Protection On Bone Loss Induced By Simulated Weightlessness Through Targeting Inhibition Of MiR-132-3p In Bone Tissues Of Mice

In spaceflight, the body’s homeostasis established by the long term adaptation to the ground normal gravity environment is disturbed by the weightlessness, resulting in disorders of multiple system functions which greatly affect the astronaut health and in-orbit operation. Among them, sustained bone loss induced by weightlessness will lead to excessive accumulation of minerals in body, increasing risk of bone fractures and kidney stones, and even irreversible damages of bones. It has become one of the three major medical problems to hinder humans’ long term space exploration. Therefore, aerospace medicine researchers have paid close attention to the study of effective prevention measures against bone loss in spaceflight. In the past 50 years of aerospace practice, several protective measures including physical intervention, medication therapy and diet adjustment were used to prevent bone loss but still not able to effectively solve the problem. In the opinion of spacefaring powers such as America and Russia, it’s necessary to recognize the intrinsic changes at cell and molecular level and further to clarify the mechanism of signal perception, transduction, and response to gravity so as to develop effective and well-targeted prevention measures. Genomic studies have confirmed that the expressions of hundreds of genes in osteoblast were altered in response to weightlessness or simulated weightlessness. The role of miRNA played in the regulation of gene expression should not be overlooked. With the development of cellular and molecular medicine, researchers have also come to realize that miRNA may play an important role in the regulation of biological response to weightlessness and begin to undertake related studies.Our previous research found that simulated weightlessness could lead to the significant increase of miR-132-3p expression in the femur bone tissue of rats. Over-expression of miR-132-3p could inhibit the proliferation, differentiation and mineralization of rat primary osteoblasts while the decrease of miR-132-3p expression could promote the function of osteoblasts. This indicates that miR-132-3p may play an important role in the bone loss caused by simulated weightlessness. Based on the above results, we studied the effect of miR-132-3p on the osteogenic differentiation of bone mesenchymal stem cells(BMSC), then by intervening of miR-132-3p expression, we further tested the prevention effects on the weakened osteogenic differentiation capacity of BMSC caused by simulated weightlessness. Finally, by using molecular targeting technology and gene modified technology, we injected the bone-targeted delivery system(AspSerSer)6 cationic liposomes with the package of miRNA-132-3p inhibitor “antagomir-132” into the tail suspended mice through tail intravenous injection, and inhibited miR-132-3p expression of bone tissue in specially to prevent bone loss induced by simulated weightlessness. The main findings and results of the research are as following:1. miR-132-3p can inhibit osteogenic differentiation of BMSC in mice. There are serial osteoblast-like cells differentiated from BMSC nearby the bone formation area. In order to achieve the ideal “therapeutic effect” by targeted inhibition of miR-132-3p expression nearby bone formation area in bone tissue, we firstly confirmed that miR-132-3p could regulate osteogenic differentiation process of BMSC. Primary BMSC of mice were isolated and then cultured with osteogenic medium. The gene and protein expression of osteogenic differentiation markers including Runx2 and Osx, the gene and protein activity of ALP, and the mineralized nodules of external matrix were all significantly increased, which indicated that the BMSC were able to differentiate to osteoblast cells in vitro. In the osteogenic differentiation process of BMSC, qRT-PCR analysis showed the expression of miR-132-3p was gradually declined. And over-expression of miR-132-3p in BMSC by transfecting its mimic could block the osteogenic differentiation of BMSC cultured with osteogenic medium, while declining expression of mi R-132-3p in BMSC by transfecting its inhibitor promoted the osteogenic differentiation process. The above results suggested that the targeted mRNA of miR-132-3p may be the key gene in the osteogenic differentiation signal pathway and the intervention on the expression of mi R-132-3p could initiate or block the osteogenic differentiation process.2. Inhibition of miR-132-3p expression partially attenuates the negative effects of simulated weightlessness on osteogenic differentiation of BMSC. The third generation BMSC cultured with osteogenic medium were exposed to simulated weightlessness environment by clinorotation for 72 h. The gene expressions of Runx2、Osx and ALP were gradually decreased, indicating the osteogenic differentiation process of BMSC was blocked by clinorotation. And the results were consistent with the previous studies. In this process, the expression of miR-132-3p gradually increased in a time-dependent manner of clinorotation, indicating that simulated weightlessness could promote the expression of miR-132-3p in BMSC. To test whether therapeutic inhibition of miR-132-3p could rescue the osteogenic differentiation decreased by clinorotation, BMSC were transfected with the inhibitor of miR-132-3p for 12 h and then exposed to simulated weightlessness for 48 h. The results showed that inhibition of miR-132-3p in BMSC could significantly promote gene and protein expression of osteogenic differentiation makers and effectively attenuate the negative effects of simulated weightlessness on osteogenic differentiation of BMSC.3. Inhibition of miR-132-3p expression in the bone of hindlimb unloading mice could effectively alleviate bone loss induced by simulated weightlessness. In this study, we delivered the inhibitor of mi R-132-3p “antagomir-132” to the bone formation area using the bone tissue targeting delivery system(AspSerSer)-6 cationic liposomes. qRT-PCR test showed that miR-132-3p expression in the bone tissues of experimental mice significantly decreased while no significant changes were observed in other major parts, such as heart, liver, kidney, and lung. This suggested that the targeting system could specifically deliver the inhibitor of miR-132 into the bone tissue. Then we further detected the effect of decreasing the miR-132-3p expression in bone tissue on bone morphology, micro structure and mechanical performance. Compared to negative control group, genes expression of Runx2, Osx, ALP and COL-Ⅰ, along with the new bone formation, were all increased. And the micro structure and mechanical performance of femur were well ameliorated. These results indicated that targeting inhibition of miR-132 expression in bone tissues could effectively alleviate bone loss induced by simulated weightlessness, which provided new reference for the future development of protection measures on bone loss induced by weightlessness.In conclusion, we studied the effect of miR-132-3p on the osteogenic differentiation of BMSC in normal and simulated weightlessness environment respectively, and further observed the prevention effects on bone loss by targeted inhibition of miR-132-3p expression in bone tissue of hindlimb unloading mice. The above results provided new reference for further elucidation of the cellular mechanism and development of preventive measures on bone loss induced by weightlessness.