MicroRNA miR-874-3p inhibits osteoporosis by targeting leptin (LEP)

Figures & data

Table 1. The sequence of PCR primers in this study

Primer	Sequences
LEP	Forward: 5′-CACACGCAGTCAGTCTCCTC-3′
	Reverse: 5′-CGGAGGTTCTCCAGGTCAT-3′
ALP	Forward: 5′-ATCTTTGGTCTGGCTCCCATG-3′
	Reverse: 5′- TTTCCCGTTCACCGTCCAC-3′
RUNX2	Forward: 5′-GTAGATGGACCTCGGGAACC-3′
	Reverse: 5′-GAGGCGGTCAGAGAACAAAC-3′
OCN	Forward: 5′-GCAATAAGGTAGTGAACAGACTCC-3′
	Reverse: 5′-GTTTGTAGGCGGTCTTCAAGC-3′
OSX	Forward: 5′-ATGGGCTCCTTTCACCTG-3′
	Reverse: 5′-GGGAAAAGGGAGGGTAATC-3′
miR-874-3p	Forward: 5ʹ-GAACTCCACTGTAGCAGAGATGGT-3’
	Reverse: 5ʹ-CATTTTTTCCACTCCTCTTCTCTC-3’
GAPDH	Forward: 5ʹ-TCCTCTGACTTCAACAGCGA-3’
	Reverse: 5ʹ-GGGTCTTACTCCTTGGAGGC-3’
U6	Forward: 5ʹ-CTCGCTTCGGCAGCACA-3’
	Reverse: 5ʹ-AACGCTTCACGAATTTGCGT-3’

Figure 1. The identification of genes of interest in osteoporosis. (a). The top 20 enriched GO terms of the differentially expressed genes (DEGs) from GSE37558 data series. DEGs selection criteria: adjusted P < 0.05, log|FC|≥2. FC: fold change. (b). The six AMPK signaling pathway-related DEGs and their expression levels in GSE37558 data series. (c). The key network that involves LEP, LEPR, and IRS2 from AMPK signaling pathway

Figure 2. MiR-874-3p promotes the differentiation of hBMSCs into osteoblasts for the treatment of osteoporosis. (a). Determination of the expression of miR-874-3p in femoral neck trabecular bones of OP patients (n = 35) and non-OP patients (n = 35) by qRT-PCR. (b). The expression levels of miR-874-3p were quantified by qRT-PCR on days 0, 3, 7, and 16 after the osteogenic differentiation of hBMSCs. (c). The expression of miR-874-3p was determined by qRT-PCR in hBMSCs transfected with the miR-874-3p mimic or negative control (NC). (d). After 24, 48 and 72 h cultured, cell proliferation was detected by CCK-8 assay in hBMSCs transfected with miR-874-3p mimic. E-H. The expression levels of ALP, RUNX2, OCN and OSX were determined by qRT-PCR in hBMSCs transfected with the miR-874-3p mimic. I. The hBMSCs were overexpressed with miR-874-3p after 16 days of osteogenic differentiation and underwent ARS staining to detect calcium deposition. n = 3, repetition = 3. * P < 0.05, ** P < 0.001. Student’s t-test, One-way or two-way ANOVA were used in statistical analysis. Control, blank control

Figure 3. MiR-874-3p negatively regulates the expression of LEP in hBMSCs. (a). Determination of the expression of LEP mRNA in femoral neck trabecular bones of OP patients (n = 35) and non-OP patients (n = 35) by qRT-PCR. (b). Pearson analysis of correlation between miR-874-3p and LEP in femoral neck trabecular bones of OP patients. (c). The expression levels of miR-874-3p were quantified by qRT-PCR on days 0, 3, 7, and 16 during hBMSCs osteogenic differentiation. (d). Schematic diagram of predicted binding sites of LEP in 3ʹ-UTR of miR-874-3p. (e). Detection of LEP enrichment on miR-874-3p in BMSCs by RNA pull-down. (f). Determination of the dual-luciferase activity of hBMSCs transfected with LEP-MUT or LEP-WT and miR-874-3p mimic or mimic NC. (g). Determination of the mRNA expression levels of LEP in hBMSCs transfected with miR-874-3p mimic or/and oe-LEP by qRT-PCR. (h). Determination of the protein expression of LEP in BMSCs transfected with miR-874-3p mimic or/and oe-LEP by Western blot. n = 3, repetition = 3. * P < 0.05, ** P < 0.001 compared with NC group; ## P < 0.001 compared with mimic+oe-LEP group. One-way ANOVA was used in statistical analysis. Control, blank control

Figure 4. Overexpression of LEP reverses the effect of miR-874-3p mimic on osteogenic differentiation of hBMSCs. (a). After 24, 48 and 72 h culture, cell proliferation was detected by CCK-8 assay in hBMSCs transfected with miR-874-3p mimic or/and oe-LEP. (b)-(e). In the hBMSCs transfected with miR-874-3p mimic or/and oe-LEP, the expression levels of ALP, RUNX2, OCN and OSX were determined by qRT-PCR. (f). The hBMSCs were overexpressed with miR-874-3p mimic or/and LEP after 16 days of osteogenic differentiation and underwent ARS staining to detect calcium deposition. n = 3, repetition = 3. * P < 0.05, ** P < 0.001 compared with NC group; # P < 0.005, ## P < 0.001 compared with mimic+oe-LEP group. One-way or two-way ANOVA was used in statistical analysis. Control, blank control

Data availability statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.