MiR-6835 promoted LPS-induced inflammation of HUVECs associated with the interaction between TLR-4 and AdipoR1 in lipid rafts

<div><p>Background</p><p>High mortality rate of critically-ill patients could be induced by sepsis and septic shock, which is the extremely life threatening. The purpose of this work is to identify and evaluate the potential regulatory mechanism of LPS-induced inflammation associated with miR-6835 and lipid rafts in HUVECs.</p><p>Methods</p><p>The 3’ UTR luciferase activity of AdipoR1 was detected, which was predicted the potential target gene of miR-6835. Moreover, the treated HUVECs with or without inhibitors or mimics of miR-6835 were used. Furthermore, the bio-functions of HUVECs were explored. The protein expression levels of SIRT-1, AMPK, and AdipoR1 were assessed, which were involved in the AdipoR1 signaling pathway. Then, the interaction between TLR-4 and AdipoR1 in lipid rafts and its mediation role on LPS-induced inflammation was investigated in HUVECs.</p><p>Results</p><p>MiR-6835 targeted directly on AdipoR1, and suppressed its expression in mRNA (mimics of miR-6835: 0.731±0.016 vs control: 1.527±0.015, <i>P</i><0.001) and proteins levels, then regulated protein expression of SIRT-1 and AMPK, which were the downstream target genes of AdipoR1 signaling pathway. MiR-6835 enhanced LPS-induced inflammation process in HUVECs (TNF-α: LPS+mimics of miR-6835: 1638.51±78.43 vs LPS: 918.73±39.73, <i>P</i><0.001; IL-6: LPS+mimics of miR-6835: 1249.35±69.51 vs LPS: 687.52±43.64, <i>P</i><0.001), which was associated with the interaction between TLR-4 and AdipoR1 in lipid rafts.</p><p>Conclusions</p><p>MiR-6835 is the key regulator of LPS-induced inflammation process in HUVECs. The interaction between TLR-4 and AdipoR1 mediated by lipid rafts at membrane of HUVECs with inflammation process induced by miR-6835. Our results demonstrated a hopeful strategy for treatment on sepsis by aiming at lipid rafts and miR-6835.</p></div

AdipoR1 could bond with TLR-4.

<p>(A) The results CO-IP’d (co-immunoprecipitated) assay was perfomed, and identified the interaction between AdipoR1 and TLR-4. (B) The confocal images demonstrated that both the two recombinant proteins of AdipoR1 and TLR-4 localized at cell membrane of HUVECs with overlaid exhibition.</p

MiR-6835 inhibited clonogenicity and growth of HUVECs.

<p>(A) The proliferation of HUVECs was restrained by miR-6835 compared to control group. Furthermore, the inhibitors of miR-6835 promoted proliferation of HUVECs. (B and C) The clonogenicity of HUVECs was restrained by miR-6835 compared to control group. Furthermore, the inhibitors of miR-6835 promoted clonogenicity of HUVECs. The data are presented as means±SD from three independent experiments. *<i>P</i><0.05, **<i>P</i><0.01.</p

Predicted consequential pairing of target region of AdipoR1 (top) and miRNA-6835 (bottom).

<p>Predicted consequential pairing of target region of AdipoR1 (top) and miRNA-6835 (bottom).</p

MiR-6835 suppressed genes expression of AdipoR1 pathway in HUVECs.

<p>Our results suggested that the mRNA and protein expression of AdipoR1 was suppressed by miR-6835, respectively (A and B). Moreover, mRNA expressions of AMPK, SIRT-1, and TLR-4 could not be influenced by miR-6835, but their proteins level (B). Additionally, the mRNA expression level of AdipoR1 could be promoted by inhibitors of miR-6835, however, which could not affect mRNAs expression level of AMPK, SIRT-1, and TLR-4 (A). The data are presented as means±SD from three independent experiments. *<i>P</i><0.05, **<i>P</i><0.01.</p

MiR-6835 targeted at AdipoR1 in HUVECs.

<p>The results showed the obviously down-regulated 3’ UTR activities of AdipoR1 in HUVECs, but no alternations were found while AdipoR1 with mutation. Moreover, miR-6835 could not directly target at AMPK, SIRT-1, and TLR-4, respectively. The data are presented as means±SD from three independent experiments. *<i>P</i><0.05, **<i>P</i><0.01.</p