Animal Biotechnology

ISSN: 1049-5398 (Print) 1532-2378 (Online) Journal homepage: http://www.tandfonline.com/loi/labt20

Differential Expression of microRNAs and their Regulatory Networks in Skin Tissue of Liaoning Cashmere Goat during Hair Follicle Cycles Wen L. Bai, Yun L. Dang, Rong H. Yin, Wu Q. Jiang, Ze Y. Wang, Yu B. Zhu, Shi Q. Wang, Ying Y. Zhao, Liang Deng, Guang B. Luo & Shu H. Yang To cite this article: Wen L. Bai, Yun L. Dang, Rong H. Yin, Wu Q. Jiang, Ze Y. Wang, Yu B. Zhu, Shi Q. Wang, Ying Y. Zhao, Liang Deng, Guang B. Luo & Shu H. Yang (2016) Differential Expression of microRNAs and their Regulatory Networks in Skin Tissue of Liaoning Cashmere Goat during Hair Follicle Cycles, Animal Biotechnology, 27:2, 104-112 To link to this article: http://dx.doi.org/10.1080/10495398.2015.1105240

Published online: 25 Feb 2016.

Submit your article to this journal

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=labt20 Download by: [University of California, San Diego]

Date: 26 February 2016, At: 19:07

ANIMAL BIOTECHNOLOGY 2016, VOL. 27, NO. 2, 104–112 http://dx.doi.org/10.1080/10495398.2015.1105240

Differential Expression of microRNAs and their Regulatory Networks in Skin Tissue of Liaoning Cashmere Goat during Hair Follicle Cycles Wen L. Baia, Yun L. Danga, Rong H. Yina, Wu Q. Jianga, Ze Y. Wanga, Yu B. Zhua, Shi Q. Wangb, Ying Y. Zhaoa, Liang Denga, Guang B. Luoa and Shu H. Yangb College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China; bInstitute of Animal Husbandry Science of Liaoning Province, Liaoyang, China

Downloaded by [University of California, San Diego] at 19:07 26 February 2016

a

ABSTRACT

KEYWORDS

MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules that negatively regulate gene expression. Herein, we investigated a selective number of miRNAs for their expression in skin tissue of Liaoning Cashmere goat during hair follicle cycles, and their intracellular regulatory networks were constructed based on bioinformatics analysis. The relative expression of six miRNAs (mir-103-3p, -15b-5p, 17-5p, -200b, -25-3p, and -30c-5p) at anagen phase is significantly higher than that at catagen and/or telogen phases. In comparison to anagen, the relative expression of seven miRNAs (mir-148a-3p, -199a-3p, -199a-5p, -24-3p, -30a-5p, -30e-5p, and -29a-3p) was revealed to be significantly up-regulated at catagen and/or telogen stages. The network analyses of miRNAs indicated those miRNAs investigated might be directly or indirectly involved in several signaling pathways through their target genes. These results provided a foundation for further insight into the roles of these miRNAs in skin tissue of Liaoning Cashmere goat during hair follicle cycles.

Hair follicle cycles; liaoning cashmere goat; microRNA expression; regulatory networks; skin tissue

Introduction Liaoning Cashmere goat is an indigenous breed in China (1). It is well adapted to the local environment at altitudes ranging from 500 to 1340 m with annual average temperature of 7 to 8°C, precipitation of 700 to 900 mm, and frost free period of 150 to 170 days. The local people rely heavily on this breed, and the main purpose of this breed is the production of cashmere (2). The cashmere fiber growth is controlled by the seasonal hair follicle activity being a cyclic biological system in the skin of Cashmere goat, and all mature hair follicles undergo a growth cycle consisting of anagen, catagen and telogen phases (3–5). This process is well characterized morphologically (6, 7). It was also demonstrated that many genes were involved in the regulation of hair follicle cycle (4, 8), each phase of which was characterized by unique patterns of gene activation and silencing (9). In fact, the hair follicle cycle might be regulated through a series of complex mechanisms at multiple levels, such as transcription and translation (4, 10, 11). In recent years, a novel mechanism of post-transcriptional regulation has emerged as a critical regulator of hair follicle cycle that

is mediated by a new class of factors called microRNA (miRNA; 9, 12–14). MiRNAs are endogenous non-coding RNA molecules, 18-22 nucleotides (nt) long, which negatively regulate the expression of target mRNA mainly through repressing translation or in some cases via cleaving mRNA transcripts (15, 16). Over the past few years, numerous miRNAs were identified in skin tissue of Cashmere goat (17, 13, 14, 18), but their expression characterizations in cashmere skin during hair follicle cycle have not yet been fully understood, and thus are still open for further investigation. On the other hand, from the functional perspective, a single miRNA can regulate the expression of more than a hundred mRNA targets, and/or conversely, the expression of a single mRNA can be controlled cooperatively by several different miRNAs (19, 20). These findings indicated that miRNAs and their mRNA targets appear to represent remarkably diverse regulatory networks (9). Thus, the aims of this study were: 1) to identify expression pattern of a selected number of miRNAs in skin of Liaoning Cashmere goat during hair follicle cycle, and 2) to reveal their regulatory network by bioinformatics tools.

