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      RNA-Seq Reveals Function of Bta-miR-149-5p in the Regulation of Bovine Adipocyte Differentiation

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          Abstract

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          This is a novel study that explored the function of bta-miR-149-5p in the lipid metabolism of bovine adipocytes. Previously, we explored the role of bta-miR-149-5p gene in proliferation and differentiation of bovine adipocytes; however, the underlying mechanism of bta-miR-149-5p function in the regulation of lipid metabolism and adipogenesis in bovine adipocytes is unexplored. A transcriptomic study with integration of bioinformatics study was performed to fully explore the function of bta-miR-149-5p microRNA in bovine adipogenesis. This study will help the scientific community deal with lipogenesis for the breed improvement program of cattle for the provision of healthy meat to consumers.

          Abstract

          Intramuscular fat is a real challenge for the experts of animal science to improve meat quality traits. Research on the mechanism of adipogenesis provides invaluable information for the improvement of meat quality traits. This study investigated the effect of bta-miR-149-5p and its underlying mechanism on lipid metabolism in bovine adipocytes. Bovine adipocytes were differentiated and transfected with bta-miR-149-5p mimics or its negative control (NC). A total of 115 DEGs including 72 upregulated and 43 downregulated genes were identified in bovine adipocytes. The unigenes and GO term biological processes were the most annotated unigene contributor parts at 80.08%, followed by cellular component at 13.4% and molecular function at 6.7%. The KEGG pathways regulated by the DEGs were PI3K-Akt signaling pathway, calcium signaling pathway, pathways in cancer, MAPK signaling pathway, lipid metabolism/metabolic pathway, PPAR signaling pathway, AMPK signaling pathway, TGF-beta signaling pathway, cAMP signaling pathway, cholesterol metabolism, Wnt signaling pathway, and FoxO signaling pathway. In addition to this, the most important reactome enrichment pathways were R−BTA−373813 receptor CXCR2 binding ligands CXCL1 to 7, R−BTA−373791 receptor CXCR1 binding CXCL6 and CXCL8 ligands, R−BTA−210991 basigin interactions, R−BTA−380108 chemokine receptors binding chemokines, R−BTA−445704 calcium binding caldesmon, and R−BTA−5669034 TNFs binding their physiological receptors. Furthermore, the expression trend of the DEGs in these pathways were also exploited. Moreover, the bta-miR-149-5p significantly ( p < 0.01) downregulated the mRNA levels of adipogenic marker genes such as CCND2, KLF6, ACSL1, Cdk2, SCD, SIK2, and ZEB1 in bovine adipocytes. In conclusion, our results suggest that bta-miR-149-5p regulates lipid metabolism in bovine adipocytes. The results of this study provide a basis for studying the function and molecular mechanism of the bta-miR-149-5p in regulating bovine adipogenesis.

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          Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

          In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-seq, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. We present DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression. The DESeq2 package is available at http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0550-8) contains supplementary material, which is available to authorized users.
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            Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

            The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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              Fast gapped-read alignment with Bowtie 2.

              As the rate of sequencing increases, greater throughput is demanded from read aligners. The full-text minute index is often used to make alignment very fast and memory-efficient, but the approach is ill-suited to finding longer, gapped alignments. Bowtie 2 combines the strengths of the full-text minute index with the flexibility and speed of hardware-accelerated dynamic programming algorithms to achieve a combination of high speed, sensitivity and accuracy.
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                Author and article information

                Contributors
                Role: Academic Editor
                Role: Academic Editor
                Journal
                Animals (Basel)
                Animals (Basel)
                animals
                Animals : an Open Access Journal from MDPI
                MDPI
                2076-2615
                22 April 2021
                May 2021
                : 11
                : 5
                : 1207
                Affiliations
                [1 ]Medical College, Xuchang University, Xuchang, Henan 461000, China; guohongfangkl@ 123456126.com
                [2 ]Livestock Management, Breeding and Genetics, The University of Agriculture, Peshawar 25120, Khyber Pakhtunkhwa, Pakistan; smsuhail@ 123456aup.edu.pk (S.M.S.); hamayunkhan@ 123456aup.edu.pk (H.K.); sbvetdr@ 123456yahoo.com (S.B.K.)
                [3 ]College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China; haiderraza110@ 123456nwafu.edu.cn
                [4 ]College of Veterinary Sciences, The University of Agriculture, Peshawar 25120, Khyber Pakhtunkhwa, Pakistan
                [5 ]Department of Animal Husbandry and Animal Wealth Development, Faculty of Veterinary Medicine, Damanhour University, Damanhour 22511, Egypt; ayman.sadaka@ 123456vetmed.dmu.edu.eg
                [6 ]National Beef Cattle Improvement Research Center, Yangling 712100, China
                Author notes
                [* ]Correspondence: rajwalikhan@ 123456aup.edu.pk (R.K.); zanlinsen@ 123456163.com (L.Z.); Tel.: +92-91-922-1262 (R.K.); +86-29-8709-1923 (L.Z.); Fax: +86-29-8709-2164 (L.Z.)
                [†]

                These authors contributed equally to this manuscript.

                Author information
                https://orcid.org/0000-0001-6032-9420
                https://orcid.org/0000-0001-5191-3457
                https://orcid.org/0000-0002-0961-1911
                https://orcid.org/0000-0002-8306-0011
                Article
                animals-11-01207
                10.3390/ani11051207
                8145242
                33922274
                42915add-04ff-4fe2-a025-8ac97b175612
                © 2021 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/).

                History
                : 18 February 2021
                : 18 April 2021
                Categories
                Article

                bta-mir-149-5p,bovine adipocytes,lipid metabolisms,rna-seq

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