ pressure transducer (BD™, Franklin Lakes, NJ, USA), was inserted into the cavernous tissue to monitor ICPs. CNES was performed with an A-M Systems, Inc. 2100 isolated pulse generator (Carlsborg, WA, USA) using 0.2-second pulses of 1.5-mA current at a rate of 20 Hz. The ICPs were amplified and recorded using the PolyView 2.1 data acquisition and analysis software system (Grass Technologies, West Warwick, RI, USA).

Main Outcomes Measured

miR Microarray Analysis CTs were collected from C57Bl/6J mice that were either consuming normal chow (control) or an HFD (n = 3 per group). The HFD-fed mice were on the diet for 22 weeks and controls were agematched. RNA was isolated with a TRIzol Total Transcriptome Isolation protocol and PureLink RNA mini kits according to the manufacturer’s instructions (Life Technologies, Carlsbad, CA, USA). An unbiased miR profiling technique termed nCounter® miR Expression Assay (NanoString Technologies) was used to determine the expression profiles of 614 different miRs. NanoString’s nCounter® uses a novel digital color-coded barcode technology, with each colorcoded barcode attached to a single target-specific probe corresponding to the miR of interest. This multiplexed measurement offers high levels of precision and sensitivity (>1 copy per cell). The miR nCounter® assay allows for highly multiplexed, direct digital detection, and counting of miRs in a single reaction without amplification. This assay system, at single-base resolution, can accurately distinguish between highly similar miRs with extreme specificity and sensitivity and requires no amplification that might introduce bias into the results. Statistical Analysis of NanoString Array Data NanoStringNorm R package was used to normalize the raw data. The method recommended by NanoString Technologies, sensible defaults, was utilized. The data were then subjected to variance stabilizing transformation, which facilitated the implementation of exploratory analysis. Subsequent principal component analysis, heat map and sample matrix generation, and volcano plot were generated. NanoString data are digital, discrete, count-based data, not a continuously distributed data like microarray intensity data. Therefore, differential expression analysis was performed using a negative binomial regression model for countbased data. Overall mean across all samples, the mean for controls, HFD, fold change in expression between groups, P value, and false discover rate (FDR)-corrected P value were generated. Validation of miR Expression by Quantitative Polymerase Chain Reaction In a separate series of experiments, we chose to validate the microarray expression data of randomly selected miRs. RNA was isolated from the J Sex Med 2015;12:621–630

624 CT of mice on HFD for 22 weeks and agematched control mice on normal chow diet (n = 12 per group). Five miRs that were upregulated, four that were downregulated, and four that were unchanged according to the miR microarray NanoString assay were chosen at random and were validated via quantitative polymerase chain reaction (Q-PCR). Taqman primer/probes for each respective candidate miR were utilized to validate the expression levels (Life Technologies, Grand Island, NY, USA).

Limitations of Methods In the current study, we employed a late-stage model of diet-induced ED; indeed, in another model of diabetes-induced ED, changes in gene expression were noted as early as 8 weeks [16]. Because a single time point was used to examine miR expression, a time on diet dependence change in miR expression could not be determined. Also, by study design, only a single ICP recording could be performed on an individual animal. The nCounter® miR Expression Assay enabled us to examine the expression of 614 different miRs in the CT. At the time of writing this manuscript, limited information exists on the functions of many of these miRs. Statistical Analysis Two-group analysis was performed by the paired two-sample for means t-test, using Microsoft Excel. Data are expressed as mean ± standard error of the mean. Results

Validation That Feeding Mice with an HFD Induces Vasculogenic ED Previous studies from our laboratory have found that feeding mice with an HFD causes obesity, increases blood glucose levels, and leads to a decrease in ICPs following cavernous nerve stimulation and a decrease in vasoreactivity [12,15]. In the current study, to ensure we were isolating miRs from mice with ED, we confirmed these previous results. Significantly higher levels of blood glucose were achieved 2 hours postglucose challenge, 208 ± 5.9, in mice fed with an HFD compared with 138 ± 3.8 in control mice (Figure 1A). Also, a significant decrease in the change of ICP from baseline to tumescence was observed, 7.3 ± 0.9 mm Hg, in mice fed with an HFD compared with 17.9 ± 1.3 mm Hg in ageJ Sex Med 2015;12:621–630

Barbery et al. matched control mice (Figure 1B). Vasoreactivity studies revealed that mice on the HFD had significantly impaired endothelial-dependent vasoreactivity (Figure 1C). Taken together, these measures confirm that the mice fed with an HFD for 22 weeks develop ED.

