Reprod Dom Anim 49, 913–919 (2014); doi: 10.1111/rda.12400 ISSN 0936–6768

Characterization of miR-126-3p and its Target Talin2 in the Bovine Corpus Luteum during the Oestrus Cycle L Dai*, J Xu*, S Liu, T Ma, Y Zhu, F Xu, Y Gao, B Yuan, S Wang, Y Zhang, G Sun and J Zhang Laboratory Animal Center, College of Animal Sciences, Jilin University, Changchun, China

Contents Although clear advances have been made in understanding of gene transcriptional regulation in the corpous luteum (CL) during the oestrous cycle, little is known about the involvement of microRNAs (miRNAs) in this physiological process. In the present study, expression of miR-126-3p was preliminarily detected in bovine CL throughout the oestrous cycle, while the expression during the middle or late stages was significantly more abundant (p < 0.01) than that during the early or regressed stages. Localization of miR-126-3p was subsequently observed in large and small luteal cells during the oestrous cycle in cattle. Meanwhile, to further investigate the function of miR-126-3p, its potential targets and responsive elements were determined and evaluated. It was revealed that miR-126-3p could target talin2 (TLN2) directly by binding the responsive element within the 30 -UTR. Quantitative analysis of TLN2 at mRNA and protein level also suggested that there was an inverse correlation between miR-126-3p and TLN2 at the developmental luteal stages in cattle. Collectively, these results demonstrated that difference in the expression pattern and location of miR-126-3p occurred at different stages of the bovine CL and that miR-126-3p acted as an important regulator of TLN2, suggesting their putative involvement in the development of bovine CL during the oestrous cycle.

Introduction The corpus luteum (CL) is an ephemeral endocrine gland that forms from the wall of a graafian follicle after ovulation. The major secretory product of CL is progesterone, which is required to establish and maintain the early intrauterine pregnancy by attenuating the pituitary release of gonadotropins in mammals (Stocco et al. 2007; Rekawiecki et al. 2008). CL can quickly develop into the most vascularized and steroidogenically active tissue in the relatively short lifespan of a normal luteal phase, reach functional maturity by the middle luteal phase and rapidly regress into non-functional avascular fibrous tissue in the absence of pregnancy with a 21-day lifespan in cattle (Hansel and Blair 1996; Skarzynski et al. 2013). Although clear advances have been made in understanding of gene transcriptional regulation involved in the CL during the oestrous cycle, the post-transcriptional gene regulatory mechanisms are not well-defined yet. However, the role of microRNAs (miRNAs) in the CL during the oestrous cycle has recently been the focus of several studies (Otsuka et al. 2008; Donadeu et al. 2012; McBride et al. 2012). MiRNAs are a class of endogenous non-coding small RNA that plays an essential role in cell proliferation, differentiation and

*Contribute equally to this work. © 2014 Blackwell Verlag GmbH

apoptosis either by controlling mRNA degradation or by translational repression in a sequence-dependent manner (Nilsen 2007; Fabian et al. 2010). Previously, it was identified that dozens of miRNAs were differentially expressed between the developmental and regressed stages in bovine CL, of which miR-126-3p was much more abundant in the developmental stage than regressed stage (Ma et al. 2011). MiR-126-3p has been shown to be involved in governing of vascular integrity and angiogenesis through targeting vascular endothelial growth factor A, Spred-1 and so on (Fish et al. 2008; Liu et al. 2009; Nicoli et al. 2010; de Giorgio et al. 2013). However, molecular mechanisms of miR126-3p in the bovine CL during the oestrous cycle remain unclear. Talin2 (TLN2) is a large dimeric cytoskeletal protein that functions to regulate the affinity of integrin for extracellular matrix ligands (Petrich 2009; Ellis et al. 2013), which plays a key role in the cell adhesion, migration, proliferation and differentiation. All of these cellular processes occur during the development and regression of the CL (Irving-Rodgers et al. 2006). However, the expression pattern of TLN2 and its regulation pathways in the bovine CL during the oestrous cycle have not been elucidated. In the present study, the expression profile and localization of miR126-3p at different stages of bovine CL were investigated. Meanwhile, we provided evidence that miR-1263p could directly target TLN2, implying their putative roles in the development of bovine CL during the oestrous cycle.

