| Many serious adverse physiological changes occur during space?ight. Some of these include bone deterioration, muscle loss, immune system dysfunction, cardiovascular deconditioning and so on. Some of these pathophysiological adjustments can be counteracted adequately with physical exercise or nutritional supplementation. But many of the changes cannot be overcome. In particular, microgravity(MG)-induced osteoporosis is one of the most severe harms and the mechanism is not clear, which have always been the critical issue in this field. In the most severe forms of MG-induced bone loss, there is an approximately 2 % decrease in bone mineral density in only 1 month, equal to that of a postmenopausal woman in 1 year. Gravity and mechanical loading are known to be essential for the maintenance of skeletal integrity. It has been reported that the change induce by microgravity in bone mass is a result of the uncoupling of bone remodeling between decreased formation and normal (or increased) resorption. Osteoblast differentiation is a key step in proper skeletal development and acquisition of bone mass, and previous syudy proved that altered osteoblast function and development play an important role in MG-induced bone loss. But it is unclear that what mechanisms result in the dysfunction of osteoblast and what moleculars are involved in it. MiRNA are endogenously expressed single-stranded noncoding RNAs of 18-25 nucleotides in length which have been identified in diverse organisms, including mammalian cells. MiRNA negatively regulate the translations of specific target genes by binding to the 3'untranslated regions (3'UTR) of the genes with partially or fully complementary sequences. It has been reported that over 30% of protein coding genes in humans are regulated by miRNA, which are involved in the regulation of development and homeostatic events. MiRNA play critical roles in cell proliferation, cell differentiation, and cell death. Many studies have identified that miRNA attenuation of gene expression posttranscripted has emerged as an important regulator of mesenchymal cell differentiation into the osteoblast lineage.In this study, we determined the changes of miRNA expression partten in the BMP-2-induced differertiation of the pluripotent mesenchymal precursor cell line C2C12 simulated by microgravity exposure and further explored roles and mechanisms of miRNA in MG-induced bone loss. The main results and findings of this work are as follows:1. Osteoblast differentiation and maturation are suppressed in microgravity. To identify the alterations of osteoblast differentiation induced by microgravity at the cellular level, C2C12 cells were cultured in clinorotation condition for 72 h without or with 300ng/ml BMP2. Real-time PCR for mRNA of the typical osteoblastic markers Alkaline phosphatase(ALP), osteocalcin(OC), osteoprotegerin(OPG), and runt-related transcription factor 2(Runx2)results showed the inhibition of osteogenesis either the BMP-2 absence or not. There are many pathways involved in osteogenesis. We investigated the expression levels of the critical moleculars related to osteoblast differentiation and maturation. We treated C2C12 cells under the same conditions for 72 hr and collected cell lysis. Western blot analysis revealed that the expressions of FGFR2, BMPR2, and Runx2 were reduced in microgrativity with or without BMP-2. The rat model of tail-suspended to make hind limb unloading has been used for detection the effects of microgrativity. The bone formation of tail-suspended rats was investigated by radionuclide bone scintigraphy, which has always been used to examine bone metabolism. We found that compard with the control group the accumulation of 99mTc-MDP was significantly lower either in bones or joints of the tail-suspended rats. And the di?erences became more and more significcant along with the time prolongation. These findings suggested that microgrativity resulted in decrease of bone metabolism and osteoblastogenesis.2. Microarray analysis reveals the differential expression partten of microRNAs in microgravity. To detect the potential involvement of miRNA in osteoblast differentiation program in microgravity, microarray was performed by using total RNA from C2C12 cells cultured in clinorotation condition for 72h. The expression levels of 2 miRs (ratio?2.0) increased and 5 miRNA decreased between stationary control and microgravity group. To validate the results of microarray profiling, Real-time PCR was performed to analyze miRNA in C2C12 cells cultured in microgravity condition for 72hr. Precursor of miR-494 (mmu-miR-494) was robustly up-regulated (>2-fold), and precursors of miR-18*, miR-122a, miR-301, and miR-340 (mmu-miR-18*, mmu-miR-122a, mmu-miR-301, and mmu-miR-340) were significantly down-regulated (>2-fold). However, precursor of miR-143 (mmu-miR-143) did not show significant change between stationary control and microgrativity group, which was not coincidence with the data from microarray analysis. Among the differentially expressed miRNA screened, we focused on the up-regulation of miR-494, because the potential target genes of miR-494 predicted by bioinformatic method play positive roles in osteoblast differentiation. To clarify the time-dependent expression of miR-494, we collected RNA from stationary control and microgrativity-treated samples at 2, 4, 8, 12, 24, 48 and72 h. Real-time PCR was performed to further analyze the expression pattern of miR-494 during microgravity of C2C12 cells. We found that miR-494 began to upregulate at 2h and kept increasing along with the time of microgravity. In addition, we isolated osteoblasts from the proximal femurs of tail-suspended rats to detecte the expression levels of miR-494. Real-time PCR analysis showed the