biostudies-literatureMa XNational Natural Science Foundation of China1744754https://www.ebi.ac.uk/biostudies/studies/S-EPMC8422159biostudies-literatureUnknown2021<h4>Background</h4>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.<h4>Methods</h4>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) <i>in vitro</i> and <i>in vivo</i> 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.<h4>Results</h4>In this study, we observed that Gram-negative bacteria, represented by <i>Escherichia coli</i>, 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-1<i>β</i>, IL-6, and TNF-<i>α</i> by activating the phosphorylation of NF-<i>κ</i>B p65. Furthermore, IL-1<i>β</i> upregulated miR-34a transcription and downregulated LGR4 expression in an IL-1<i>β</i>-dependent manner.<h4>Conclusions</h4>Taken together, our study confirmed that miR-34a is regulated by IL-1<i>β</i> 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.Oxidative medicine and cellular longevityEnhanced Expression of miR-34a Enhances <i>Escherichia coli</i> Lipopolysaccharide-Mediated Endometritis by Targeting LGR4 to Activate the NF-<i>κ</i>B Pathway.PMC84221593197274431772816Wu ZLiu JUmar TZhou QYin BMa XGuo SZahoor ADeng GfalseEnhanced Expression of miR-34a Enhances <i>Escherichia coli</i> Lipopolysaccharide-Mediated Endometritis by Targeting LGR4 to Activate the NF-<i>κ</i>B Pathway.<h4>Background</h4>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.<h4>Methods</h4>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) <i>in vitro</i> and <i>in vivo</i> 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.<h4>Results</h4>In this study, we observed that Gram-negative bacteria, represented by <i>Escherichia coli</i>, 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-1<i>β</i>, IL-6, and TNF-<i>α</i> by activating the phosphorylation of NF-<i>κ</i>B p65. Furthermore, IL-1<i>β</i> upregulated miR-34a transcription and downregulated LGR4 expression in an IL-1<i>β</i>-dependent manner.<h4>Conclusions</h4>Taken together, our study confirmed that miR-34a is regulated by IL-1<i>β</i> 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.2021-01-01T00:00:00Z20212024-11-13T04:14:54.556Z2022-02-11T10:47:30.68ZS-EPMC84221593450463910.1155/2021/1744754