Materials and Methods Collection of the bovine corpus luteum Ovaries with CL were collected from healthy nonpregnant Simmental cattle at a local abattoir within 20 min after slaughter and transported on ice to the laboratory. Stages of CL (n = 6/stage) were classified as of early (days 1–4 after ovulation), middle (days 5–10), late (days 11–16) or regressed (days 17–21) stage as described previously (Sakumoto et al. 2011; Chouhan et al. 2013). Luteal tissues were separated from ovaries and frozen in liquid nitrogen until further analysis. Cellular RNA and protein were extracted from these same samples if necessary. RNA isolation Total RNA was extracted from the samples of four stages of CL using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) as per the standard protocol. RNA

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quality was assessed by electrophoresis in ethidium bromide-stained agarose gels, while RNA concentrations were determined with Nanodropâ ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). In prior to analysis, RNA samples were pretreated with the RNase-free DNase-I (Takara, Dalian, China) according to the manufacturer’s instructions. Stem-loop RT-qPCR Hundred nanogram of total RNA was reverse-transcribed with 50 nM of either miR-126-specific stem-loop primer or U6 small nuclear RNA primer, following instructions of the ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). MiR-126 levels were detected in triplicate using the SYBRâ Green Real-time PCR Master Mix (Toyobo) on the Eppendorf realplex2 Mastercycler (Eppendorf, Hamburg, Germany) with the following conditions: 5 min at 95°C followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. MiRNA levels were normalized to U6 small nuclear RNA; fold change was determined by 2MMCt. Sequences for miR126-specific and U6 primers were listed in Table S1. Histological analysis and in situ hybridization Different stages of frozen CL were embedded in OCT compound and cryosectioned at 10 lm thickness. The sections were mounted on poly L-lysine-coated slides and initially fixed in 4% paraformaldehyde at room temperature for 15 min. For histological analysis, sections were stained with haematoxylin–eosin (H&E). For in situ hybridization, Enhanced Sensitive ISH Detection (Boster, Wuhan, China) was performed following the manufacturer’s protocols. In brief, sections were treated with 0.5% H2O2/methanol for 30 min to block endogenous peroxidase and subsequently treated with pepsin in 3% citric acid for 1 min so as to expose the RNA. Fixed sections were pre-hybridized for 3 h at 37°C in hybridization buffer and hybridized overnight at 52°C in the presence of 40 nM locked nucleic acid (miRCURY LNATM, Exiqon, Vedbaek, Denmark) modified and digoxigenin (DIG)-labelled miR-126 probe (50 -DIGCGCATTATTACTCACGGTAG-30 ) or scramble probe (50 -DIG-GTGTAACACGTCTATATCGCCCA-30 ; Exiqon, Denmark). After incubation with blocking solution for 30 min at 37°C, biotin-labelled antidigoxigenin antibody was added for 60 min at 37°C. The sections were treated with streptavidin–biotin–peroxidase complex (SABC-POD) for 20 min and reacted with biotinlabelled peroxidase for 20 min at 37°C. Following the final wash, diamido-benzidine (DAB) was used to detect the hybridization signals, while haematoxylin was used for cell staining. Developed sections were then dehydrated, cleared and mounted. Images were acquired using an Olympus Fv1000 confocal microscope (Japan). MiR-126 target prediction Putative targets of miR-126-3p were in silico predicted by comparing results of different miRNA prediction programs including TargetScan (http://www.targetscan. org/), Microcosm Targets (http://www.ebi.ac.uk/en-