CONTACT Rong H. Yin [email protected] College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang 110866, China. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/labt. © 2016 Taylor & Francis

ANIMAL BIOTECHNOLOGY

Materials and methods

kit (no. D350; TaKaRa, Dalian, China) following the manufacturer’s instructions. A negative control was included for each synthesis without template RNA.

Downloaded by [University of California, San Diego] at 19:07 26 February 2016

Sample, RNA extraction, and cDNA synthesis In the present study, all experimental procedures were reviewed and approved by the Animal Experimental Committee of Shenyang Agricultural University. Six female adult Liaoning Cashmere goats were randomly selected, and the animals used were clinically healthy without common traceable genetic relationships. The skin tissue biopsies of approximately 1 cm2 were collected from each individual in October (anagen), January (catagen), and March (telogen) as described in our previous study (21). The tissue biopsy samples collected were immediately put into a Sample Protector (TaKaRa, Dalian, China) and were transported to the laboratory in low temperature conditions (approximately 4°C) within 12 hours. The Sample Protector can prevent RNA degradation at 25°C for 7 days and 4°C for 30 days according to the manufacturer’s instructions. Subsequently, the samples were stored at -80°C until the isolation of total RNA. We isolated the total RNA from each sample with the RNAiso reagent kit (TaKaRa, Dalian, China) following the manufacturer’s instructions. In order to exclude the contamination from residual genomic DNA, we treated the isolated RNA using DNase I (TaKaRa, Dalian, China). The integrity of total RNA was verified by gel electrophoresis of 1.5% agarose, and the purity and quantity of total RNA were assessed by ultraviolet spectrometer with the optical density (OD) ratio of OD260/OD280 ranging from 1.8 to 2.0 for the samples. For miRNAs expression analysis, the synthesis of first strand of cDNA was performed using the One Step PrimeScript microRNA (miRNA) cDNA synthesis Table 1.

Expression detection of miRNA in skin tissue during hair follicle cycle A list of miRNAs was chosen based on the previous investigations (17, 9, 13, 14). In the present study, the miRNAs were chosen mainly based on the following considerations: first, they were identified in skin tissue of Cashmere goat, and second, those miRNAs were found to have significantly differential expression in mice skin tissue during hair follicle cycle. Therefore, we hypothesize that these miRNAs might play roles in the regulation of hair follicle cycles of skin in Liaoning Cashmere goat. Herein, the expression of these miRNAs was analyzed by Real-time PCR (qPCR) technique. The mature sequence of miRNAs were retrieved from miRNA database (http:www.mirbase.org), and the miRNA specific sense primers was designed according to the principle described by Benes and Castoldi (22) (Table 1). The corresponding anti-sense primer for miRNA was obtained from the kit (Code: D350, TaKaRa, Dalian, China) being universal miRNA reverse primer. On a LightCycler 480 Real Time PCR system (Roche Diagnostics, Mannheim, Germany), the qPCR was carried out with the SYBR Premix Ex Taq II kit (TaKaRa, Dalian, China). Six-point standard curve was produced for each miRNA by the 10-fold serial dilutions of cDNA. We performed the reactions in a final volume of 20 µL containing 10.0 µL SYBR Premix Ex Taq II (2 �), 2.0 µL first-strand cDNA, 0.8 µL sense primer

Characteristics of the primers and the real-time PCR conditions.

MiRNA Chi-let-7b-5p Chi-mir-103-3p Chi-mir-10a-5p Chi-mir-148a-3p Chi-mir-15b-5p Chi-mir-17-5p Chi-mir-199a-3p Chi-mir-199a-5p Chi-mir-200b Chi-mir-21-5p Chi-mir-24-3p Chi-mir-25-3p Chi-mir-29a-3p Chi-mir-30a-5p Chi-mir-30c-5p Chi-mir-30e-5p Chi-let-7d-5p Chi-miR-26a-5p Chi-miR-15a-5p a

105

Reference in miRBase

Primer Sequence (50 -30 )a

Primer Length (nt)

Tab (°C)

