miR-181c-5p mediates simulated microgravity-induced impaired osteoblast proliferation by promoting cell cycle arrested in the G2 phase

Abstract

Impaired osteoblast proliferation plays fundamental roles in microgravity-induced bone loss, and cell cycle imbalance may result in abnormal osteoblast proliferation. However, whether microgravity exerts an influence on the cell cycle in osteoblasts or what mechanisms may underlie such an effect remains to be fully elucidated. Herein, we confirmed that simulated microgravity inhibits osteoblast proliferation. Then, we investigated the effect of mechanical unloading on the osteoblast cell cycle and found that simulated microgravity arrested the osteoblast cell cycle in the G₂ phase. In addition, our data showed that cell cycle arrest in osteoblasts from simulated microgravity was mainly because of decreased cyclin B1 expression. Furthermore, miR-181c-5p directly inhibited cyclin B1 protein translation by binding to a target site in the 3'UTR. Lastly, we demonstrated that inhibition of miR-181c-5p partially counteracted cell cycle arrest and decreased the osteoblast proliferation induced by simulated microgravity. In conclusion, our study demonstrates that simulated microgravity inhibits cell proliferation and induces cell cycle arrest in the G₂ phase in primary mouse osteoblasts partially through the miR-181c-5p/cyclin B1 pathway. This work may provide a novel mechanism of microgravity-induced detrimental effects on osteoblasts and offer a new avenue to further investigate bone loss induced by mechanical unloading.

Keywords: cell cycle; cell proliferation; cyclin B1; miR-181c-5p; osteoblast; simulated microgravity.

Figure 1

Simulated microgravity (MG) inhibits cell growth of primary mouse osteoblasts. A, EdU incorporation experiments were analysed using an inverted microscope linked to a confocal scanning unit. Proliferating primary mouse osteoblasts were labelled with EdU. Osteoblasts were stained with the nucleic acid dye Hoechst (blue) and EdU (green). B, Histogram of the average percentage of EdU‐positive cells from the two groups. The EdU incorporation rate was expressed as the ratio of EdU‐positive cells to total Hoechst positive cells (n = 4). C, Comparison of changes in cell growth between control (Con) and MG groups. Cells were seeded on 96‐well plates at a density of 2000 cells/well. Cell proliferation was evaluated by a CCK‐8 assay at 24‐96 h (n = 3). D, Western blot analysis of PCNA expression in cells treated with simulated microgravity. The total protein loaded per lane was 40 μg. Detection of GAPDH on the same blots was used to verify equal loading amongst the various lanes (upper). Histogram illustrated average data for the relative expression of PCNA present in cells from each group quantified by camera‐based detection of emitted chemiluminescence (lower) (n = 4). The results were expressed as the mean ± SD. Two‐tailed Student's t test was performed for each sample against control samples. *P < 0.05 and **P < 0.01, when compared with the stationary control.

Figure 2

Cell cycle of osteoblasts is arrested in the G₂ phase (as opposed to the M phase) in response to simulated microgravity. A and B, Flow cytometry analysis of primary mouse osteoblasts treated with simulated microgravity was performed to test the cell cycle distribution. A, Representative histograms indicate the cell cycle distribution in different groups. The relative DNA contents of cells were determined by PI staining. B, The percentage of cells in each cycle stage was quantified (n = 5). C‐E, The effect of simulated microgravity on the mitosis index of osteoblasts was detected by immunofluorescence for histone H₃ (phospho Ser10). C, Cells were seeded onto glass coverslips and, after simulated microgravity treatment for 48 h, cells were fixed, permeabilized and subjected to staining with Hoechst (blue) to visualize nuclei and with anti‐histone H₃ (phospho Ser10) primary antibody and Alexa Fluor 488 conjugated secondary antibody (green) to visualize cells undergoing mitosis. Images were analysed using a confocal microscope. D, Histogram of the percentage of histone H₃ (phospho Ser10)‐positive cells from these groups. The mitotic index was expressed as the ratio of histone H₃ (phospho Ser10)‐positive cells to total Hoechst positive cells (n = 3). E, Western blot analysis of histone H₃ (phospho Ser10) expression was determined in cell lysates from primary mouse osteoblasts. The total protein loaded per lane was 40 μg. Detection of GAPDH on the same blots was used to verify equal loading among the various lanes (upper). Histogram of the relative expression of histone H₃ (phospho Ser10) present in cells from each group quantified by camera‐based detection of emitted chemiluminescence (lower) (n = 4). Cells treated with 0.5 μg/mL nocodazole (a mitotic inhibitor) for 24 h were used as a positive control. The results were expressed as the mean ± SD with a one‐way ANOVA with a SNK‐q test. *P < 0.05 and **P < 0.01, compared with the stationary control.

