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. 2023 Feb 8;12:e79768. doi: 10.7554/eLife.79768

Figure 4. The loss of MIR27a alone causes osteoporotic defects.

(A) Diagrams illustrate the Mir23a cluster (WT), and the creation of mouse strains deficient for Mir27a (∆Mir27a) by CRISPR/Cas9 genome editing. Broken lines and arrows indicate the deleted genomic regions and primers used for PCR genotyping analysis, respectively. (B) PCR-based genotyping identifies the wild-type (+/+), heterozygous (+/–), and homozygous (–/–) for miR-27a mice showing the mutant (∆) alleles with deletion of Mir27a result in the generation of shorter PCR products. (C) RT-PCR analysis reveals the disruption of MIR27a but not MIR23a and MIR24-2 RNAs in ∆Mir27a mutants. The analysis of small non-coding RNA U6 is used as an internal control. Femurs of the 3-month-old (3 M) wild-type (+/+) and mutant (–/–) males and females were analyzed by μCT scanning (E–F), followed by sectioning and von Kossa staining (D). The three dimensional (3D) rendered μCT images of the distal femur (E) and femoral metaphysis (F) were subject to quantitative analysis for trabecular bone volume (G). (H) Spines of the 3-month-old (3 M) ∆Mir27a males and females were analyzed by μCT scanning. Images show the μCT scanned wild-type (+/+) and mutant (–/–) L5 vertebrae (top) and 3D-rendered trabecular bone (bottom). Quantitative analyses of trabecular bone volume per total volume in the femurs (BV/TV, n=5 for female and n=4 for male, mean ± SD; student t-test) and vertebrates (BV/TV, n=3, mean ± SD; student t-test) are shown in graphs. Images (D–F and H) are representatives of three independent experiments. Scale bars, 500 µm (D–F and H).

Figure 4—source data 1. Statistical data for Figure 4G–H.
Figure 4—source data 2. Raw gel image of Figure 4B.
Figure 4—source data 3. Uncropped gel image of Figure 4B with labels.
Figure 4—source data 4. Raw gel image of Figure 4C.
Figure 4—source data 5. Uncropped gel image of Figure 4C with labels-1.
Figure 4—source data 6. Uncropped gel image of Figure 4C with labels-2.
Figure 4—source data 7. Uncropped gel image of Figure 4C with labels-3.
Figure 4—source data 8. Uncropped gel image of Figure 4C with labels-4.

Figure 4.

Figure 4—figure supplement 1. Identification of miRNA candidates in osteoclast differentiation pathway.

Figure 4—figure supplement 1.

The diagram illustrates the top candidates identified by the miRPath Reverse-Search module based on the target accumulation in osteoclast differentiation-related genes in the Kyoto Encyclopedia of Genes ad Genomics (KEGG: mmu04380).
Figure 4—figure supplement 2. The loss of MIR27a does not affect cortical bone thickness.

Figure 4—figure supplement 2.

Femurs of the 3-month-old (3 M) and ∆Mir27a males and females were analyzed by μCT scanning. Images show the reconstructed cortical bone of wild-type (+/+) and mutant (–/–). Quantitative analyses of cortical (Ct.) thickness (Th) are shown in graphs (n=5 for females and n=4 for males, mean + SD; student t-test, ns, non-significant). Images are representatives of five independent experiments. Scale bars, 500 µm.
Figure 4—figure supplement 2—source data 1. Statistical data for Figure 4—figure supplement 1.