MIMAT0035882 MIMAT0035902 MIMAT0035911 MIMAT0035977 MIMAT0035992 MIMAT0035998 MIMAT0036037 MIMAT0036036 MIMAT0036046 MIMAT0036061 MIMAT0036091 MIMAT0036100 MIMAT0036113 MIMAT0036121 MIMAT0036125 MIMAT0036128 MIMAT0035886 MIMAT0036101 MIMAT0035990

F:CGTGAGGTAGTAGGTTGTGTGGTT F:TAAGCAGCATTGTACAGGGCTATGA F:CGTACCCTGTAGATCCGAATTTGT F:CGCTCAGTGCACTACAGAACTTTGT F:CGTAGCAGCACATCATGGTTTACA F:CTCAAAGTGCTTACAGTGCAGGTAGT F:CGGACAGTAGTCTGCACATTGGTT F:GACCCAGTGTTCAGACTACCTGTTC F:CGCTAATACTGCCTGGTAATGATGA F:CGCTAGCTTATCAGACTGATGTTGAC F:CGTGGCTCAGTTCAGCAGGAAC F:TACATTGCACTTGTCTCGGTCTGA F:CGTAGCACCATCTGAAATCGGTT F:CTGTAAACATCCTCGACTGGAAGCT F:CGTGTAAACATCCTACACTCTCAGC F: CTGTAAACATCCTTGACTGGAAGCT F:CGAGAGGTAGTAGGTTGCATAGTT F:CGTTCAAGTAATCCAGGATAGGCT F: CGTAGCAGCACATAATGGTTTGTG

24 25 24 25 24 26 24 25 25 26 22 24 23 25 25 25 24 24 24

61 63 62 63 62 62 63 61 63 62 64 62 63 63 61 61 62 61 63

F ¼ Sense primer, and the corresponding anti-sense primer was obtained from the kit (Code: D350, TaKaRa, Dalian, China) being universal microRNA reverse primer. b Ta ¼ annealing temperature.

106

W. L. BAI ET AL.

Downloaded by [University of California, San Diego] at 19:07 26 February 2016

(10 µM), 0.8 µL anti-sense primer (10 µM), and 6.4 µL PCR grade water. The thermal cycling conditions were as follows: initial template denaturation at 95°C for 4 minutes, followed by 40 cycles of 95°C for 30 seconds, 61-64°C for 30 seconds (Table 1), and 72°C for 30 seconds, with a final extension at 72°C for 5 minutes. In order to confirm the presence of single specific peak for each reaction, the last cycle was followed by a melting curve analysis ranging from 65°C to 95°C. Each reaction was performed in triplicate. A negative control was included in each measurement without the cDNA template. Bioinformatics analysis of differential expression miRNAs for regulatory network Firstly, the DIANA-mirPath was used to identify molecular signaling pathways altered potentially by the expression of miRNAs on given pathways (23). This software is a web-based computational tool that performs an enrichment analysis of predicted target genes of one or multiple miRNA via comparing each set of miRNA target genes to known biological pathways from Kyoto Encyclopedia of Genes and Genomes (KEGG). The enrichment analysis of input dataset is carried out via a Pearson’s chi-squared test: {χ2 ¼ [(O–E)2/E]}, where O indicates the number of genes in the input dataset identified to be involved in a given pathway, and E indicates the number of genes expected by chance to be member of the given pathway. The resultant Pvalues were corrected for multiple tests with an adjusted value of < 0.05 being considered statistically significant. Secondly, the ToppCluster (http://toppcluster.cchmc. org/) was used for further functional enrichment and regulatory networks analysis. The ToppCluster is a web server application that leverages a powerful enrichment analysis and underlying data environment for comparative analyses of multiple gene lists (24). It generates connectivity networks that reveal functional features shared or specific to multiple gene lists. In the present study, the putative target genes of miRNAs from DIANA-mirPath analysis were further used to generate regulatory network in ToppCluster with the hierarchical clustering. For this analysis, the P-values were also corrected for multiple tests by setting the FDR to 0.05. The resulted files from ToppCluster were saved as xgmml format, and then were subjected to Cytoscape (version 2.8.3; 25) for the visualizable networks. In the network, circles, hexagons, and boxes were respectively used for representing miRNAs, target genes, and signaling pathways in different size based on their relative importance which means the number

of edges joined to particular miRNAs, target genes and signaling pathways. Statistical analysis In our preliminary experiments, three miRNAs: let7d-5p, miR-26a-5p, and miR-15a-5p were verified to be suitable normalizers for studying microRNA expression in skin tissue of Liaoning Cashmere goat during hair follicle cycling (unpublished data). In this study, therefore, the geometric mean of let-7d-5p, miR-26a-5p, and miR-15a-5p were used to normalize the expression level of microRNAs. The normalized qPCR data are further processed as the method described in our previous study (26). That is that the normalized qPCR data was reported as log2n-fold change relative to anagen stage. In order to estimate standard errors at the anagen stage and prevent possible biases in statistical analysis, we transformed normalized data to obtain a perfect average of 1.0 at the anagen stage, leaving the proportional difference between the biological replicates. We calculated the same proportional change at catagen and telogen stages to obtain a fold change relative to the anagen stage. In order to assess the effect of differential stages of hair follicle cycle, we analyzed the dataset by a mixed model in SPSS (SPSS Inc., Chicago, IL) that consisted of the fixed effect of different three stages of hair follicle cycle (anagen, catagen, and telogen) and the random effect of goat. Means of different stages of hair follicle cycle were compared with the Tukey test, and the differences were considered to be significant when P < 0.05.