right-srv/microcosm/cgi-bin/targets/v5/search.pl) and RNA22 (http://cbcsrv.watson.ibm.com/rna22.html). Based on their high prediction scores in different programs, several candidates such as a disintegrin and metalloproteinase with thrombospondin motifs 9 (ADAMTS9), apelin receptor (APLNR), LIM domain containing preferred translocation partner in lipoma (LPP), receptor-interacting serine–threonine kinase 3 (RIPK3) and TLN2 were selected for further validating. DNA construct To construct luciferase reporter plasmids, a series of wild-type or mutated sequences corresponding to the predicted miRNA response elements (MREs) were synthesized as sense and antisense oligomers, annealed and cloned into psiCHECK-2 vector (Promega, Madison, WI, USA) at restriction sites XhoI and NotI directly in the downstream of Renilla luciferase. The nucleotide sequences of the constructs were confirmed by DNA sequencing analyses. The oligonucleotide sequences used for constructing were shown in Table S1. Cell cultures and transfection NIH3T3 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% foetal bovine serum, 100 units/ml penicillin and 100 lg/ml streptomycin at 37°C with 5% CO2. Cell culture reagents and plasticware were purchased from Gibico and Corning Inc., respectively. For transfection, approximately 5 9 103 cells/well were seeded in 96-well plates at 24 h before transfection. MiR-126 mimics or scrambled RNA (GenePharma, Shanghai, China) was cotransfected at a final concentration of 15 nM with 2 ng of the target gene construct or vector only using lipofectamine 2000 according to the manufacturer’s instructions. Dual luciferase assay Cells were lysed 48 h post-transfection with passive lysis buffer (Promega) after washing with PBS in 96-well plates. Firefly and Renilla luciferase activities were tested consecutively with the dual luciferase reporter assay system (Promega) using a luminometer (Mithras LB960; Berthold Technologies, Bad Wilbad, Germany). In all cases, Renilla luciferase activity was used to assess the effect of the MREs on translation efficiency, while constitutively expressed firefly luciferase in psiCHECK2 was used as a normalization control for transfection efficiency. The ratio of Renilla luciferase to firefly luciferase was calculated for each well. All luciferase assays were repeated a minimum of three independent experiments with triplicate wells in each group. Real-Time PCR for mRNA quantitative First strand cDNA synthesis was performed with 1 lg of total RNA using the ReverTra Ace qPCR RT kit (Toyobo) following the manufacturer’s protocols. The relative quantitation of mRNA was detected using the SYBRâ Green Real-time PCR Master Mix (Toyobo) with specific primers on the Eppendorf realplex2 © 2014 Blackwell Verlag GmbH

MiR-126-3p and its Target Talin2 in Bovine CL

Mastercycler (Eppendorf). The PCR reaction was carried out using the cDNA (1 : 10 diluted) as templates and Ex Taq polymerase (Takara) with the following conditions: 5 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. All test samples were run in triplicate, along with negative control. The efficiencies of primers were determined by amplification of a standardized dilution series. The specificity of PCR products was evaluated by melting curves and agarose gel electrophoresis. The results were normalized by GAPDH, and fold change was determined by the comparative threshold method (2MMCt). The mRNA-specific primers are listed in Table S1. Western blot Protein was extracted from samples of four stages of CL using lysis buffer (10 mM Tris-HCl, pH 8, 100 mM NaCl, 1% Nonidet P-40, 2 mM sodium orthovanadate) containing protease inhibitors and phosphatase inhibitors (Sangon, Shanghai, China) as per the manufacture’s instruction. Cell lysates were cleared by centrifugation at 4°C for 15 min at 16 000 9 g to remove cellular debris. Protein concentrations were determined with Braford Protein Assay Kit (Sangon) at the absorbance of 595 nm in the Synergy MultiMode Microplate Reader (BioTek, Winooski, VT, USA). For Western blot analysis, 30 lg of total protein was size-fractionated using SDS-PAGE on 10% gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) by electrophoresis. The membrane was blocked for 1 h at room temperature in 5% milk and probed with primary antibodies including goat anti-talin2 polyclonal antibody (sc-7534; Santa Curz, Santa Curz, CA, USA) or mouse anti-b-actin monoclonal antibody (sc-47778; Santa Cruz) overnight at 4°C. The membranes were washed 3–4 times followed by incubation with horseradish peroxidase-conjugated donkey antigoat or goat anti-mouse IgG (Santa Cruz) for 60 min at room temperature. Protein bands were visualized by the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA). The density of each band was determined by chemiluminescent bio-imaging system (Micro Chemi, Jerusalem, Israel).

