RNA-Seq Reveals the Role of miR-29c in Regulating Inflammation and Oxidative Stress of Bovine Mammary Epithelial Cells

Abstract

Healthy mammary gland is essential for milk performance in dairy cows. MicroRNAs (miRNAs) are the key molecules to regulate the steady state of mammary gland in dairy cows. This study investigated the potential role of miR-29c in bovine mammary epithelial cells (bMECs). RNA sequencing (RNA-seq) was used to measure the transcriptome profile of bovine mammary epithelial cells line (MAC-T) transfected with miR-29c inhibitor or negative control (NC) inhibitor, and then differentially expressed genes (DEGs) were screened. The results showed that a total of 42 up-regulated and 27 down-regulated genes were found in the miR-29c inhibitor group compared with the NC inhibitor group. The functional enrichment of the above DEGs indicates that miR-29c is a potential regulator of oxidative stress and inflammatory response in bMECs through multiple genes, such as forkhead box O1 (FOXO1), tumor necrosis factor-alpha (TNF-α), and major histocompatibility complex, class II, DQ alpha 5 (BoLA-DQA5) in the various biological process and signaling pathways of stress-activated mitogen-activated protein kinase (MAPK) cascade, Epstein-Barr virus infection, inflammatory bowel disease, etc. The results imply that miR-29c plays an important role in a steady state of bMECs or cow mammary gland and may be a potential therapeutic target for mastitis in dairy cows.

Bta-miR-223 Targeting the RHOB Gene in Dairy Cows Attenuates LPS-Induced Inflammatory Responses in Mammary Epithelial Cells

“…The bMECs (an immortalized MAC-T cell line) were cultured in DMEM/F12 medium (Ge Healthcare Life Sciences Hyclone Laboratories, South Logan, Utah) containing 10% fetal bovine serum (FBS) (Biological Industries Israel Beit Haemek Ltdkibbutz Beit Haemek, Israel) [17]. The purity of the bMECs was assessed by immunofluorescence staining to detect the expression of Cytokeratin 18 (a marker protein for epithelial cells) when the bMECs reached 80-90% confluence based on previously reported methods [17].…”

“…The bMECs (an immortalized MAC-T cell line) were cultured in DMEM/F12 medium (Ge Healthcare Life Sciences Hyclone Laboratories, South Logan, Utah) containing 10% fetal bovine serum (FBS) (Biological Industries Israel Beit Haemek Ltdkibbutz Beit Haemek, Israel) [17]. The purity of the bMECs was assessed by immunofluorescence staining to detect the expression of Cytokeratin 18 (a marker protein for epithelial cells) when the bMECs reached 80-90% confluence based on previously reported methods [17]. Human embryonic kidney (HEK)-293T cells were recovered and cultured in high-glucose DMEM (Ge Healthcare Life Sciences Hyclone Laboratories, South Logan, Utah) containing 5% FBS (Biological Industries Israel Beit Haemek Ltdkibbutz Beit Haemek, Israel).…”

H3K27Me3 abundance increases fibrogenesis during endothelial-to-mesenchymal transition via the silencing of microRNA-29c

“…Additionally, a growing body of evidence indicates that EZH2 can directly methylate non-histone proteins independent of H3K27Me3, although these non-histone substrates are mostly transcription factors and chromatin associated proteins ( 48 ). Also, prediction of potential target genes of miR-29c using a target scan database shows an enormous amount of predicted target genes ( 49 ). We do not investigate all extracellular matrix proteins, but rather focus on collagen I and III, which could serve as an example for how EZH2 targets miR-29c and is thereby involved in the regulation of extracellular matrix proteins.…”

Bta-miR-199a-3p Inhibits LPS-Induced Inflammation in Bovine Mammary Epithelial Cells via the PI3K/AKT/NF-κB Signaling Pathway

“…293T cells were thawed and cultured in Dulbecco’s modified eagle medium (DMEM) (Hyclone, Logan, UT, USA) supplemented with 5% fetal bovine serum (FBS) (System Biosciences, Mountain View, CA, USA). Laboratory-identified frozen MAC-T cells were thawed and cultured in DMEM/F12 (Hyclone, Logan, UT, USA) containing 10% FBS [ 11 , 20 ] ( Figure S1 ). The 293T cells and MAC-T cells were maintained at 37 °C, in 5% CO 2 concentration, and at 100% saturated humidity.…”