111 ALTERATIONS IN BOVINE ENDOMETRIAL MESSENGER AND micro RNA EXPRESSION PROFILE DUE TO CLINICAL ENDOMETRITIS

Abstract

Endometritis is a postparturition infection of endometrium which is known to reduce fertility of dairy cows. However, the molecular and biochemical changes incurred in the endometrium as a result of such inflammations are not yet known. Therefore, the present study was conducted to investigate transcritome (mRNA and microRNA) changes associated with the incidence of clinical endometritis. For this, Holestein Friesian cows were grouped as clinically sick (CS) or healthy (HY) based on clinical symptoms and histological evaluation. Following this, total RNA, including small RNAs, was isolated from endometrial biopsies taken from each group of cows and subsequently used for mRNA and microRNA expression studies. The human miRCURY LNA Universal RT microRNA PCR array system (Exiqon A/S, Vedbaek, Denmark) (with 750 miRNA probes) was used for miRNA expression profiling, while the bovine GeneChip Genome Array (Affymetrix, Santa Clara, CA) was used for expression profiling of mRNAs from the same RNA pools. Following the miRNA PCR array run, data analysis was performed using a comparative threshold cycle (CT) method. A total of 21 miRNAs were found to be differentially expressed (fold change ≥2 and P ≤ 0.05) between CS and HY cows. Among these, 11 miRNAs including mir-526b*, mir-339-3p, mir-608, mir-10a, mir-943 and mir-19b-1* were enriched and 10 miRNAs including mir-1265, mir-658, mir-196b, mir-498 and mir-192 were downregulated in CS compared with the HY animals. The global endometrial mRNA expression analysis between CS and HY cows was performed using 250 ng of total RNA as a starting material. After RNA amplification, fragmented and biotin-labelled cRNA samples were hybridized to Bovine Genome Array (Affymetrix). The microarray data normalization and background correction were performed using GCRMA. A total of 67 genes were found to be differentially expressed (with fold change ≥2 and P ≤ 0.05) between CS and HY cows. Among those, the transcript level of 30 genes including PTHLH, INHBA, P2RY14, MAOB and KCNB2 were upregulated and 37 genes including CHRDL1, PTGDS, F5, ALPL, FOLH1v and TRIB1 were downregulated in CS compared with HY cows. The Ingenuity Pathway Analysis (Ingenuity Systems Inc., Redwood City, CA) showed that some of those genes to be involved in induction of fibrosis (CXCR4, PLOD2, POSTN, PTHLH and TIMP2), growth of ovarian tumour (KLF6), genital tumour (FOLH1, INHBA, KLF6, MGP and PTGDS), proliferation of epithelial cells (EFNA1, INHBA, MAGED1, PTHLH and SNAI2) and chemotaxis of mononuclear leukocytes (CXCR4, INHBA and RGS1). In addition, target prediction of candidate miRNAs using web-based tools (http://mirecords.biolead.org/prediction_query.php) showed that the majority of the differentially expressed genes are potential targets of the miRNA candidates. Therefore, the results of this study showed transcriptome profile changes associated with bovine clinical endometritis and subsequent influence of fertility in cows.