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by stem-loop RT-qPCR. MiR-126-3p could be detected in the CL throughout the oestrous cycle. The level of miR-126-3p in the middle or late stages was more abundant (p < 0.01) than that in early or regressed stages (Fig. 1). To differentiate cell type in bovine CL, the histology at the four stages was evaluated with H&E stain techniques. Large luteal cells (LLC) and small luteal cells (SLC) were identified according to their shapes and localization (Fig. 2a–d). Meanwhile, localization of miR-126-3p in the four stages of CL during the oestrous cycle was investigated by in situ hybridization. Consistent with the data obtained from stem-loop RT-qPCR, the signals of miR-126-3p were observed throughout the four luteal phase, and strong signals were observed in the middle and late stages of CL. Moreover, miR-1263p was mainly localized in the large and small luteal cells at different stages of CL during the oestrous cycle (Fig. 2e–h). No positive signal was obtained for CL tissues hybridized with scramble probes (Fig. 2i–l). Identification of miR-126-3p target genes ADAMTS9, APLNR, LPP, RIPK3 and TLN2 were predicted as the putative targets of miR-126-3p by several widely used algorithms such as TargetScan, Microcosm Targets and RNA22. Dual luciferase reporter assays were subsequently performed to assess the authenticity of candidate targets, with the constructs containing the putative miR-126-3p responsive element (Fig. 3a). It was indicated in comparison with cells transfected with scrambled RNA that the relative luciferase activity (Renilla/Firefly) in the presence of miR-126-3p mimic significantly decreased (p < 0.05) in the cells cotransfected with TLN2 constructs (Fig. 3b). However, the relative luciferase activity for empty vector, ADAMTS9, APLNR, LPP or RIPK3 constructs, did not showed significant difference (p > 0.05) between miR-126-3p mimic and scrambled RNA group (Fig. 3b). Thus, TLN2 appeared to be the target gene regulated by miR-126-3p. Analysis of miR-126-3p responsive element in TLN2 Alignment of the responsive element in TLN2 mRNA for miR-126-3p indicated that the sequence was highly

Statistics The significance of the differences between different groups was determined by Tukey’s multiple-comparison test (one-way ANOVA analysis) using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). All data are represented as mean  SEM. A p value < 0.05 or 0.01 was considered as indicating a significant or extremely significant difference, respectively.

Results Expression pattern and localization of miR-126-3p at different stages of bovine CL during the oestrous cycle The expression pattern of miR-126-3p in the four stages of bovine CL during the oestrous cycle was determined © 2014 Blackwell Verlag GmbH

Fig. 1. Quantitative assays of miR-126-3p in different stages of bovine corpous luteum (CL) during the oestrous cycle. Quantitative measurements of miR-126-3p from the four stages of bovine CL detected by stem-loop RT-qPCR. Data presented are from three independent studies each performed in triplicate (means  SE). **p < 0.01

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Fig. 2. Localization of miR-1263p at different stages of bovine corpous luteum (CL) during the oestrous cycle. (a–d) Sections at the four stages of bovine CL were stained with H&E. (e–h) In situ hybridization was performed with miR-126-3p probes at the four stages of bovine CL. (i–l) In situ hybridization was performed with scramble probes at the four stages of bovine CL. Early, middle, late and regressed stages of CL sections were presented in the longitudinal direction as indicated, respectively. Large luteal cells (LLC) and small luteal cells (SLC) were indicated in the sections. Bars in sections represent 50 lm

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conserved across species of mammalian (Fig. 4a). Moreover, the duplex structure between miR-126-3p and MRE in the TLN2 mRNA revealed that the binding site located at nt 8131-8149 (30 -UTR) of TLN2 mRNA (XM_606666) completely complementary to miR-126-3p seed region (nt 2–8; Fig. 4b). To further validate miR-126-3p responsive element in TLN2 mRNA, a mutated construct was prepared at the sequence complementary to miR-126-3p seed region and base-pairing region of the 50 end (Fig. 4c). Dual luciferase reporter assay demonstrated the relative luciferase activity in the MRE mutated group completely restored even in the presence of a miR-126-3p mimic when compared to the wild-type group (Fig. 4d). Taken together, the data suggested that miR-126-3p could target TLN2 mRNA directly by binding the MRE within the 30 -UTR.

