Baratta, Mario

Miretti, Silvia

Tewari, Rupal Shukla

English

Tewari, RUPAL SHUKLA

Institutional Research Information System University of Turin

14 December 2021AperTO - Archivio Istituzionale Open Access dell'Università di TorinoOriginal Citation:PILOT STUDY ON CIRCULATING MICRORNAS PROFILE IN PIEDMONTESE CATTLE DURING DIFFERENT GROWTHPERIODSPublisher:Terms of use:Open Access(Article begins on next page)Anyone can freely access the full text of works made available as "Open Access". Works made available under aCreative Commons license can be used according to the terms and conditions of said license. Use of all other worksrequires consent of the right holder (author or publisher) if not exempted from copyright protection by the applicable law.Availability:Black Sea PublishersThis is the author's manuscriptThis version is available http://hdl.handle.net/2318/1771091 since 2021-02-03T11:48:47Ziris-AperTO University of Turin’sInstitutionalResearch Information System and Open Access InstitutionalRepository      This is the author's final version of the contribution published as:  Rupal TEWARI1, Mario BARATTA1 and Silvia MIRETTI1 1Physiology Unit, Department of Veterinary Science, University of Turin, Italy  Pilot study on Circulating microRNAs profile in Piedmontese Cattle during different growth periods  Animal Nutrition, Aquaculture and Veterinary Science Proceedings of the ICABGEH-19 Prague, CZECHIA 11-13 September 2019 Publisher: Black Sea Publishers ISBN: 978-605-031-805-0          Thisfull textwasdownloaded from iris-Aperto: https://iris.unito.it/          iris-AperTO University of Turin’sInstitutionalResearch Information System and Open Access InstitutionalRepository Pilot study on Circulating microRNAs profile in Piedmontese Cattle during different growth periods Rupal TEWARI1, Mario BARATTA1 and Silvia MIRETTI1 1Physiology Unit, Department of Veterinary Science, University of Turin, Italy  Abstract MicroRNA (miRNA)are small (21-23nt) non-coding, highly conservative and tissue specific RNA that regulate geneexpression after transcription and their circulating levels can be measured and thus could be potentially used as biomarker for various physiological changes. However, the role of circulating miRNAs in livestock species has not been fully studied.Therefore, our pilot study aimed to explore and describe expression profile of miRNAs in plasma samples during different growth periods.Out of 24 RNA samples processed, we have used one RNA sample from each age group (newborn calf, 5 month/weaning, 11 month and 16 month of age) for sequencing.Using small RNA sequencing (NGS), we have identified total 1031 miRNAs of which369 miRNAs were expressed in all samples (n = 4 bovine).Among miRNAs identified in plasma, we have found a lot of miRNAs closely related to myoblast proliferation or myocyte differentiation such as miR-1, miR-206, miR-26a and miR-27b.These results provide novel information on bovine circulating miRNAs in Piedmontesebreed but this is pilot study and further analysis is needed.  Key words:cattle, circulating miRNA, skeletal muscle  INTRODUCTION  Piedmontese cattle is a native-breed of North-West Italy and is characterized by a peculiar muscular hypertrophic development called double-muscle. Genesis of skeletal muscle is a complex process comprising of proliferation, differentiation, and formation of myotubes and myofibers. A set of postnatal muscle stem cells (satellite cells) produce myoblast cells that can fuse with the existing myofibers, thus increasing their size(Robelin et al, 1993). These molecular events are regulated by myogenic regulatory factors (e.g. MyoD) and microRNAs (miRNAs)(Bukingham et al, 2014). MiRNAs are small non-coding RNAs, 21-23 nt in length, that regulate gene expression and play key roles in various physiological and developmental processes(Bartel et al, 2004). miRNAs highly enriched in muscle are called myomiRNAs. The main known myomiRNAs are miR-1, miR-133a/b and miR-206. These miRNAs have important roles in regulating myogenesis and differentiation of skeletal muscle (Kim et al., 2006). Other miRNAs such as miR-24, miR-26a, miR27b, miR125b, miR148a, miR-181, and miR-214 have been reported to regulate myoblast physiology but are not particularly abundant in the muscle tissue (Liu et al., 2010). MiRNAs can be also released from the cell and circulate in the blood or other body fluids in a stable cell-free form (ci-miRNAs). Recent studies show that ci-miRNAs participate in cell-to-cell communication to regulate normal and pathological processes indicating that ci-miRNAs can be used as biomarkers of physiological or pathological processes(Stenfeldt et al, 2017; Turchinovich et al, 2012). In livestock, ci-miRNAs have not been totally explored. So, studying the role of ci-miRNAs associated with the skeletal muscle growth could be useful for developing strategies to improve production performance in livestock. Previous studies indicated that circulating levels of tissue specific miRNAs could be measured and used as biomarker