2.1 The expression of LGR4 in cow uterus tissues.
Endometritis leads to endometrium injury and early loss of embryos . Fig. 1a showed apparent inflammatory damage in infected cow uterine tissues, with a large number of inflammatory cell infiltration, visible swelling. qRT-PCR results showed more secretion of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, while decrease of anti-inflammatory mediators IL-10 in endometritis tissue (Fig. 1b). To confirm the expression of LGR4 in cow uterus, immunohistochemistry displayed that LGR4 is mainly placed in plasma membrane, extracellular, cytoskeleton, and nucleus (Fig. 1c). Meantime, our western blot protein expression and qRT-PCR mRNA levels of LGR4 were reduced in infected uterus relative to control uterus tissues (Figs. d-f). Thus, our results reflected that LGR4 was down-regulated in the presence of uterus inflammation, suggesting that lack of LGR4 may lead to enhancing inflammation.
2.2 LGR4 suppresses the secretion of inflammatory cytokines in BENDs.
Given the above results, to further investigate whether the reduction of LGR4 will induce the secretion of large number of inflammatory cytokines. Different doses of LPS (0, 0.5, 1.0, 1.5, 2.0 μg/ml) treated on the BENDs at different time points (0, 3, 6, 12, 24 h). The data indicated that LGR4 decreased both in a dose- and time-dependent manner (Figs. 2a-b). In addition, CCK-8 was used to analyzed the effect of LPS on BENDs viability. The cell viability of BENDs was found to be largely unaffected with LPS (1.0 μg/ml ) stimulation, as detected by CCK-8 assay in Fig. 2c. The result appeared that the level of LGR4 was dramatically lowered 3-fold change was verified by qRT-PCR in Fig. 2f. It also revealed that knockdown LGR4 up-regulated the production of IL-1β, IL-6 and TNF-α (Fig. 2g).
2.3 LGR4 inhibits the inflammatory response by blocking NF-κB phosphorylation.
Given that LGR4/Gpr48 was a negative regulator in TLR2/4-associated immune responses. Therefore, we speculate that LGR4 negative feedback regulates the activation of NF-κB p65 phosphorylation, enhance the transcription of pro- inflammatory cytokines to cause inflammation. To verify the above hypothesis, si-LGR4 or si-NC was transfected into cells to knock down LGR4 expression, and the transfection efficiency was verified by western blot (Figs. 3a-b). Fig. 3a and Fig.3c showed that the phosphorylation level of NF-κB p65 after transfection of si-LGR4 was apparent inhibited. Meantime, immunofluorescence also confirmed this result (Figs. 3d-e). Taken together, these data confirmed that knockdown of LGR4 significantly suppresses the activity of phosphorylated NF-κB p65.
2.4 MiR-34a inversely correlates with the expression of LGR4.
Recent studies have shown that miRNA is involved in the embryonic development and various biological processes of ruminants. Based on the finding that LGR4 has anti-inflammatory properties, four online websites (miRcode, miRDB, Targetscan and ENCORI ) were used to predict the putative microRNA that may target LGR4. By taking the intersection of the prediction results of all websites, as shown in Fig. 4a, it was found that 5 microRNAs (miR-34a, miR-34a, miR-34c, miR-302a and miR-218) have the potential to target LGR4.
Based on this result, qRT-PCR was performed to verify the level of putative microRNA under the stimulation of LPS (1.0 μg/ml) (Fig. 4b) and in infected cow uterus tissues (Fig. 4c). The findings indicated that miR-34a, miR-34b, miR-34c, miR-218 and miR-302a were up-regulated, particularly, miR-34a was increased about 5-fold change.
To further determine the regulatory relationship between miR-34a and LGR4, miR-34a mimic or inhibitor were transfected into BENDs and then stimulated the cells with LPS (1.0 μg/ml) for 6 h. The transfection efficiency was verified by qRT-PCR (Fig. 4d). Figs. 4e-f appeared that the expression of LGR4 mRNA and protein in the mimic group was markedly lowered, but the inhibitor group achieved the opposite result. The data further displayed that miR-34a may directly negatively regulates LGR4 mRNA and then inhibits its translational level, which also confirmed the interaction between miR-34a and LGR4.