Fig. 3. Identification of miR-1263p targets using dual luciferase assay. (a) Schematic of luciferase constructs. A series of wild-type or mutated sequences corresponding to the predicted miR-126-3p miRNA response elements (MREs) were cloned into psiCHECK-2 vector directly in the downstream of Renilla luciferase at the restriction sites XhoI and NotI. (b) Target genes regulated by miR126-3p were identified using dual luciferase assay with putative MRE constructs in the presence of miR-126-3p mimics or scrambled RNA (NC). Results are the means  SE derived from three independent experiments with triplicates each. *p < 0.05

Inverse correlation between miR-126-3p and TLN2 in bovine CL during the oestrous cycle Quantitative assays of TLN2 mRNA in the four stages of bovine CL showed that TLN2 was expressed throughout the oestrous cycle, and the expression level in the middle stage was significantly more abundant (p < 0.05) than the others (Fig. 5a). The level of TLN2 protein in the four stages of CL detected by Western blot demonstrated that it was also expressed throughout the oestrous cycle; however, expression of TLN2 at protein level in the middle stage was much lower (p < 0.05) than the others (Fig. 5b). Considering the data obtained from Fig. 1, it was concluded that there was an inverse correlation between miR-126-3p and its target TLN2 protein in the developmental stages (especially in the middle stage) of bovine CL during the oestrous cycle. © 2014 Blackwell Verlag GmbH

MiR-126-3p and its Target Talin2 in Bovine CL (a)

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Fig. 4. Analysis of miR-126-3p responsive element in the TLN2 mRNA. (a) MiR-126-3p MRE in the TLN2 mRNA showing species conservation. The sequence complementary to miR-126-3p seed region was highlighted. (b) Duplex structure between miR-126-3p and MRE in the TLN2 mRNA predicted by mfold program (http://mfold.rna.albany.edu/?q=mfold). (c) Mutation diagram of miR-126-3p MRE in the TLN2 mRNA. The sequence complementary to miR-126-3p seed region and base-pairing region at the 50 end was mutated as indicated. (d) Dual luciferase assay with TLN2 MRE wild-type (WT) or mutant (MU) constructs in the presence of miR-126-3p mimics or scrambled RNA (NC). Data are indicated as the means  SE derived from triplicate transfectants of three independent experiments. *p < 0.05

Discussion It has been demonstrated that miRNAs were associated with the follicular–luteal transition and were important for the development and function of the ovarian CL (Otsuka et al. 2008; McBride et al. 2012). However, the role of individual miRNAs in the CL during the oestrous cycle has not been extensively investigated so far. To explore the role of miRNAs in the CL, it is necessary to determine the mRNAs targeted by miRNAs. This study is for the first time to characterize miR-126-3p and its target TLN2 in the different stages of bovine CL during the oestrous cycle. MiR-126-3p is highly expressed in the endothelial cells, recognized as an essential regulator of angiogenesis through regulating angiogenic growth factor signalling (Fish et al. 2008; Wang et al. 2008; Nicoli et al. 2010). As the CL is accompanied by intensive vascularization during the development, it was expected that miR-126-3p would be detected in the bovine CL. Indeed, our previous microarray analysis (Ma et al. 2011) and current quantitative study demonstrated that miR-126-3p was expressed throughout the oestrous cycle and more abundant in the middle and late stages of bovine CL. Moreover, in this study, it was observed that miR-126-3p was also mainly localized in large and small bovine luteal cells. It had demonstrated that vasoactive and angiogenic factors, which are produced in the developmental CL, participated in interactions between the endothelial cells and steroidogenic luteal cells (Berisha et al. 2000; Miyamoto et al. 2009).Also, recent studies had suggested that miR-126-3p could influence the expression of apoptosis markers and suppress the release of steroid hormones including progesterone, testosterone and estradiol in the human primary ovarian granulosa cells (Sirotkin et al. 2009, 2010). Hence, it was suggested from these studies and our findings that miR-126-3p produced by these cells possibly acts as an autocrine regulator of luteal cell function. © 2014 Blackwell Verlag GmbH