for tissue functions and production traits and also for the early diagnosis of animal diseases(Ioannidis, 2018; Farrell, 2015).Therefore, we hypothesize that iris-AperTO University of Turin’sInstitutionalResearch Information System and Open Access InstitutionalRepository plasma miRNA expression profiles are affected by age and associated with production traits in beef cattle and that ci-miRNAs could be promising biomarkers for the genetic selection of meat-purpose animals for future breeding. The aim of this pilot study was to explore and define a panel of expression of ci-miRNAs in the plasma samples collected from the Piedmontese breed cattle just after the birth and during the course of skeletal muscle growth until slaughter. This study is part of a larger project that aims to identify if molecular factors such as specific ci-miRNAs could be effective biomarkers associated with skeletal muscle growth and production traits.   MATERIALS AND METHODS  Animals and collection of samples For our project, blood samples are to be collected from 72 animals falling into four age groups (new born calf, 5 month/weaning, 11 month and 16 month of age) with 18 animals in each group and so far samples have been collected from 24 animals (6 samples for each age group) in Plasma EDTA vacutainers. Sample size was calculated using the online software- Power and Sample Size Calculation-PS, Vanderbilt University. Animals were housed in the animal facility of the Dept of Veterinary Science, University of Turin, under the same management conditions. All the experiments involving Piedmontese cattle were approved by the Ethical Animal Welfare Committee of the Dept. of Veterinary Science, UniTO (Prot. No. 663).   MiRNA and total RNA extraction Blood samples were processed within 2 hr of collection according to a standardized operating procedure (centrifugation at 3500 rpm for 10 min at 22°C) to collect plasma. MiRNA and total RNA were extracted with miRNeasy Plasma/serum kit (Qiagen, Germany) according to the manufacturer’s protocol where Ce_miR39 was added as spike-in control. Quantification of miRNA was done using QuBit 3.0 fluorometer (Invitrogen, Life technologies, USA) and the samples with sufficient quantity of miRNA were immediately reverse transcribed into cDNA (Qiagen/Exiqon). The quality of RNA extraction and reverse transcription was checked with Real-time qPCR using Ce_miR39 primer assay.  Library preparation and sequencing TruSeqSmallRNA Sample Prep kit (Illumina, San Diego, CA) was used for library preparation following the manufacturer’s instructions. Just before the library preparation, RNA samples were again quantified and quality tested by Agilent 2100 Bioanalyzer RNA (Agilenttechnologies, Santa Clara, CA) or by Caliper RNA LabChip GX (Caliper Life Sciences, Hopkinton, MA). Libraries were then prepared for sequencing and sequenced on single-end 75 bp mode on NextSeq500 (Illumina, San Diego, CA). Final libraries were quantified using the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and quality tested by Agilent 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA) or by Caliper RNA LabChip GX (Caliper Life Sciences, Hopkinton, MA).  Sequencing data analysis  The Bcl2Fastq 2.0.2 version of the Illumina pipeline was used to process raw data for both format conversion and de-multiplexing. Whole data was normalized using the standard method by DESeq2.  iris-AperTO University of Turin’sInstitutionalResearch Information System and Open Access InstitutionalRepository RESULTS AND DISCUSSION Out of 24 RNA samples processed, we used one RNA sample from each age group for next generation sequencing (NGS). The quality of sequencing reads was assessed by the read length (Fig 1)  Figure 1The length distribution of all sequence reads (example of one sample)  Identified miRNAs matching sequence reads as determined with up to two mismatches allowed are as follows Age group Known miRNAs Known matched Matched % Newborn 3424 1932 56.43 05 month 3424 2327 67.97 11 month 3424 895 26.14 16 month 3424 1171 34.20  Total 1031 miRNAs were identified and 369 miRNAs were expressed in all samples. The top 20 most abundant miRNAs of total miRNA expression in bovine blood plasma are listed here  bta-miR-286    bta-miR423-5p  bta-miR-191  bta-miR-25  bta-miR-92a  bta-miR-30d  bta-miR-148a  bta-miR-186  bta-miR-27b  bta-miR-30e-5p  bta-miR-151-3p  bta-miR-146b  bta-miR-26a  bta-miR-660  bta-miR-6529a Age in months 0 month or newborn 05 month 11 month 16 month miR-1 (Koutsoulidou et al, 2011)  32.14 10.02 35.3 14.5 miR-206 (Kim et al, 2006) 9.84 0.52 76.49 1.61 miR-133a (Koutsoulidou et al, 2011) 30.18 3.16 76.48 5.23 miR-27b (Christ et al, 2009) 85319.92 121582.8 72960.99 122924.14 miR-221 7699.87 7435.64 6724.81 29248.14 miR-181 b 9322.38 7613.92 6654.21 5923.51 miR-26a 263241.72 333975.31 46997.20 582001.53 miR-214 33.46 20.04 5.88 27.80 miR-24 349.70 504.78 141.20 408.89 iris-AperTO University of Turin’sInstitutionalResearch Information System and Open Access InstitutionalRepository  bta-miR-150  bta-miR-30a-5p  bta-miR-451  bta-let-7i  bta-let-7c Among miRNAs identified in plasma in all samples sequenced, we have found miRNAs involved in either myoblast proliferation or myocyte differentiation. (Table 1)  Table 1Expression (reads in million) of three myo-miRNAs and other miRNAs based on NGS results during four periods of muscle growth.  