Enhanced Expression of miR‐34a Enhances Escherichia coli Lipopolysaccharide‐Mediated Endometritis by Targeting LGR4 to Activate the NF‐κB Pathway

Abstract

Background. Persistent endometritis caused by bacterial infections has lethal effects on the reproductive performance of dairy cattle, which compromises animal welfare and delays or prevents pregnancy. The microRNA (miRNA) miR-34 family plays a pivotal role in the inflammatory process; however, the precise mechanism of miR-34a in endometritis has not been thoroughly elucidated to date. Methods. In this study, the endometrium of cows diagnosed with endometritis was harvested for bacterial culture and Gram staining to evaluate bacterial contamination of the uterus. Based on this, a bovine endometrial epithelial cell (BEND) inflammation model and a mouse model stimulated with lipopolysaccharide (LPS) in vitro and in vivo were constructed. Cell viability was assessed by CCK-8, trypan blue staining, and flow cytometry. H&E was applied to histopathological analysis. Immunohistochemical, immunofluorescence, qRT-PCR, and western blot assays were performed to measure the mRNA and protein expression of relevant genes. Online databases, plasmid construction, and dual-luciferase reporter gene assays were used to predict and validate the interaction between miR-34a and its target gene LGR4. Finally, mice were injected vaginally with a local antagomir to validate the role of miR-34a in murine uterine inflammation. Results. In this study, we observed that Gram-negative bacteria, represented by Escherichia coli, are the predominant pathogenic agents responsible for the recurrent occurrence of endometritis in dairy cows. Further, miR-34a was found to repress the expression of LGR4 by targeting the 3 ′ untranslated region (3 ′ UTR) of LGR4. miR-34a was upregulated in bovine uterine tissues and bovine endometrial epithelial cells stimulated with LPS. miR-34a induced the release of the proinflammatory cytokines IL-1β, IL-6, and TNF-α by activating the phosphorylation of NF-κB p65. Furthermore, IL-1β upregulated miR-34a transcription and downregulated LGR4 expression in an IL-1β-dependent manner. Conclusions. Taken together, our study confirmed that miR-34a is regulated by IL-1β and suppresses the level of the LGR4 3 ′ UTR, which in turn exacerbates the inflammatory response. Thus, the knockdown of miR-34a might be a new direction for the treatment of endometritis.

Emerging Roles for LGR4 in Organ Development, Energy Metabolism and Carcinogenesis

“…MiR-449a could promote caprine endometrial receptivity by targeting 3′-untranslated region of LGR4 ( An et al, 2017 ). By using a bovine endometrial epithelial cell inflammation model and a mouse lipopolysaccharide-mediated endometritis model, the author confirmed that miR-34a/miR-193a-3p was upregulated by IL-1β and suppressed the level of the LGR4 3′UTR, which in turn amplified the inflammatory response through activating the phosphorylation of NF-κB p65 pathway, suggesting miR-34a/miR-193a-3p-LGR4 playing a pivotal role in endometritis ( Ma et al, 2021 ; Yin et al, 2021 ). Furthermore, the gene of LGR4 modulated a WNT-NR5A2 signaling cascade facilitating the secretion, maturation and steroidogenesis of oviduct epithelial cells to safeguard the development and function of oviduct in mice ( Tan et al, 2021 ).…”

Syringin Prevents 6-Hydroxydopamine Neurotoxicity by Mediating the MiR-34a/SIRT1/Beclin-1 Pathway and Activating Autophagy in SH-SY5Y Cells and the Caenorhabditis elegans Model

“…The pro-inflammatory transcription factor NF-κB can bind to the gene promoter region to increase the expression of miR-34a [ 60 ]. Similarly, the upregulation of miR-34a can also enhance the activity of NF-κB [ 61 ]. In addition, the overexpression of miR-34a can promote the apoptosis of cancer cells [ 25 ], hinder the cell cycle, and inhibit metastasis [ 62 ] and angiogenesis [ 58 ].…”

MicroRNA‐34a in coronary heart disease: Correlation with disease risk, blood lipid, stenosis degree, inflammatory cytokines, and cell adhesion molecules

“…Apart from being closely related to blood lipid, miR‐34a also discloses a positive correlation with inflammatory factors in many diseases such as rheumatoid arthritis and endometritis. 18 , 38 For instance, one study finds that miR‐34a induces the release of the proinflammatory cytokines including IL‐1β, IL‐6, and TNF‐α in endometritis. 38 However, no relevant study reports the association between miR‐34a and inflammation cytokines in CHD patients.…”

“… 18 , 38 For instance, one study finds that miR‐34a induces the release of the proinflammatory cytokines including IL‐1β, IL‐6, and TNF‐α in endometritis. 38 However, no relevant study reports the association between miR‐34a and inflammation cytokines in CHD patients. Our study found that miR‐34a was positively correlated with inflammatory‐related indexes such as CRP, TNF‐α, IL‐1β, and IL‐17A.…”