In addition to other known targets of miR-126, TLN2 was identified a novel target of miR-126-3p in the present study, and this regulation may be broadly (a)

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Fig. 5. Quantitative assays of TLN2 mRNA and protein in different stages of bovine corpous luteum (CL) during the oestrous cycle. (a) Quantitative measurements of TLN2 mRNA at the four stages of bovine CL detected by real-time RT-qPCR. Results presented are from three independent studies each performed in triplicate. (b) Western blot analyses with anti-TLN2 antibody and anti-b-actin (internal control) in the four stages of bovine CL. Signals from three independent assays were quantified and normalized by b-actin (low panel). Data are presented as the means  SE (*p < 0.05)

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conserved in the different species of mammalian. TLN2 is widely viewed as a key structural protein of integrin adhesion complexes that both regulates the affinity of integrin for extracellular matrix ligands and links adhesions to the actin cytoskeleton (Moser et al. 2009; Das et al. 2014). The cyclic development of the CL and the appropriate remodelling of CL structures over the lifespan are likely to be heavily dependent on the correct extracellular environment (Curry and Smith 2006; Irving-Rodgers et al. 2010). In this study, we demonstrated that TLN2 highly expressed in the bovine CL and inversely correlated with miR-126-3p during the oestrous cycle, suggesting miR-126-3p might play an important role in the development of luteal cell through targeting TLN2. In the early stage of CL, high expression of TLN2 may define specialized microenvironments for extracellular matrix that directly or indirectly influence luteal cell process. The fact that TLN2 is downregulated significantly by miR-126-3p in the middle stage, may be important for the maintenance of the luteal cell phenotype and is crucial to the proper function of the CL. Extensive investigation is necessary to precisely define the effect of miR-126-3p on the function of luteal cells and CL development in vitro and in vivo, especially how miR-126-3p modulation in the CL would affect TLN2 expression in vivo. In conclusion, this study suggests that miR-126-3p is present both in the large and small luteal cells in bovine CL throughout the oestrous cycle. The concentration of miR-126-3p at the middle or late luteal stages is greater than that of early or regressed stages. Furthermore,

References Berisha B, Schams D, Kosmann M, Amselgruber W, Einspanier R, 2000: Expression and tissue concentration of vascular endothelial growth factor, its receptors, and localization in the bovine corpus luteum during estrous cycle and pregnancy. Biol Reprod 63, 1106–1114. Chouhan VS, Panda RP, Yadav VP, Babitha V, Khan FA, Das GK, Gupta M, Dangi SS, Singh G, Bag S, Sharma GT, Berisha B, Schams D, Sarkar M, 2013: Expression and localization of vascular endothelial growth factor and its receptors in the corpus luteum during oestrous cycle in water buffaloes (Bubalus bubalis). Reprod Domest Anim 48, 810–818. Curry TE Jr, Smith MF, 2006: Impact of extracellular matrix remodeling on ovulation and the folliculo-luteal transition. Semin Reprod Med 24, 228–241. Das M, Subbayya Ithychanda S, Qin J, Plow EF, 2014: Mechanisms of talin-dependent integrin signaling and crosstalk. Biochim Biophys Acta 1838, 579–588. Donadeu FX, Schauer SN, Sontakke SD, 2012: Involvement of miRNAs in ovarian follicular and luteal development. J Endocrinol 215, 323–334. Ellis SJ, Goult BT, Fairchild MJ, Harris NJ, Long J, Lobo P, Czerniecki S, Van Petegem F, Schock F, Peifer M, Tanentzapf G, 2013: Talin autoinhibition is required for morphogenesis. Curr Biol 23, 1825–1833.

TLN2 is a direct target of miR-126-3p and inversely correlated with miR-126-3p at the developmental stages of bovine CL. These results indicate that miR-126-3p produced by luteal cells may play an important role in regulating luteal function during the oestrous cycle in bovine. Acknowledgements This work was financial supported by the Earmarked Fund for Modern Agro-industry Technology Research System (CARS-38), National Natural Science Foundation (31030058 and 31201076) and National Key Technology R&D Program (2011BAD19B04) in China.