Arrow shows the higher expression of a miRNA at a particular period of growth. Based on the expression profile of different miRNAs, validation will be done Real time qPCR. All these miRNAs are known to be involved in skeletal muscle growth and differentiation processes. MyomiRNA such asmiR-1 and miR-206have specific roles in modulating bovine skeletal muscle satellite cell proliferation and differentiationthrough the positive regulation of Pax7 andthe downregulation of HDAC4 (Dai et al, 2016) High expression levels of miR-26a are required to promote differentiation of satellitecells (Wang  et al., 2018).The miR-27 family influences many cellular processes and plays an important role in down regulation of Myostatin, a negative regulator of muscle growth, thus regulation of post-natal myogenesis and promotes skeletal muscle satellite cell proliferationin animal production species (Huang et al, 2012;Zhang et al, 2018).  CONCLUSION MiRNAs are necessary for skeletal muscle development and have regulatory roles throughout growth and differentiation. Little is known about role of ci-miRNA in cell-to-cell communication to regulate normal skeletal muscle processessuch as the regulation ofmuscle differentiation. Nevertheless, to our knowledge, no studies have examined ci-miRNA expression profiles during different growth periods in bovine. Thus, the primary objective of this study was to identify if a panel of miRNA-centered on regulation in the context of bovine skeletal muscle cell growth and differentiation was represented.  ACKNOWLEDGEMENT This work was supported by Fondazione Cassa di Risparmio di Cuneo and Local Found of the University of Turin (ex 60%).  REFERENCES Dai et al., The role of microRNA-1 and microRNA-206 in the proliferation and differentiation of bovine skeletal muscle satellite cells. In Vitro Cell Dev Biol Anim. 2016 Jan;52(1):27-34  Zhang et al, MiR-27b promotes sheep skeletal muscle satellite cell proliferation bytargeting myostatin gene. Journal of Genetics, Vol. 97, No. 5, December 2018, pp. 1107–1117  J Robelin et al., Myosin expression in semitendinosus muscle during fetal development of cattle: immunocytochemical and electrophoretic analyses. ReproduNutr Dev (1993) 33, 25-41  Buckingham M., Peter W.J. Gene Regulatory Networks and Transcriptional Mechanisms that Control Myogenesis. Developmental Cell (2014) 28;3, 225-238  iris-AperTO University of Turin’sInstitutionalResearch Information System and Open Access InstitutionalRepository Bartel DP (1). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. (2004) 23;116(2):281-97.  Stenfeldt C, Arzt J, Smoliga G, LaRocco M, Gutkoska J and Lawrence P. Proof-of-concept study: profile of circulating microRNAs in Bovine serum harvested during acute and persistent FMDV infection. Virology Journal (2017) 14:71.   Turchinovich A, Weiz L and Burwinkel B. Extracellular miRNAs: the mystery of their origin and function. Trends in biochemical sciences. (2012) 37:460‐465  Miretti S, Martignani E, Accornero P and Baratta M.Functional effect of mir-27b on myostatin expression: a relationship in piedmontese cattlewith double-muscled phenotype. BMC Genomics 2013, 14:194.   Huang Z, Chen X, Yu B, He J, Chen D: MicroRNA-27a promotes myoblastproliferation bytargeting myostatin. Biochem Biophys Res Commun 2012,423:265–269.  Crist CG, Montarras D, Pallafacchina G, Rocancourt D, Cumano A, Conway SJ,Buckingham M: Muscle stem cell behavior is modified by microRNA-27regulation of Pax3 expression. Proc Natl Acad Sci USA 2009, 106(32):13383–13387.  Kim HK, Lee YS, Sivaprasad U, Malhotra A, Dutta A: Muscle-specificmicroRNA miR-206 promotes muscle differentiation. J Cell Biol 2006,174(5):677–687  Koutsoulidou A, Mastroyiannopoulos NP, Furling D, Uney JB, Phylactou LA:Expression of miR-1, miR-133a, miR-133b and miR-206 increases duringdevelopment of human skeletal muscle. BMC Dev Biol 2011, 11:3  Dmitriev et al. Simultaneous miRNA and mRNA transcriptomeprofiling of human myoblasts reveals a novel setof myogenic differentiation-associated miRNAsand their target genes. BMC Genomics 2013, 14:265.  Luo et al. MicroRNAs Involved in Skeletal Muscle DifferentiationMarch 2013Journal of Genetics and Genomics 40(3):107-16             

PILOT STUDY ON CIRCULATING MICRORNAS PROFILE IN PIEDMONTESE CATTLE DURING DIFFERENT GROWTH PERIODS

Rupal Shukla Tewari

Mario Baratta

Silvia Miretti

Archivio istituzionale della Ricerca -  Università degli Studi di Parma

https://iris.unito.it/bitstream/2318/1771091/1/Tewari_Full%20text%20con%20copertina%20OA.pdf

PILOT STUDY ON CIRCULATING MICRORNAS PROFILE IN PIEDMONTESE CATTLE DURING DIFFERENT GROWTH PERIODS

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