Profile of Baseline miR Expression in C57BL/6 Mice A total of 602 miRs were evaluated by the NanoString microarray. The array generated count-based expression levels and had intrinsic load and background controls. In the CT of normal control C57BL/6 mice, 181 of the 602 miRs, thus 30%, were expressed above background levels. Figure 2 displays the number of miRs according to their relative expression level. Examples of miR with relatively high expression values in the normal CT are miR-125b-5p, miR145, miR-125a-5p, miR-720, and let-7b (Table 1). miRs Differentially Expressed in the CT of Mice With Diet-Induced ED Compared with Control Mice Differential expression analysis between miR expression from control CT and from the CT of mice fed with an HFD revealed a general downregulation of miR expression in mice fed with the HFD. Sixty-five miRs were downregulated with a fold change greater than 1 in the CT of mice with diet-induced ED, whereas only five miRs were increased with a fold change greater than 1 and a miR with a fold change greater than 2 in expression. Figure 3 displays a volcano plot, which plots miRs as a function of both FDR-corrected P value and log2-fold change in expression between the two conditions. Five miRs that demonstrated an increase in expression in the CT of HFD-fed mice were miR-720, miR-1937a, miR-1937c, miR205, and miR-151-5p. Also noteworthy is that three miRs went from below background values to above background in the HFD-fed group; they were miR-15b, let-7c, and miR-1944. Examples of miRs that were downregulated are miR-550, miR425, miR-134, miR-153, and miR-26b. Validation of Microarray by Q-PCR To validate the miR microarray results, we performed a separate series of experiments whereby five random upregulated miRs, four random downregulated miRs, and four random unchanged miRs (fold change difference less than 1) were chosen for Q-PCR validation from CT from 12 control mice and 12 mice fed with an HFD for 22 weeks. The five upregulated miRs were miR-720,

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Figure 1 Validations that an HFD induces vasculogenic ED. (A) Glucose tolerance testing revealed significantly higher blood glucose levels in mice fed with an HFD compared with mice fed with a normal chow diet. n = 6 per group. (B) Electrical stimulation of the cavernous nerve and ICP monitoring revealed that mice fed with an HFD had a significantly lower maximal ICP than mice fed with normal chow. n = 7 per group. (C) Mice fed with an HFD had impaired endothelium-dependent vasoreactivity compared with mice fed with normal chow. Asterisk indicates P < 0.05, n = 6 per group. ED = erectile dysfunction; HFD = high-fat diet; ICP = intracorporal pressure

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Barbery et al. establish the NanoString array as a useful and effective method for identifying differentially expressed miRs. Conclusions

Figure 2 Relative expression of miRs in corporal tissue. Of the 602 miRs evaluated from RNA from the corporal tissue, 181 were expressed at levels above background. The histogram displays the number of miRs at different expression levels. n = 3. miR = microRNA

miR-1937a, miR-1937c, miR-205, and miR-1515p. The four downregulated miRs were miR-26b, miR-425, miR-134, and miR-153, and the four unchanged miRs were miR-125b, miR-541, miR214, and miR-149. Of the five miRs that were upregulated in the microarray data, all trended to be upregulated via Q-PCR, but only miR-1937c and miR-151-5p were significantly upregulated in the CT of HFD mice compared with control (Figure 4A). Three out of the four miRs trended to be downregulated in the Q-PCR experiment compared with the microarray data and of these three miRs, miR-153 and miR-425 were significantly downregulated in the CT of HFD mice when compared with control (Figure 4B). With regard to the miRs that were identified as unchanged in the microarray data, all four of these miRs were also unchanged as analyzed by Q-PCR (Figure 4C). These results help

