Effect of CUMS on body weight, behavioral performance, and serum metabolism. (A) Schematic of the procedure used to establish a CUMS model in mice. (B) Body weight (n= 9). (C) Distance moved and (D) Time spent in the central area of OFT (n= 9). (E) Total distance moved in OFT (n=9). (F) Frequency and amplitude of body movements during struggle. (G) Immobility in TST (n=9). (H) Sucrose preference index (n=9). (I) Serum CORT levels (n=6). (J) Serum NE levels (n=5). All data are presented as means ± SD; ns, P>0.05, *, P < 0.05, **, P < 0.01, ***, P < 0.001, by unpaired Student’s t-test.

Bone mass of the femur decreased in CUMS model. (A) Three-dimensional reconstruction images of femurs from control and CUMS mice. (B) Representative X-ray images of femurs from control and CUMS mice. Quantitative micro-CT analysis of femur (C) trabecular bone and (D) cortical bone. (E)H&E staining, scale bar, 200um. All data are presented as mean ± SD, n=7; *, P < 0.05, **, P < 0.01, ***, P < 0.001, by unpaired Student’s t-test.

Osteogenic and osteoclastic metabolism of mouse femur under psychological stress. (A) Masson staining (B-C) Representative IHC images for OSX, OCN expression in the femur. (D) Calcein double labeling of cortical bone with quantification of MAR (E) qRT-PCR quantification analysis of osteogenic markers including Osx, Ocn, Opn, Dmp1, Runx2, Alp. (F) Representative TRAP staining images of the femur with quantitative analysis of (G) osteoclast surface per bone surface (Oc.S/BS) and (H) number per bone surface (N.OC/B). (I) qRT-PCR quantification analysis of osteogenic markers including Ca2, Mmp9, Nfatc1, Acp5. (J) Representative IHC images for MMP9 in the femur. All data are presented as mean ± SD, n=3; *, P < 0.05, **, P < 0.01, ***, P < 0.001, by unpaired Student’s t-test. Scale bar, 100 um. Figure 3— figure supplement 1 (A) Serum Elisa assay of osteogenic and osteoclastic markers. (B) Serum biochemical assay of osteogenic and osteoclastic markers. All data are presented as mean ± SD, n=4; ns, P>0.05, *, P < 0.05, **, P < 0.01, by unpaired Student’s t-test. Figure 3— figure supplement 2 qRT-PCR quantification analysis of osteoclastic markers including Oscar, Dastamp, Calar, Ctsk, Rank/Opg, Clcn7. All data are presented as mean ± SD, n=3; ns, P>0.05, by unpaired Student’s t-test.

Screening of key miRNAs. (A) The heatmap and (B) Volcano plot for visualization of differentially expressed miRNAs in the femur proximal to the distal femoral growth plate. (p < 0.05) (C-D) Gene Ontology (GO) terms associated with targeted mRNA of differentially expressed miRNAs between groups obtained by database (miRDB and miRTarBase) prediction. (E) Validation by Real-Time expression analysis of miRNAs with statistically significant differences (Con, n=3; CUMS, n=5). (F) Real-Time expression of miR-335-3p in five stress related brain regions (n=4) and (H) serum (n=3). All data are presented as mean ± SD, n=5; *, P < 0.05, **, P < 0.01, ***, P < 0.001, by unpaired Student’s t-test.

Initial validation of the function of miR-335-3p. (A) Representative cytochemical TRAP staining images, Scale bar, 200um. (B) The amounts of osteoclasts (multinucleated TRAP-positive cells), n=5. (C) The positive TRAP area of osteoclasts, n=5. (D) Representative resorption pits, Scale bar, 200um. (E) The area of resorbed surface, n=6. (F) qRT-PCR quantification analysis of Acp5, Nfatc1, Mmp9 expression of RANKL-induced RAW264.7 transfected with mimic-NC, mimic-miR-335-3p, inhibitor-NC, or inhibitor-miR-335-3p, n=3. (G) Representative IF images for FOS, NFATC1, and HOCHEST expression, Scale bar, 200um. (H) Three-dimensional reconstruction images of femurs from agomir NC, agomir-mir-335-3p, antagomir NC and antagomir-miR-335-3p mice. (I) Quantitative micro-CT analysis of femur trabecular bone, n=3; Scale bar, 1mm. All data are presented as mean ± SD; *, P < 0.05, **, P < 0.01, ***, P < 0.001, by unpaired Student’s t-test.

Seek and verification mechanism of miR-335-3p in vivo and in vitro. (A) The IPA analysis of miR-335-3p and its predictive target gene. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) terms associated with targeted mRNA of differentially expressed miRNAs between groups obtained by database (miRDB and miRTarBase) prediction. (D) Firefly luciferase activity (n=3). (C) Sequence alignment of miR-335-3p and its predictive target sites in 3′UTR of Fos. (E) Representative IF images for FOS, CTSK, and HOCHEST expression in the femur (scale bar, 50um). (F) Representative cytochemical TRAP staining images (scale bar, 200um). (G) The area and (H) the number of osteoclasts (multinucleated TRAP-positive cells) (n=4). (I) qRT-PCR quantification analysis of Acp5, Nfatc1, Mmp9 expression of RANKL-induced RAW264.7 transfected with inhibitor-NC, miR-335-3p-inhibitor, or miR-335-3p-inhibitor+T5224 (n=3). All data are presented as mean ± SD; *, P < 0.05, **, P < 0.01, ***, P < 0.001,****, P < 0.0001, by one-way ANOVA with Tukey’s post hoc test.

Schematic model of miR-335-3p as a regulator of osteoclast in psychological stress induced osteoporosis. This study demonstrates that miR-335-3p levels in NAC, serum, and bone tissue are decreased under psychological stress, which reduces its targeting of Fos. These promote Fos translation and binding to NFATC1, increasing osteoclast activity and ultimately leading to bone loss.

(A) Serum Elisa assay of osteogenic and osteoclastic markers. (B) Serum biochemical assay of osteogenic and osteoclastic markers. All data are presented as mean ± SD, n=4; ns, P＞0.05, *, P < 0.05, **, P＜0.01, by unpaired Student's t-test.

qRT-PCR quantification analysis of osteoclastic markers including Oscar, Dastamp, Calar, Ctsk, Rank/Opg, Clcn7. All data are presented as mean ± SD, n=3; ns, P＞0.05, by unpaired Student's t-test.

Reads per million mapped reads (RPMM) expression of miR-335-3p in different tissues.

Cell transfection efficiency. All data are presented as mean ± SD; *, P < 0.05, ****, P<0.0001, by unpaired Student's t-test.

qRT-PCR quantification analysis of Fos expression in femur of control and CUMS mice. All data are presented as mean ± SD, n=3; ns, P＞0.05, by unpaired Student's t-test.