miR-375-3p damaged osteogenesis by inducing cell apoptosis.

<p>(A) Relative expression levels of RUNX2 in MC3T3-E1 cells treated with miR-375-3p mimics. (B) Relative expression levels of SOST in MC3T3-E1 cells treated with miR-375-3p mimics. (C) Representative TUNEL staining images of MC3T3-E1 cells. More TUNEL positive staining cells (Red) were found after the cells were transfected with miR-375-3p mimics. (D) Quantification of TUNEL-positive cells. * p < 0.05, ** p < 0.01. n = 3/group.</p

The examples of region of interests.

<p>The examples of region of interests.</p

Sequences of all primers.

<p>Sequences of all primers.</p

The baseline of mice.

<p>The baseline of mice.</p

miR-375-3p arrested the expression of LRP5 and β-catenin.

<p>(A) Relative expression levels of LRP5 in MC3T3-E1 cells upon transfection of miR-375-3p mimics or inhibitors. (B) Relative expression levels of β-catenin in MC3T3-E1 cells upon transfection of miR-375-3p mimics or inhibitors. (C) Representative staining images of MC3T3-E1 cells. Less green cells were found after the cells were transfected with miR-375-3p mimics. * p < 0.05. ** p < 0.01. n = 3/group.</p

miR-375-3p targeted LRP5 and β-catenin.

<p>(A) The binding sites between miR-375-3p and the 3’UTR of LRP5 and β-catenin. (B) The relative luciferase activity of MC3T3-E1 cells transfected with luciferase-wild type (or mutant) LRP5 plasmids and miR-375-3p mimics. (C) The relative luciferase activity of MC3T3-E1 cells transfected with luciferase-wild type (or mutant) β-catenin plasmids and miR-375-3p mimics. * p < 0.05. ** p < 0.01, NS, not significant. n = 3/group.</p

Loss of function of LRP5 resulted in damaged osteogenesis in vitro and in vivo.

<p>(A) The relative absorbance of MC3T3-E1 cells transfected with a serial dilution of siLRP5 in the WST-1-based colorimetric assay. (B) Representative 3D images of bone microstructure in the knee-joints in LRP5 WT and KO mice. (C) Quantification of trabecular bone volume fraction (BV/TV) in the knee-joints in WT or KO mice was determined from the μCT measurement. (D) Relative expression levels of SMAD7 in the bone tissues of mice. * p < 0.05, ** p < 0.01, n = 3/group.</p

The expression dynamics of effectors of miR-375-3p during osteogenesis.

<p>(A) Relative expression levels of LRP5 during MC3T3-E1 differentiation and osteogenesis. (B) Relative expression levels of β-catenin during MC3T3-E1 differentiation. (C) Relative expression levels of β-catenin in bone tissues from osteoporotic patients and their controls. * p < 0.05. ** p < 0.01.</p

The seven sequences identified in Dap-seq.

Iron is an essential element required for all organisms. Iron response regulator (Irr) is a crucial transcriptional regulator and can affect the growth and iron uptake of Brucella. The growth rate of Brucella melitensis M5-90 irr mutant was significantly lower than that of B. melitensis M5-90 under normal or iron-sufficient conditions, however, the growth rate of the B. melitensis M5-90 irr mutant was significantly higher than that of B. melitensis M5-90 under iron-limited conditions. In addition, irr mutation significantly reduced iron uptake under iron-limited conditions. Previous studies suggested that the Irr protein has multiple target genes in the Brucella genome that are involved in iron metabolism. Therefore, in the present study, a Dap-seq approach was used to investigate the other iron metabolism genes that are also regulated by the Irr protein in Brucella. A total of seven genes were identified as target genes for Irr in this study and the expression levels of these seven genes were similar in both the RNA-seq and qRT-PCR results. The electrophoretic mobility shift assay confirmed that six out of the seven genes, namely rirA (BME_RS13665), membrane protein (BME_RS01725), hypothetical protein (BME_RS09560), ftrA (BME_RS14525), cation-transporting P-type ATPase (zntA) (BME_RS10660), and 2Fe-2S binding protein (BME_RS13655), interact with the Irr protein. Furthermore, the iron utilization and growth assay experiments confirmed that rirA was involve in iron metabolism and growth of Brucella. In summary, our results identified six genes regulated by the Irr protein that may participate in iron metabolism, and the rirA was identified as a regulon of Irr and it also plays a role in iron metabolism of Brucella. Collectively, these results provide valuable insights for the exploration of Brucella iron metabolism.</div

S7 Fig -

The number of CFUs of M5-90 and M5-90 rirA mutant grown in (A) normal TSB, (B) iron-limited TSB, and (C) iron-sufficient TSB. The asterisk positioned atop each time point denotes the statistically significant contrast in growth between M5-90 and M5-90 irr mutant across various temporal intervals. (TIF)</p