Conflict of interest None of the authors have any conflict of interest to declare.

Author contributions Dai L, Xu J, Liu S, Ma T, Zhu Y, Xu F, Wang S, Zhang Y and Sun G conducted the experimental work and data analysis; Liu S helped in drafting and revision of the manuscript; Ma T, Gao Y and Yuan B helped with design of the study, and Zhang J supervised all the aspects of the research work.

Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1 Oligonucleotide sequences for RT-qPCR or constructing.

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Microarray analysis of differentially expressed microRNAs in non-regressed and regressed bovine corpus luteum tissue, microRNA-378 may suppress luteal cell apoptosis by targeting the interferon gamma receptor 1 gene. J Appl Genet 52, 481–486. McBride D, Carre W, Sontakke SD, Hogg CO, Law A, Donadeu FX, Clinton M, 2012: Identification of miRNAs associated with the follicular-luteal transition in the ruminant ovary. Reproduction 144, 221–233. Miyamoto A, Shirasuna K, Sasahara K, 2009: Local regulation of corpus luteum development and regression in the cow: Impact of angiogenic and vasoactive factors. Domest Anim Endocrinol 37, 159– 169. Moser M, Legate KR, Zent R, Fassler R, 2009: The tail of integrins, talin, and kindlins. Science 324, 895–899. Nicoli S, Standley C, Walker P, Hurlstone A, Fogarty KE, Lawson ND, 2010: MicroRNA-mediated integration of haemodynamics and Vegf signalling during angiogenesis. Nature 464, 1196– 1200. Nilsen TW, 2007: Mechanisms of microRNA-mediated gene regulation in animal cells. Trends Genet 23, 243–249. Otsuka M, Zheng M, Hayashi M, Lee JD, Yoshino O, Lin S, Han J, 2008: Impaired microRNA processing causes corpus luteum insufficiency and infertility in mice. J Clin Invest 118, 1944–1954.

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MiR-126-3p and its Target Talin2 in Bovine CL Petrich BG, 2009: Talin-dependent integrin signalling in vivo. Thromb Haemost 101, 1020–1024. Rekawiecki R, Kowalik MK, Slonina D, Kotwica J, 2008: Regulation of progesterone synthesis and action in bovine corpus luteum. J Physiol Pharmacol 59 (Suppl 9), 75–89. Sakumoto R, Vermehren M, Kenngott RA, Okuda K, Sinowatz F, 2011: Localization of gene and protein expressions of tumor necrosis factor-a and tumor necrosis factor receptor types I and II in the bovine corpus luteum during the estrous cycle. J Anim Sci 89, 3040–3047. Sirotkin AV, Ovcharenko D, Grossmann R, Laukova M, Mlyncek M, 2009: Identification of microRNAs controlling human

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ovarian cell steroidogenesis via a genome-scale screen. J Cell Physiol 219, 415– 420. Sirotkin AV, Laukova M, Ovcharenko D, Brenaut P, Mlyncek M, 2010: Identification of microRNAs controlling human ovarian cell proliferation and apoptosis. J Cell Physiol 223, 49–56. Skarzynski DJ, Piotrowska-Tomala KK, Lukasik K, Galvao A, Farberov S, Zalman Y, Meidan R, 2013: Growth and regression in bovine corpora lutea: regulation by local survival and death pathways. Reprod Domest Anim 48(Suppl 1), 25–37. Stocco C, Telleria C, Gibori G, 2007: The molecular control of corpus luteum formation, function, and regression. Endocr Rev 28, 117–149.

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Submitted: 13 Mar 2014; Accepted: 16 Jul 2014 Author’s address (for correspondence): J Zhang, Laboratory Animal Center, College of Animal Sciences, Jilin University, Changchun 130062, China. E-mail: [email protected]

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Characterization of miR-126-3p and its target talin2 in the bovine corpus luteum during the oestrus cycle.

Although clear advances have been made in understanding of gene transcriptional regulation in the corpous luteum (CL) during the oestrous cycle, littl...
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