The Effect And Its Mechanism Of MiR-33-5p In The Mice Osteopenia Induced By Simulated Weightlessness

BackgroundWith the fast development of manned space technology in our country, the project of mid-long term manned space flight has entered the preliminary implementation phase. The harmful effects to astronauts induced by weightlessness need to be researched deeply. The long-term exposure to weightlessness can diminish the bone mass rapidly and severely, and it is the main medical factor to constrain the ability of human body to perform the long-distance space flight. Some therapeutic methods have been found to attenuate the weightlessness-induced osteopenia tentatively, such as artificial gravity, nutritional supplement, physical exercise, physical therapy, etc. However, their therapeutic effects are still unsatisfactory. In order to solve this problem, the pathogenesis of weightlessness-induced osteopenia need to be clarify adequately.miRNA is a kind of small single non-coding RNA, which could regulate broad-spectrum physiological and pathological processes. In recent years, the roles of miRNAs in many diseases have became more clearly, and the miRNA research have been turned from mechanism research in vitro to clinical application in vivo, such as the new targets for drug intervention and the biomarkers of pathological processes. There are many miRNAs have been proved to participate in bone development, growth and occurrence of bone diseases. The cells in bone tissue, such as osteoblast and osteoclast, are also under the control of many miRNAs. While the role of miRNA in weightlessness-induced osteopenia is still rarely studied, it has been found that the functional inhibition of osteoblast is the main reason of weightlessness-induced osteopenia. So our team focus on the effect of miRNA in weightlessness-induced osteoblast inhibition. And through massive researches, we have found some miRNAs can regulate the proliferation, differentiation and electrophysiological activities of osteoblast under simulated weightlessness, and we also explored their mechanisms. This study is a part of our research.ObjectiveThis research aimed to screen out the significant differentially expressed miRNA from our candidate miRNAs in osteoblasts cultured in simulated weightlessness, clarify its effects on osteoblast function and further discuss the mechanism, and then implement the intervention study to explore its potential ability in the target therapy of weightlessness-induced osteopenia in vivo.Methods1. SYBR Green method q RT-PCR and Taq Man assay were performed to select the most differentially expressed miRNA from our candidate miRNAs in osteoblasts cultured in simulated weightlessness, and assess the time course change of its expression.2. By the transfection of its mimic/inhibitor, we explored the miR-33-5p gain/loss-of-function in osteoblasts. We selected Runx2, Osx and ALP as the makers of osteoblast differentiation. q RT-PCR tested their m RNA levels, Western blot tested Runx2 and Osx protein levels, ALP assay and stain tested ALP protein activity. CCK-8 assay was performed to represent the activity of osteoblast proliferation, and PCNA protein levels were also tested by Western blot as a maker of cell proliferation. Alizarin red stain and assay tested osteoblast ability in mineralization. Then we assessed whether the over-expression of miR-33-5p could resist the simulated weightlessness-induced inhibition of osteoblast differentiation.3. Luciferase assay was performed to prove the direct combination of miR-33-5p and its predicted target gene Hmga2. q RT-PCR and Western blot detected the regulative effect of miR-33-5p on Hmga2 m RNA and protein level. Change of Hmga2 expression under simulated weightlessness was also tested. Over-expression vector and si RNA were used to over-express and knock down the Hmga2 expression, then we tested the effect of Hmga2 to osteoblast differentiation. Furthermore, we verified whether the regulative effect of miR-33-5p on osteoblast differentiation is dependent on Hmga2.4. We over-expressed miR-33-5p in osteoblasts by(Asp Ser Ser)6-liposome osteoblast-targeting delivery system in vivo, and tested its therapeutic effect on mice osteopenia induced by simulated weightlessness by the means of micro CT, three-point bending test, double calcein labeling assay, Masson’s trichrome stain and osteoblast differentiation makers detection.Results 1. The expression level of miR-33-5p in osteoblasts continuously decreased over time in response to simulated weightlessness.2. miR-33-5p increased expression levels of Runx2, Osx and ALP in osteoblasts, and its expression was increased during the osteoblast differentiation process. Supplement of miR-33-5p partially weakened inhibition effects of simulated weightlessness on the expression of Runx2, Osx and ALP. miR-33-5p promoted the production of mineralized nodule. However, miR-33-5p affected insignificantly on osteoblast proliferation activity.3. miR-33-5p bonded with the 3’UTR of Hmga2 directly. miR-33-5p inhibited the protein expression of Hmga2 without influencing its m RNA experssion. The expression of Hmga2 increased significantly in response to simulated weightlessness.Hmga2 down-regulated the expressions of Runx2, Osx and ALP in osteoblasts, and its expression was gradually diminished during osteoblast differentiation. Elimination the changes of Hmga2 could weaken the regulative effect of miR-33-5p on the expression of Runx2, Osx and ALP in osteoblasts.4. Targeting improve miR-33-5p in osteoblasts in vivo partially alleviated the decrease of osteoblast activity, the reduction of new bone, the destruction of trabecular architecture and the diminution of mechanical property in hindlimb unloading mice.Conclusion 1. Simulated weightlessness down-regulates miR-33-5p in osteoblasts.2. miR-33-5p promotes the differentiation and mineralization of osteoblasts, while affects osteoblast proliferation insignificantly. Supplement of miR-33-5p partially weakens inhibition effects of simulated weightlessness on osteoblast differentiation.3. Hmga2 is the target gene of miR-33-5p, and negatively regulates Hmga2 expression at the post-transcriptional level. Hmga2 inhibits the differentiation of osteoblasts, and its expression increases under simulated weightlessness. Induction of osteoblast differentiation by miR-33-5p is partially dependent on Hmga2.4. miR-33-5p partially counteracts the bone loss in hindlimb unloading mice.Taken together, our study shows that miR-33-5p modulates osteoblast differentiation in response to weightlessness in vitro. And miR-33-5p functions by inhibiting its direct target, Hmga2, at the post-transcriptional level to negatively affect osteoblast differentiation. Finally, over-expression of miR-33-5p in osteoblasts partially counteracts the bone loss in hindlimb unloading mice. Our findings provide new insights into the cytological mechanisms of weightlessness-induced osteopenia and theoretical foundation of new therapeutic measures.