Results and discussion Expression characterization of miRNAs in skin liaoning cashmere goat during hair follicle cycle Hair follicle cycle is a unique biological process, which consists of three distinct phases including anagen, catagen, and telogen. In the present study, based on qPCR technique, a total of sixteen miRNAs was investigated in skin of Liaoning Cashmere goat during hair follicle cycle. The results were presented in Fig. 1. Our data indicated that all the 16 miRNAs analyzed were found to be expressed in skin of Liaoning Cashmere goat in three phases of hair follicle cycle (anagen, catagen, and telogen). No significant difference was observed in the relative expression of three miRNAs: let-7b-5p, mir-10a-5p, and mir-21-5p (Fig. 1a, c, and j). In mice skin, however, it was reported that these three miRNAs exhibited differential expression during distinct hair cycle stages with significant differences (9). Thus, our

107

Downloaded by [University of California, San Diego] at 19:07 26 February 2016

ANIMAL BIOTECHNOLOGY

Figure 1. Relative expression of miRNA in skin of Liaoning Cashmere goat during hair follicle cycle compared with anagen stage, taking the geometric mean of 3 miRNAs (let-7d-5p, miR-26a-5p, and miR-15a-5p) as references. Error bar indicated SEM within the group. Different symbols (#, *, and §) among anagen, catagen, and telogen stages indicate significant difference (P < 0.05). No any symbol was marked in the relative expression of three miRNAs: let-7b-5p (a), mir-10a-5p (c), and mir-21-5p (j) because of no significant difference was recorded in their relative expression during differential hair follicle stages.

Downloaded by [University of California, San Diego] at 19:07 26 February 2016

108

W. L. BAI ET AL.

results from Liaoning Cashmere goat appear to go against that reported in mice. We did not investigate the expression status for these three miRNAs in other tissues of Liaoning Cashmere goat because of our research interest. Further studies, including in vitro experiments, should be performed for their potential roles in the hair follicle cycle of Cashmere goat. On the other hand, considering the relatively stable expression of these three miRNAs in skin of Liaoning Cashmere goat, they also have potential value as normalizers for microRNA quantitative analysis in skin of this breed during the hair follicle cycle. The published data showed that miRNA expression in the skin changes remarkably during distinct stages of the hair follicle cycle in humans (27) and mice (9), suggesting that miRNAs may participate in the regulation of hair follicle transition from one stage to another. In the present study, the remaining thirteen miRNAs analyzed exhibited significant differences (P < 0.05) in relative expression between the distinct hair follicle cycle stages (Fig. 1). For these miRNAs, on the whole, there is a greater expression difference between the anagen and telogen stages, such as mir-199a-3p and mir-30a-5p, whereas a relatively lower difference in relative expression was observed between anagen and catagen, as well as, between the catagen and telogen stages of the hair follicle cycle. Similar difference in miRNA expression was also observed in the skin of mice during hair follicle cycle (9). For the hair follicle cycling of Liaoning Cashmere goat, the anagen phase lasts for 4 months (from September to December), whereas the catagen is a short transition phase lasting for only one month (January) that is followed by the telogen phase lasting 3 months (from February to April). Moreover, compared with catagen and telogen phases, the anagen is an active growth phase of hair follicles that is characterized by most dramatic changes in expression of a large number of genes in the hair follicle of skin (4, 28). In the present study, therefore, we mainly analyzed the expression differences of miRNAs in Cashmere goat skin during the hair follicle cycle through grouping into anagen against catagen and/or telogen. Herein, we found that the relative expression of six miRNAs (mir-103-3p, -15b-5p, 17-5p, -200b, -25-3p, and -30c-5p) at the anagen phase is significantly higher than that at catagen and/or telogen phases, of which two miRNAs (mir-103-3p and -17-5p) had significantly higher relative expression at both anagen and catagen phases (Fig. 1b and f). Recently, in Inner Mongolia Cashmere goat skin, two miRNAs (mir-421 and -421*) were also found to exhibit high relative expression at anagen phase but low at catagen and telogen phases