2.5 MiR-34a directly targets the 3′UTR of the LGR4.
LGR4 is a member of the leucine-rich repeat domain-containing G protein-coupled receptors, and it has been predicted that its interaction with miR-34a is highly conserved among species as shown in Fig. 5a. Based on website prediction results, miR-34a may target the 3'UTR of LGR4, and the interaction map is shown in Fig. 5b. Furthermore, RNAhybrid 2.2 was also shown that miR-34a has the potential to bind LGR4 3'UTR according to the calculation of minimum free energy (Fig. 5c). To further explore the interaction mechanism between miR-34a and LGR4, LGR4 3'UTR (WT-3'UTR or MuT-3'UTR) were amplified and cloned into psiCHECK-2 vector to synthesis the psiCHECK-2-LGR4 3'UTR plasmid (Fig. 5d), then co-transfected with miR-34a mimics or mimics NC or negative control into the 293T cells, the results of the dual-luciferase reporter gene assay described that the fluorescent activity of the WT-3'UTR was drastically decreased, while MuT-3'UTR group had no notable difference (Fig. 5e), reflecting that miR-34a directly targets the 3'UTR of LGR4 mRNA.
2.6 MiR-34a regulates inflammation through NF-κB in BENDs.
In this study, we determined that miR-34a was up-regulated in both the infected cow uterine tissue and the endometrial epithelial cells stimulated by LPS (1.0 μg/ml), contrary to the expression of LGR4. Based on the anti-inflammatory potential of LGR4. To further explain the function of miR-34a in the inflammatory response, miR-34a gain-of-function or loss-of-function experiments were performed. The data displayed that overexpression of miR-34a not only suppressed the expression of LGR4 (Figs. 6a-b), but also promoted the phosphorylation of NF-κB p65(Figs. 6c and 6d), conversely, the miR-34a inhibitor group had the opposite result, in line with the results of immunofluorescence assay (Figs. 7a-b). At the meantime, the production of IL-1β, IL-6 and TNF-α were strongly higher in miR-34a mimic group (Fig. 7c), but remarkably lower in miR-34a inhibitor group (Fig. 7d). Taken together, it further implied that miR-34a has the potential to promote the progression of inflammation.
2.7 Inhibition of miR-34a suppresses endometritis in mice.
miR-34a is highly conserved among species. To furher investigate the role of miR-34a on endometritis in vivo, we established a mouse endometritis model following the previous laboratory method. The groups as follows: blank group, LPS group, LPS+ miR-34 antagomir NC , and LPS + miR-34a antagomir. As shown in Fig. 8a, Kunming mice were infused with LPS ( 50 μl, 1 mg/ml ) in each uterine horn, and the day of injection was recorded as day 0 (D0). 24 h later, miR-34a antagomir or NC (intrauterine injection of 0.5 μmol/ kg)  was treated into the uterus on D1, D4, D7 and uterine tissues were collected on D10. qRT-PCR (Fig. 8b) verified the expression of miR-34a, showing a significant reduction after miR-34a antagomir treatment. Furthermore, LGR4 was remarkably inhibited after LPS stimulation, while the expression was upregulated after miR-34a antagomir injectsion (Fig. 8c-d). Consistently, miR-34a antagomir suppressed the entry of NF-κB into the nucleus (Fig. 8e-f) and lowered the production of IL-1β，IL-6 and TNF-α (Fig. 8g). Overall，our data reveal that knockdown of miR-34a could suppress the inflammatory cytokines and thus allesviate endometritis.
2.8 IL-1β suppresses LGR4 expression by enhancing miR-34a.
A et al. has shown that IL-6 and TNF-α-activated p65 to bind to the miR-34a promoter and promote its transcription to enhance its activity. Therefore, we speculate that LPS induced the high expression of miR-34a in BENDs, which is most likely caused by the regulation of the pro-inflammatory cytokines IL-1β induced by activated NF-κB p65. To evaluate the effect of IL-1β on the expression of miR-34a and LGR4, different concentrations of recombinant IL-1β (0, 1, 5, 10 ng/ml) were used to treat BENDs. As shown in the Fig. 9a, miR-34a was enhanced under the incubation of IL-1β for 6 h in a dose manner (0, 1, 10 ng/ml). Moreover, BENDs was transfected with miR-34a inhibitor in the presence or absence of 5 ng/mL IL-1β treatment to further investigate the effect of IL-1β on miR-34a and LGR4. qRT-PCR (Fig. 9b) showed that down-regulation of miR-34a by inhibitor was partially reversed by IL-1β stimulation. As we expected, the mRNA and protein expression of LGR4 were notably aggravated by miR-34a inhibitor, which was rescued in the incubation of IL-1β, evidenced by Figs. 9c-e. Immunofluorescence results showed that IL-1β induced NF-κB p65 into the nucleus, while silencing miR-34a apparently rescued this change (Figs. 9f-g). Consequently, these above data together implied that IL-1β is an inducer of miR-34a enhancement which can also mediate LGR4 expression.
In summary, our results demonstrated that IL-Iβ mediates the transcription of miR-34a, and further positively regulates the secretion of inflammatory mediators via the LGR4-NF-κB pathway, causing an excessive inflammatory response, which in turn damages the endometrium (as shown Fig. 10).