Table 1

According to the Centers for Disease Control and Prevention, data from 2010 indicated that over 37.5 million, approximately 36%, of American men over the age of 20 are obese. Alarmingly, approximately 7 million, almost 19% of boys in the United States, are also obese. It is predicted that these numbers will continue to grow as males continue on a Western diet and sedimentary lifestyle. Thus, a substantial number of American men suffer from diet-induced ED [8,20]. Studies from our laboratory have developed and characterized a preclinical murine model of diet-induced ED that takes into account many factors seen in the human population [12,15]. We sought to examine miRs in diet-induced ED. To gain insight into the possible mechanisms of diet-induced ED, we now report that specific miRs are differentially expressed in the CT of mice with diet-induced ED and further studies are necessary to determine if specific miRs may contribute to the pathology and offer possible new therapeutic options. In the current study, we employed an nCounter® miR expression to provide a highthroughput screen of miRs from the CT. This screen allowed us to profile the miR transcriptome of the normal murine corpora as well as in the corpora of mice with diet-induced ED. Of the 602 miRs in the microarray, 181 were detected above background values in the normal corpora suggesting that mRNA regulation by miRs may play an important role in normal physiologic function of the CT. Of these 181 miRs, the majority are expressed at relatively low expression values, whereas several are expressed at relatively higher levels (Figure 2). Inclusive of these more abundant

Highly expressed miRs in the normal corporal tissue

MicroRNA

NanoString count

125b-5p

13,880

145 125a-5p 720 Let-7b

5,086 4,843 3,759 3,610

miR = microRNA

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Previous reports Angiogenesis and negative correlates with VEGF (He et al. [2]) Targets p53 (Le et al. [19]) Vascular smooth muscle cell differentiation (Zeng and Childs [21]) Angiogenesis and negative correlates with VEGF target (Fan et al. [16]) Endothelial cell development and regulation of endothelial progenitor cells (Wang et al. [22]) Endothelial angiogenesis via regulation of the anti-angiogenic factor TIMP1 (Otsuka et al. [20]) Let-7 family highly expressed in endothelial cells (Kuehbacher et al. [18])

miRs in Diet-Induced ED

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Figure 3 Volcano plot that displays miRs as a function of both FDRcorrected P value and log2-fold change in expression between mice fed with an HFD and normal chow. As evident in the plot, the majority of miRs trended to decrease in expression and 65 miRs displayed a decrease (red arrow) in expression with a fold change greater than 1 in the CT of mice with dietinduced ED. Five miRs were increased (green arrow) with a fold change greater than 1 and only one miR was increased with a fold change greater than 2. n = 3. CT = corporal tissue; ED = erectile dysfunction; FDR = false discover rate; HFD = high-fat diet; miR = microRNA

miRs are miR-125b-5p, miR-145, miR-125a-5p, miR-720, and let-7b (Table 1). Interestingly, the two most abundantly expressed miRs are miR125b-5p, which has been reported in endothelial cells, and miR-145, which has been reported in vascular smooth muscle cells. miR-125b-5p, which demonstrates the highest expression level, has been linked to angiogenesis in tumors and levels miR-125b are negatively correlated with VEGF expression [17,21]. Recently, miR-145 has not only been found in vascular smooth muscle but also found to promote vascular smooth muscle cell differentiation [19]. We hypothesize that with the abundance of endothelial cells and smooth muscle cells in CT, strict control of their proliferation and migration may be essential for proper corporal endothelial and smooth muscle cell function. Previous work from our group using the same mouse model of diet-induced ED described an increase in p53 expression, and this was correlated with a significant increase in apoptotic cell death in the CT as well as an increase in phosphodiesterase type 5 (PDE5) levels [15]. Le et al. [18] recently demonstrated that miR-125b is an important regulator of p53 and p53-induced apoptosis. Thus again, the highly expressed miRs in the normal CT may function to control normal CT homeostasis and/or may limit a stress or injury-induced response.

Mice fed with an HFD for 22 weeks became obese and hyperglycemic, and developed ED. Previous studies using this diet-induced ED model have focused on the well-characterized cGMP/NO pathway [12,15]. It is now widely accepted that up to half of men with ED do not respond well to the current PDE5 inhibitor oral therapy; thus, the pursuit of new therapies is essential. Targeting specific miRs offers a potentially new therapeutic option as an individual miR may target multiple members of one signaling pathway. In the present study, using miR microarray analysis and validation by Q-PCR, we now demonstrate that in response to the HFD, approximately one-third of the CT miR transcriptome is downregulated while only 3% (six miRs) upregulated. Because miRs bind to mRNA targets and cause their downregulation, a decrease in miR levels may possibly result in an increase of specific target proteins. Indeed, an increase in certain proteins has been reported in this model of dietinduced ED. Of the miRs that exhibited the greatest degree of downregulation (miR-550, miR-425, miR-134, miR-153, and miR-26b), miR-425, miR-153, and miR-26b may be noteworthy to this present study. miR-425 has been reported to be expressed in human atria and ventricles and negatively regulates atrial natriuretic J Sex Med 2015;12:621–630