(18) where the authors suggested that mir-421 and miR-421* may acts to promote cashmere production. It was well known that the transition of hair follicle from the telogen to the anagen phase leads to a formation of hair shaft and is accompanied by the activation of many signaling pathways that regulate the expression of genes encoding hair-specific molecules, such as keratins, and components of the inner root sheath (29, 30, 28). Therefore, we speculated that the six miRNAs analyzed in this study might play roles in hair follicle development, new hair-fiber formation and cashmere production. On the other hand, in comparison to anagen, the relative expression of seven miRNAs (mir-148a3p, -199a-3p, -199a-5p, -24-3p, -30a-5p, -30e-5p, and -29a-3p) was revealed to be significantly up-regulated at catagen and/or telogen stages, of which three miRNAs (mir-148a-3p, -199a-5p, and -30e-5p) had significantly higher relative expression only at telegen phase (Fig. 1d, h, and p). A recent investigation by Su et al. (18) reported that miR-1839, -374b, and -2284n exhibit the highest relative expression in skin of Inner Mongolia cashmere goat in February when the hair follicle goes into telogen from catagen. Therefore, they speculated that the miRNAs (miR-1839, -374b, and -2284n) expression may repress their targets to promote the transition of cashmere goat hair follicle from catagen to telogen. The well-known catagen is a short transition phase that occurs at the end of the anagen phase and signals the termination of anagen-specific signaling interactions between the epithelium and the mesenchyme leading to apoptosis in the hair follicle epithelium (31, 32). The telogen is a relative resting phase of the hair follicle that is characterized by the silencing of a large number of genes involved in the regulation of proliferation and differentiation of keratinocyte (4; 31, 33). Therefore, it can be suggested that those seven miRNAs up-regulated at catagen and/or telogen might play significant roles in the apoptosis-driven involution of hair follicle with the transition of the dermal papilla into a resting period, as well as the retaining of the hair follicle being in a quiescent state. Regulatory network analysis for the miRNAs with higher expression at anagen phase The findings from human and mouse demonstrated that several signaling pathways widely participate in the development, morphogenesis, and every part of the hair follicle cycle, such as Wnt, Hedgehog, and TGF-beta (34, 35). Our results from the present study indicated that a set of six miRNAs has significantly

Downloaded by [University of California, San Diego] at 19:07 26 February 2016

ANIMAL BIOTECHNOLOGY

higher relative expression at anagen compared with that of catagen and/or telogen phases. Using the bioinformatics tool, DIANA mirPath, we analyzed targets of those six miRNAs (i.e., mir-103-3p, -15b-5p, 17-5p, -200b, -25-3p, and -30c-5p) in the aforementioned signaling pathways. For a better understanding of the biological functional relationships of these miRNAs and their putative targets in various pathways altogether, we used the ToppCluster to generate a regulatory network, and the Cytoscape was used for the visualization of the network. The obtained results are presented in Fig. 2. As observed from Fig. 2, the network distinctly showed the six miRNAs and their related target genes and signaling pathways. The relative importance of the miRNAs,

109

target genes, and signaling pathways was shown by the relative size of circles, hexagons, and boxes, respectively. As an example, the regulatory network of JUN has been shown clearly by red color lines, and mir-200b was indicated to target JUN that was involved in Wnt, EGF, FGF, and MAPK signaling pathways (Fig. 2). There was evidence that mir-200b was involved in the regulation of hair follicle development and was a target for the Wnt signaling pathway (36). However, the Wnt pathway was found to be one of the key pathways associated with hair follicle development (37). The inhibiting of Wnt pathway led to the failure of structural maintenance of hair follicle (38). Recently, the functional roles of these miRNAs have been reported in different disciplines. MiR-103-3p was

Figure 2. Regulatory network of putative targets of miRNAs (mir-103-3p, -15b-5p, 17-5p, -200b, -25-3p, and -30c-5p) with higher expression at anagen stage in major signaling pathways involved in hair follicle cycle. Circles, hexagons, and boxes were respectively used for representing miRNAs, target genes, and signaling pathways in different size based on their relative importance, which means the number of edges joined to particular miRNAs, target genes, and signaling pathways.