Figure 3

Cellular localization, expression levels and activity of Cdc2 kinase are unchanged under simulated microgravity conditions. A and B, Immunocytochemistry assay was analysed using an inverted microscope linked to a confocal scanning unit (n = 3). Proliferating primary mouse osteoblasts in different groups were stained with the nucleic acid dye Hoechst (blue), anti‐Cdc2 antibody (A) or anti‐p‐Cdc2 antibody (B) and Alexa Fluor 488 conjugated secondary antibody (green). C, Western blot analysis of Cdc2 and p‐Cdc2 expression in cell lysates from osteoblasts in different groups. The total protein loaded per lane was 40 μg. Detection of GAPDH on the same blots was used to verify equal loading among the various lanes. Histogram of the relative expression of Cdc2 (D) and p‐Cdc2 (E) present in cells from the Con, MG and Nocodazole groups quantified by camera‐based detection of emitted chemiluminescence (n = 5). F, Cell lysates obtained from different groups were incubated with the anti‐cyclin B1 antibody reversible immunoprecipitation system. Then, Cdc2‐cyclin B1 kinase activity was measured using a Cdc2‐cyclin B kinase assay kit (n = 4). Nocodazole is a mitotic inhibitor that served as a positive control treatment. Bars represent the mean ± SD with a one‐way ANOVA with a SNK‐q test. **P < 0.01 compared with the stationary control.

Figure 4

Simulated microgravity does not change the cellular localization of cyclin B1, but decreases its expression. A, Immunostaining staining experiments were conducted using a confocal microscope (n = 3). Primary mouse osteoblasts in different treatment groups were labelled with the nucleic acid dye Hoechst (blue), anti‐cyclin B1 and Alexa Fluor 488 conjugated secondary antibody (green). B, Western blot analysis of cyclin B1 expression in cell lysates from cells in Con, MG and Nocodazole groups. The total protein loaded was 40 μg per lane. Detection of GAPDH on the same blots was used to verify equal loading among the various lanes (upper). Histogram of the relative expression of cyclin B1 present in different conditions quantified by camera‐based detection of emitted chemiluminescence (lower) (n = 3). C, qPCR of relative cyclin B1 mRNA levels in osteoblasts treated with simulated microgravity (n = 6). Primary mouse osteoblasts treated with 0.5 μg/mL nocodazole for 24 h served as a positive control. The results were expressed as the mean ± SD with a one‐way ANOVA with a SNK‐q test. *P < 0.05 and **P < 0.01, compared with the stationary control.

Figure 5

Cyclin B1 overexpression restores the cell cycle arrest in the G₂ phase and partially counteracts the decrease of osteoblast proliferation induced by simulated microgravity. A, Western blot to test the efficiency of the pcDNA3.1‐cyclin B1 vector in primary mouse osteoblasts under normal gravity and simulated microgravity conditions. Cell lysates were obtained after transfection with pcDNA3.1‐cyclin B1 or pcDNA3.1 empty vector in the Con and MG groups. The total protein loaded was 40 μg per lane. Detection of GAPDH on the same blots was used to verify equal loading among the various lanes (upper). Histogram of the relative expression of cyclin B1 in different treatment groups quantified by camera‐based detection of emitted chemiluminescence (lower) (n = 3). B and C, FCM analyses of osteoblasts transfected with pcDNA3.1‐cyclin B1 or pcDNA3.1 empty vector in the Con and MG groups. B, Representative histograms indicating cell cycle distribution in different groups. The relative DNA content of cells was determined by PI staining. C, The percentage of cells in each cycle stage was quantified (n = 4). D, EdU labelling assays were analysed using an inverted microscope linked to a confocal scanning unit. Proliferating osteoblasts were loaded with EdU. Osteoblasts were stained with the nucleic acid dye Hoechst (blue) and EdU (green). E, Histogram of the percentage of EdU‐positive cells from different groups. The EdU incorporation rate was expressed as the ratio of EdU‐positive cells to total Hoechst positive cells (n = 3). F, Comparison of cell growth changes in different treatment groups. Cells were seeded on 96‐well plates at a density of 2000 cells/well. Cell proliferation was evaluated by a CCK‐8 assay at 24‐96 h (n = 4). G, Western blot analysis of PCNA expression in cells transfected with pcDNA3.1‐cyclin B1 or pcDNA3.1 empty vector in Con and MG groups. The total protein loaded per lane was 40 μg. Detection of GAPDH on the same blots was used to verify equal loading among the various lanes. H, Histogram of the relative expression of PCNA present in cells from each group as quantified by camera‐based detection of emitted chemiluminescence (lower) (n = 4). The results were expressed as the mean ± SD with a one‐way ANOVA with a SNK‐q test. *P < 0.05 and **P < 0.01, compared with the stationary control.