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Figure 4 Q-PCR validation of randomly selected miRs that were either upregulated, downregulated, or unchanged between the two groups. (A) The five upregulated miRs as determined by NanoString data all trended to show an increase via Q-PCR but only two were significantly increased, miR1937c and miR-151-5p. (B) Of the four downregulated miRs validated, three trended to be downregulated in the Q-PCR experiment compared with the microarray data and two of these three miRs, miR-153 and miR-425, were significantly downregulated in the CT of HFD mice when compared with control. (C) Of the four unchanged miRs via microarray data, all four remained unchanged with Q-PCR validation. All expression levels were normalized to Sno202. Asterisk indicates P < 0.05, n = 12. CT = corporal tissue; HFD = high-fat diet; miR = microRNA; Q-PCR = quantitative polymerase chain reaction

peptide, a powerful vasodilator [22] that has been shown to have a role in smooth muscle tone in the corpus cavernosum [23–26], while miR-26b has been shown to respond to low levels of oxygen and suggested to suppress hypoxiainduced apoptosis [27]. Interestingly, miR-153 is embedded in the gene islet-associated protein-2, a major auto-antigen in type 1 diabetes [28]. Of the miRs that were upregulated in the CT of mice with diet-induced ED (miR-720, miR1937a, miR-1937c, miR-205, and miR-151-5p), a search of the literature revealed little information J Sex Med 2015;12:621–630

about these miRs with regard to possible targets and tissue expression. Both miR-720 and miR151-5p have been linked to different forms of cancer [29,30]. miR-205, on the other hand, has been shown to negatively regulate the androgen receptor and is associated with adverse outcome in prostate cancer patients [31]. Of the three miRs that were below background but went to above background levels after 22 weeks on the HFD, miR-15b is noteworthy. Targets for miR15b include HIF-1, VEGFR2, and SMAD7, all proteins with roles in angiogenesis [32].

miRs in Diet-Induced ED miRs are novel and potent regulators to tissue injury and their role in benign urologic pathologies, specifically ED, is unknown. Results of the present study have identified the CT miR transcriptome and report on the differential regulation of specific miRs in diet-induced ED. These results have the potential to open up a whole new field of investigation for ED. The identification of miRs involved in ED and their target mRNAs may uncover new mechanisms for the pathogenesis for ED that may lead to the development of novel therapeutic agents. Corresponding Author: Jeffrey J. Lysiak, PhD, Department of Urology, University of Virginia, Charlottesville, VA 22908, USA. Tel: (434) 924-5007; E-mail: [email protected] Conflict of Interest: The authors report no conflicts of interest. Statement of Authorship

Category 1 (a) Conception and Design Brian H. Annex; Jeffrey J. Lysiak; Parviz K. Kavoussi (b) Acquisition of Data Carlos E. Barbery; Stephen D. Turner; Frank A. Celigoj; Parviz K. Kavoussi; Ryan P. Smith (c) Analysis and Interpretation of Data Stephen D. Turner; Brian H. Annex; Jeffrey J. Lysiak

Category 2 (a) Drafting the Article Carlos E. Barbery; Frank A. Celigoj; Jeffrey J. Lysiak (b) Revising It for Intellectual Content Brian H. Annex; Jeffrey J. Lysiak

Category 3 (a) Final Approval of the Completed Article Brian H. Annex; Jeffrey J. Lysiak References 1 Gunaratne PH, Creighton CJ, Watson M, Tennakoon JB. Large-scale integration of MicroRNA and gene expression data for identification of enriched microRNA-mRNA associations in biological systems. Methods Mol Biol 2010;667: 297–315. 2 He L, Hannon GJ. MicroRNAs: Small RNAs with a big role in gene regulation. Nat Rev Genet 2004;5:522–31. 3 Ghelani HS, Rachchh MA, Gokani RH. MicroRNAs as newer therapeutic targets: A big hope from a tiny player. J Pharmacol Pharmacother 2012;3:217–27.

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