Downloaded by [University of California, San Diego] at 19:07 26 February 2016

110

W. L. BAI ET AL.

recorded to participate in pancreatic cancer (39). The mir-15b-5p was involved in human epithelial ovarian cancer (40). The miR-25-3p regulates cell cycle-related protein expression and plays an important role in the occurrence and development of squamous cell carcinoma of the tongue (41). Also, miR-17-5p modulates osteoblastic differentiation and cell proliferation (42), and promotes proliferation in gastric cancer cells (43). To date, however, a minimal amount of information is available on the roles of these miRNA in the hair follicle cycle of cashmere goat. Our results from the present study indicated these miRNAs may directly or indirectly target several signaling pathways, which are known to be important in regulating the development, morphogenesis, and the cycle of hair follicle in skin. Thus, we hypothesize that these six miRNAs may play multiple roles in cashmere goat skin at anagen. Further

investigations will be needed to elucidate their functional roles in the hair-fiber growth and development of hair follicle of cashmere goat skin. Regulatory network analysis for the miRNAs upregulated at catagen and/or telogen stages In the present work, compared with anagen, the relative expression of seven miRNAs was significantly up-regulated at catagen and/or telogen stages, including mir-148a-3p, -199a-3p, -199a-5p, -24-3p, -30a5p, -30e-5p, and -29a-3p (Fig. 1). Based on the DIANA-mirPath analysis, the putative target genes of these miRNAs were further incorporated to construct regulatory network using ToppCluster. The resulted network was presented in Fig. 3. The network figure showed the information on the relationships

Figure 3. Regulatory network of putative targets of miRNAs (mir-148a-3p, -199a-3p, -199a-5p, -24-3p, -30a-5p, -30e-5p, and -29a-3p) upregulated at catagen and/or telogen stages in major signaling pathways involved in hair follicle cycle. Circles, hexagons, and boxes were respectively used for representing miRNAs, target genes, and signaling pathways in different size based on their relative importance, which means the number of edges joined to particular miRNAs, target genes, and signaling pathways.

Downloaded by [University of California, San Diego] at 19:07 26 February 2016

ANIMAL BIOTECHNOLOGY

among the targeted genes to Wnt, TGF-beta, MAPK, Hedgehog, VEGF, EGF, FGF, and JAK-STAT signaling pathways. For example, the PIK3CD gene was involved in FGF, VEGF, and JAK-STAT signaling pathways, and it was targeted in multiple miRNAs such as mir-199a-5p, mir-148a-3p, mir-30a-5p, and mir-30e-5p (shown in Fig. 3 with red colored lines). It has been proven that FGF pathway promotes the transition of hair follicle from telogen to anagen through providing the stimulatory signals to hair follicle stem cells, whereas the activity of FGF pathway can be antagonized by BMP signaling during telogen (44). Our date indicated that the expression of mir-199a-5p, mir-148a-3p, and mir-30e-5p markedly increases at the telogen stage in comparison to anagen stage suggesting that they might be involved in the establishment of an optimal balance of gene expression in hair follicle at the telogen stage that may be required for retaining a quiescent state. In the past few years, the functions of miR-199a-3p, miR-199a-5p, miR-30a-5p, and miR-29a-3p were also reported in various cases. Some studies revealed that miR-199a-3p and miR-199a-5p inhibited the hepatocyte growth factor in renal cancer carcinoma (45), and the monocyte/macrophage differentiation, respectively (46). Also, it was reported that miR-30a-5p suppresses tumor growth in colon carcinoma (47), and miR-29a-3p inhibits the activation of pulmonary adventitial fibroblast (48). Taken altogether with our results, it can be suggested that these four miRNAs (miR-199a-3p, miR-199a-5p, miR30a-5p, and miR-29a-3p) might participate in the control of the hair follicle of cashmere goat skin being in a quiescent state (at telogen stage). However, further studies, including in vitro and in vivo experiments, would be necessary for biological role evidences of these miRNAs in the cyclic process of hair follicle of cashmere goat skin. In conclusion, herein, we for the first time identified the expression characterization of a selective number of miRNAs in skin tissue of Liaoning Cashmere goat during the hair follicle cycle, and constructed their complete regulatory network by bioinformatics tools. The results from this work contribute to further elucidating the roles of those miRNAs in skin tissue of Liaoning Cashmere goat during the hair follicle cycles. Also, they would provide useful information to reveal the molecular mechanisms of the miRNAs in regulating the development, morphogenesis, and the cycle of hair follicle in skin of cashmere goat.

Funding The work was supported financially by the Foundation of Natural Science Project of Liaoning Province, China

111

(No. 2015020758), Scientific and Technology Project of Liaoning Province, China (No. 2014036), the Foundation of Education Department of Liaoning Province, China (No. L2010507), a Foundation for University Talents of Liaoning Province of China (LJQ2013070), and the Projects of Tianzhushan Person of Outstanding Ability of Shenyang Agricultural University (Shenyang, Liaoning, China).