Figure 6

Ccnb1, the gene for cyclin B1, is the target gene of miR‐181c‐5p in primary mouse osteoblasts. miRNA target prediction tools were used to screen for cyclin B1‐targeting miRNAs, and the top ten miRNAs that received the highest composite score were selected for the expression assay. A, qPCR analysis of changes in expression of miR‐181a‐5p, miR‐181b‐5p, miR‐181c‐5p, miR‐181d‐5p, miR‐1942, miR‐6388, miR‐1954, miR‐3089‐3p, miR‐300‐5p and miR‐411‐3p in osteoblasts treated with simulated microgravity (n = 6). B, A schematic illustration of the design of luciferase reporters containing the WT Ccnb1 3′UTR (WT 3′UTR) or the site‐directed mutant Ccnb1 3′UTR (MUT 3′UTR). Sequences below indicate putative miR‐181c‐5p target sites on the WT 3′UTR, the MUT derivative, and the pairing regions of miR‐181c‐5p. C, The effects of the miR‐181c‐5p mimic and inhibitor or their negative controls on the luciferase activity of the WT Ccnb1 3′UTR or MUT Ccnb1 3′UTR reporter in 2 T3 cells. The values in the condition of WT Ccnb1 3′UTR or MUT Ccnb1 3′UTR are shown relative to that of the mimic‐NC in the same condition (n = 3). D, qPCR of miR‐181c‐5p levels in osteoblasts after treatment with mimic‐181c‐5p, inhibitor‐181c‐5p or their negative controls (n = 3). E, qPCR experiments were performed to detect changes in Ccnb1 mRNA expression in osteoblasts after treatment with mimic‐181c‐5p, inhibitor‐181c‐5p or the negative controls (n = 3). F, Western blot analyses of cyclin B1 proteins levels in primary mouse osteoblasts after treatment with mimic‐181c‐5p, inhibitor‐181c‐5p or the negative controls for 48 h. The total protein loaded per lane was 40 μg. Detection of GAPDH on the same blots was used to verify equal loading among the various lanes (upper). The histogram illustrated the relative expression of cyclin B1 present in cells from each group as quantified by camera‐based detection of emitted chemiluminescence (lower) (n = 3). The results were expressed as the mean ± SD with a one‐way ANOVA with a SNK‐q test. *P < 0.05 and **P < 0.01, compared with the stationary control.

Figure 7

Induction of cell cycle arrest in the G₂ phase and decreased proliferation in primary mouse osteoblasts treated with simulated microgravity partially depends on the up‐regulation of miR‐181c‐5p. A, qPCR of the miR‐181c‐5p levels in osteoblasts to test the efficiency of inhibitor‐181c‐5p under normal gravity and simulated microgravity conditions (n = 3). B, Western blot experiments in primary mouse osteoblasts test the effects of inhibitor‐181c‐5p on cyclin B1 expression in the Con and MG groups. Cell lysates were obtained after transfection with inhibitor‐181c‐5p or inhibitor NC in both groups. The total protein loaded was 40 μg per lane. Detection of GAPDH on the same blots was used to verify equal loading among the various lanes (upper). The histogram showed the relative expression of cyclin B1 present in different groups as quantified by camera‐based detection of emitted chemiluminescence (lower) (n = 3). C, FCM analyses of osteoblasts transfected with inhibitor‐181c‐5p or inhibitor NC in Con and MG groups to examine the cell cycle distribution. Representative histograms indicate the cell cycle distribution in the different groups. The relative DNA content of cells was determined by PI staining (left). The percent of cells in each cycle stage was quantified and showed as histograms (right) (n = 4). D, EdU labelling assays were analysed using an inverted microscope linked to a confocal scanning unit. Proliferating osteoblasts were loaded with EdU. Osteoblasts were stained with the nucleic acid dye Hoechst (blue) and EdU (green). E, Histogram of the percentage of EdU‐positive cells from different groups. The EdU incorporation rate was expressed as the ratio of EdU‐positive cells to total Hoechst positive cells (n = 3). F, Comparison of changes in cell growth among the different groups. Cells were seeded on 96‐well plates at a density of 2000 cells/well. Cell proliferation was evaluated by a CCK‐8 assay at 24‐96 h (n = 3). G, Western blot of PCNA expression in cells transfected with inhibitor‐181c‐5p or inhibitor NC in the Con and MG groups. The total protein loaded per lane was 40 μg. Detection of GAPDH on the same blots was used to verify equal loading among the various lanes. H, Histogram of the relative expression of PCNA present in cells from each group as quantified by camera‐based detection of emitted chemiluminescence (lower) (n = 4). The results were expressed as the mean ± SD with a one‐way ANOVA with a SNK‐q test. *P < 0.05 and **P < 0.01, compared with the stationary control.