expression of miR-494 was dramatically increased after 2 weeks of tail-suspension, and kept increasing with the time of unloading. These results indicited that miRNA may play important roles in abnomal osteblastogenesis induced by microgrativity.3. MiR-494 inhibits the differentition of osteoblast in normal grativity. To elucidate the role of miR-494 in osteogenesis, firstly we investigated the effects of miR-494 on proliferation and cell cycles. We transfected C2C12 cells and MC3T3-E1 cells separately with miR-494 mimics and negative contro(lN.C.), and MTT assay and flow cytometry analysis suggested miR-494 had no significant effects on cell proliferation and cycles either in C2C12 cells or MC3T3-E1 cells. Then we measured ALP activities in C2C12 cells transfected with N.C. or miR-494 mimic with and without BMP-2 treatment for 72 h. The ALP activities in the transfected with miR-494 cells were significantly suppressed in the presence of BMP-2. There was no significant difference between transfection with negative control and miR-494, because mesenchymal stem cell C2C12 did not osteogenicly differentiate without BMP-2. To further investigated whether miR-494 are directly coupled to BMP-2-induced C2C12 osteoblastogenesis, C2C12 cells were transfected with N.C. or miR-494 and treated with BMP-2 simultaneously for 72 h. Real-time PCR, ELISA, and Western Blot were performed to determine the mRNA levels ALP, OC, OPG, and Runx2. Consistent with the ALP staining analysis, the mRNA expression of ALP was reduced, and the gene expressionsof OC, OPG, Runx2 were decreased whether BMP-2 presence or not. ELISA analysis showed miR-494 reduced the secretions of OC and OPG. Runx2 protein level was enhanced by BMP-2, but the enhancement was deducted in cells transfected with miR-494 compared with cells transfected with negative control. Thus, for osteogenesis of C2C12 cells to proceed, there is a requirement for BMP-2. But the introduction of miR-494 inhibits the osteoblast differentiation whether BMP-2 exist or not. Osx acts the downstreamof Runx2, and we found miR-494 inhibited Osx mRNA expression in C2C12 cell induced by BMP-2. In addition, miR-494 could promote myogenesis and have no effect on adipogenesis.4. MiR-494 targeting Runx2, BMPR2, and FGFR2 inhibits the osteoblast differentition. More than 1,000 genes were predicted by miRanda, TargetScan, pictar or RNAhybrid databases to be potential target genes for miR-494. We selected more than 30 putative miR-494 target genes involved in osteoblastic differentiation from all the predicted genes and used a luciferase reporter assay system and transfection of miR-494 mimics to determine which targets were for miR-494. We cotransfected the luciferase reporter plasmids cloned the 3'-untranslated region (3'-UTR) sequences of the selected genes and negative control or miR-494 into 293A cells, the relative luciferase activity demonstrated whether miR-494 had effect on the genes. In 293A cells cotransfected with luciferase reporter genes carrying 3'-UTRs of BMPR2, FGFR2, and Runx2 with a putative miR-494 binding site, the level of suppression was about 40-60%. In contrast, the suppressive activity was lost when the seed sequence of miR-494 on 3'-UTRs of these genes were mutant. MiR-494 was able to down-regulated endogenous proteins of BMPR2, FGFR2, and Runx2, and able to suppress the mRNA levels of the three genes, consistent with the mechanism of miRNA regulation. To further address the hypothesis that miR-494 negatively regulated osteoblast differentiation by targeting key signal transduction factors as BMPR2, FGFR2, and Runx2, we introduced siRNAs targeted these genes which repression effects were confirmed by western blot analysis into C2C12 cells for 72 h. Real-time PCR analysis suggested knockdown of the three genes suppress the osteoblast differerntiation. Together, these results suggest that BMPR2, FGFR2, and Runx2 are targets for miR-494. On the other hand, miR-494 may indirectly downregulate Runx2 through suppressing the expressions of BMPR2 and FGFR2 which are the upstream genes of Runx2.5. Myod enhances the expression of miR-494. To investigate the transcriptional regulation mechanism of miR-494, we analyzed the conservatism of 5'terminal of miR-494 by bioinformatics method. The region about 2-3 kb upstream of 5'terminal of pre-miR-494 is highly conserved, and there are several binding sites of Myod in this sequence. We identified the expression pattern of Myod during microgravity and BMP-2 induction in C2C12 cells. Real-time PCR result suggested the expression of Myod was increasd along with the time of MG and decreased with BMP-2 induction. So we presumed that Myod promote the expression of miR-494, and miR-494 upregulate Myod mRNA.6. MiR-494 inhibitor can partly rescue the dysdifferentiation of osteoblast in microgrativity. Osteoblastic differentiation is suppressed and the expression of miR-494 is upregulated in microgrativity condition. C2C12 cells were transfected with miR-494 inhibitor or N.C.inhibitor and cultured in clinorotation condition for 72 h without or with 300ng/ml BMP-2. Real-time PCR analysis for the osteoblastic markers suggested that the inhibition of miR-494 partly enhanced ostogenesis with the presence of BMP-2 or not. Consistent with Real-time PCR array, ALP staining was increased following transfection of miR-494 inhibitor compared with transfection of N.C. inhibitor.In conclusion, we found miRNA involved in dysfunction of osteoblast during MG. Overexpression of miR-494 inhibits osteogenesis and inhibition of endogenous miR-494 promotes ostenblast differentiation. Our finding might help to provide some theoretical basis to the protection and treatment of MG-induced Osteoporosis. |
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