References 1. McGregor BA, An M, Jiang Y. Fleece metrology of Liaoning Cashmere goats. Small Ruminant Res 1991; 4:61–71. 2. Bai WL, Yin RH, Jiang WQ, et al. Molecular characterization of prolactin cDNA and its expression pattern in skin tissue of Liaoning Cashmere goat. Biochem Genet 2012; 50:694–701. 3. Nixon AJ, Gurns MP, Betterid K. Seasonal hair follicle activity and fibre growth in some New Zealand Cashmere-bearing goats (Caprus hircus) J Zoology 1991; 224:589–598. 4. Stenn KS, Paus R. Controls of hair follicle cycling. Physiol Rev 2001; 81:449–494. 5. Wu J, Zhang Y, Zhang J, Chang Z, Li J, Yan Z Husile, Zhang W. 2012. Hoxc13/β-catenin correlation with hair follicle activity in Cashmere goat J Integr Agric 2012; 11:1159–1166. 6. Paus K, Muller-Rover S. Comprehensive guide for the recognition classification of distinct stages of hair follicle morphogenesis. J Invest Dermatol 1999; 113:523–532. 7. Zhang C, Li J, Yin J, Zhang Y, Miao X. Study on hair follicles periodical variety in Inner Mongolia Arbas white cashmere goats. Chin J Anim Sci 2005; 41:10–13. 8. Wu N, Sulpice E, Obeid P, et al. The miR-17 family links p63 protein to MAPK signaling to promote the onset of human keratinocyte differentiation. PLoS One 2012; 7: e45761. 9. Mardaryev AN, Ahmed MI, Vlahov NV, et al. Micro-RNA31 controls hair cycle-associated changes in gene expression programs of the skin and hair follicle. FASEB J 2010; 24: 3869–3881. 10. Millar SE. Molecular mechanisms regulating hair follicle development. J Invest Dermatol 2002; 118:216–225. 11. Närhi K, Järvinen E, Birchmeier W, Taketo MM, Mikkola ML, Thesleff I. Sustained epithelial β-catenin activity induces precocious hair development but disrupts hair follicle down-growth and hair shaft formation. Development 2008; 135:1019–1028. 12. Zhang, L., Stokes, N., Polak, L., Fuchs, E., 2011. Specific microRNAs are preferentially expressed by skin stem cells to balance self-renewal and early lineage commitment. Cell Stem Cell. 8, 294–308. 13. Liu Z, Xiao H, Li H, et al. Identification of conserved and novel microRNAs in cashmere goat skin by deep sequencing. PLoS One 2012; 7:e50001. 14. Yuan C, Wang X, Geng R, He X, Qu L, Chen Y. Discovery of cashmere goat (Capra hircus) microRNAs in skin and hair follicles by Solexa sequencing. BMC Genomics 2013; 14:511. 15. Bushati N, Cohen SM. MicroRNA functions. Ann Rev Cell Dev Biol 2007; 23:175–205. 16. Carthew RW, Sontheimer EJ. Origins and mechanisms of miRNAs and siRNAs. Cell 2009; 136:642–655.

Downloaded by [University of California, San Diego] at 19:07 26 February 2016

112

W. L. BAI ET AL.

17. Wenguang Z, Jianghong W, Jinquan L, Yashizawa M. A subset of skin-expressed microRNAs with possible roles in goat and sheep hair growth based on expression profiling of mammalian microRNAs. OMICS 2007; 11:385–396. 18. Su R, Fu S, Zhang Y, et al. Comparative genomic approach reveals novel conserved microRNAs in Inner Mongolia cashmere goat skin and longissimus dorsi. Mol Biol Rep 2015; 42:989–995. 19. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of mammalian microRNA targets. Cell 2003; 115:787–798. 20. Kim VN. Small RNAs: classification, biogenesis, and function. Mol Cells 2005; 19:1–15. 21. Bai WL, Yin RH, Yin RL, et al. Selection and validation of suitable reference genes in skin tissue of Liaoning Cashmere goat during hair follicle cycle. Livestock Sci 2014; 161:28–35. 22. Benes and Castoldi. Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available. Methods 2010; 50:244–249. 23. Papadopoulos GL, Alexiou P, Maragkakis M, Reczko M, Hatzigeorgiou AG. DIANA-mirPath: integrating human and mouse microRNAs in pathways. Bioinformatics 2009; 25:1991–1993. 24. Kaimal V, Bardes EE, Tabar SC, Jegga AG, Aronow BJ. ToppCluster: a multiple gene list feature analyzer for comparative enrichment clustering and network-based dissection of biological systems. Nucl Acid Res. 2010; 38 (Web Server issue):W96-W102. 25. Smoot M, Ono K, Ruscheinski J, Wang P, Ideker T. Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics 2011; 27:431–432. 26. Bai WL, Yin RH, Zhao SJ, et al. Technical note: Selection of suitable reference genes for studying gene expression in milk somatic cell of yak (Bos grunniens) during the lactation cycle. J Dairy Sci 2014; 97:902–910. 27. Schneider MR. McroRNAs as novel players in skin development, homeostasis and disease. Brit Assoc Dermatol 2012; 66:22–28. 28. Rendl M, Polak L, Fuchs E. BMP signaling in dermal papilla cells is required for their hair follicle-inductive properties. Genes Dev 2008; 22:543–557. 29. Slominski A, Wortsman J, Plonka PM, Schallreuter KU, Paus R, Tobin DJ. Hair follicle pigmentation. J Invest Dermatol 2005; 124:13–21. 30. Sharov AA, Sharova TY, Mardaryev AN, et al. Bone morphogenetic protein signaling regulates the size of hair follicles and modulates the expression of cell cycle-associated genes. Proc Natl Acad Sci USA 2006; 103:18166–18171. 31. Botchkarev VA, Kishimoto J. Molecular control of epithelial-mesenchymal interactions during hair follicle cycling. J Invest Dermatol Symp Proc 2003; 8:46–55. 32. Schneider MR, Schmidt-Ullrich R, Paus R. The hair follicle as a dynamic miniorgan. Curr Biol 2009; 19:R132–R142.

33. Alonso L, Fuchs E. The hair cycle. J Cell Sc 2006; 119:391–393. 34. Wang LC, Liu ZY, Gambardella L, et al. Conditional disruption of hedgehog signaling pathway defines its critical role in hair development and regeneration. J Invest Dermatol 2000; 114:901–908. 35. Zhang L, Zhang Y, Su R, Wang R, Li J. The regulatory mechanism of microRNAs in skin and hair follicle development. Yi Chuan 2014; 36:655–660. 36. Andl T, Murchison EP, Liu F, et al. The miRNA-processing enzyme dicer is essential for the morphogenesis and maintenance of hair follicles. Curr Biol 2006; 16:1041–1049. 37. Park PJ, Moon BS, Lee SH, et al. Hair growth-promoting effect of Aconiti Ciliare Tuber extract mediated by the activation of Wnt/β-catenin signaling. Life Sci 2012; 91:935–943. 38. Andl T, Reddy ST, Gaddapara T, Millar SE. WNT signals are required for the initiation of hair follicle development. Dev Cell 2002; 2:643–653. 39. Zhou H, Rigoutsos I. MiR-103a-3p targets the 5′UTR of GPRC5A in pancreatic cells. RNA 2014; 20:1431–1439. 40. Wang L, Zhu MJ, Ren AM, et al. A ten-microRNA signature identified from a genome-wide microRNA expression profiling in human epithelial ovarian cancer. PLoS One 2014; 9:e96472. 41. Xu JY, Yang LL, Ma C, Huang YL, Zhu GX, Chen QL. MiR-25–3p attenuates the proliferation of tongue squamous cell carcinoma cell line Tca8113. Asian Pac J Trop Med 2013; 6:743–747. 42. Jia J, Feng X, Xu W, et al. MiR-17–5p modulates osteoblastic differentiation and cell proliferation by targeting SMAD7 in non-traumatic osteonecrosis. Exp Mol Med 2014; 46:e107. 43. Wu Q, Luo G, Yang Z, et al. MiR-17–5p promotes proliferation by targeting SOCS6 in gastric cancer cells. FEBS Lett 2014; 588:2055–2062. 44. Greco V, Chen T, Rendl M, et al. A two-step mechanism for stem cell activation during hair regeneration. Cell Stem Cell 2009; 4:155–169. 45. Huang J, Dong B, Zhang J, et al. miR-199a-3p inhibits hepatocyte growth factor/c-Met signaling in renal cancer carcinoma. Tumour Biol 2014; 35:5833–5843. 46. Liu HS, Gong JN, Su R, et al. miR-199a-5p inhibits monocyte=macrophage differentiation by targeting the activin. A type 1B receptor gene and finally reducing C/EBPa expression. J. Leukoc. Biol. 2014; 96:1023–1035. 47. Baraniskin A, Birkenkamp-Demtrode, K, Maghnouj A, et al. MiR-30a-5p suppresses tumor growth in colon carcinoma by targeting DTL. Carcinogenesis 2012; 33:732–739. 48. Luo Y, Dong HY, Zhang B, 2014. MiR-29a-3p attenuates hypoxic pulmonary hypertension by inhibiting pulmonary adventitial fibroblast activation. Hypertension 2014; pii: hypertensionaha.114.04600.

Differential Expression of microRNAs and their Regulatory Networks in Skin Tissue of Liaoning Cashmere Goat during Hair Follicle Cycles.

MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules that negatively regulate gene expression. Herein, we investigated a selective number o...
2MB Sizes 0 Downloads 11 Views