The Study Of The Effect Of And Mechanism Of Simulated Microgravity Environment On MC3T3-E1 Osteoblasts

Background: In recent decades,great achievements have been made in the field of aerospace in China.It is reported that China will enter the phase of manned space station in 2020,and astronauts may stay in the space station for more than one year.However,long-term exposure in micro-gravity will cause healthy astronauts suffer from a rapid loss of bone mass which cannot return to the normal level even when they return to the earth.The prone to osteoporosis is the main factor restricting astronauts’ space operations.It is well-known that bone tissue is in a state of dynamic equilibrium under normal physiological conditions,but when the mechanical environment of bone tissue changes,such as μ-gravity or hypergravity,the existing state of equilibrium will be broken,which lead to abnormal metabolism of bone tissue and the occurrence of related diseases.The bone resorption process was inhibited in traditional treatment regimens which cannot effectively solve the problem of bone loss.Therefore,it is necessary to find new alternative therapies.Objective: To explore the differential changes of non-coding RNA under the microgravity and expand the prevention and treatment of osteoporosis from the traditional level to the level of gene regulation.This article provides a comprehensive and systematic study on the mechanism of bone tissue damage,repair and reconstruction under microgravity to offer new ideas and strategies for prevention and treatment of astronauts’ osteoporosis caused by space exploration.Methods:（1）.Cell rotating culture system（rotary cell culture system,RCCS）and the method of tail suspension feeding was used on mice to simulate micro-gravity environment for MC3T3-E1 osteoblast and C57BL/6J mice.Three point bending mechanics experiment and micro-CT scanning were applied to observe the difference of the tibia tissue microstructure and mechanics properties.Histomorphology was used to analyze the difference of the tissue.The proliferation of the osteoblast before and after the experiment was compared using the Edu staining method and MTT colorimetry.The difference of the expression of the osteogenic differentiation related genetic proteins were tested by the RT-PCR and western blot to observe the osteoblast differentiation difference.（2）.Transcriptome sequencing technology（RNA-seq）was used to seek out the difference of LncRNA,miRNA,CircRNA,mRNA in MC3T3-E1 osteoblast after 3 days culture in microgravity environment and normal conditions.Using of Bioinformatics methods to establish a co-expression network of LncRNA-mRNA,miRNA-mRNA,CircRNA-mRNA,LncRNA-miRNA-mRNA and CircRNA-miRNA-mRNA,meanwhile,predict the target gene in differential LncRNA,miRNA,CircRNA.Then,using of GO and KEGG to find out the osteoblast differentiation-related LncRNA,miRNA and CircRNA in micro-gravity environment for analysis.We also predicted the CeRNA of relevant LncRNA,CircRNA and screened signaling molecules and pathways associated with osteogenic differentiation as well as validated relevant results.（3）.Based on the predicted results from step 2),NF-κB signaling pathway was selected to validate its effect on osteoblast differentiation in micro-gravity environment by detecting the expression of critical proteins and genes and the content and the expression of IL-6 in culture solution and osteoblasts to investigate the effect of NF-κB signaling pathway on osteoblast differentiation under microgravity.（4）.CKIP-1 gene can affect bone metabolism through negative regulation of osteogenesis.CKIP-1 gene knockout cell line in MC3T3-E1 osteoblast was bulit by crisper cas 9 technology.Results:（1）.In the present study,mice tibial metaphyseal bone structure and function was significantly decreased after 4 weeks of tail suspension,mainly by reducing the bone volume fraction,trabecular number and trabecular thickness;The three-point bending test results revealed lower fracture load,fracture energy,fracture displacement and fracture stress,which make the resistant ability of bone damage decrease.At the cellular level,cellular proliferation and differentiation was markedly inhibited.Which mainly reflects in a lower proliferating rate and expression of osteogenesis-related genes.（2）.We first constructed the expression profiles of LncRNA,miRNA,and CircRNA,the results showed that 1198 LncRNA,101 miRNA,427 CircRNA and 2538 mRNAs were significantly changed in the simulated microgravity group.Then,using of coexpression network analysis to constructe the interaction network.We obtained 5 LncRNA,5 miRNA,3 CircRNA and 9 mRNA for the follow-up study,in addition,we predicted the CeRNA of LncRNA or CircRNA,and screened two relations unit: NONMMUT114186.1-let-7c-5p-Il6 ra and circ₀14154-let-7c-5p-Il6 ra.Finally,using of RT-PCR we verified the result of each the screened-genes,the results were in accordance with the results of RNA-Seq.（3）.Through the functional annotation of GO and KEGG Pathway analyses,we found that TNF signaling pathway,PI3K-Akt signaling pathway,NF-κB signaling pathway and MAPK signaling pathway were involved in the progress of osteogenic differentiation.（4）.We explored the response mechanism of NF-κB signaling pathway to “zerog” in MC3T3-E1 cells under the simulated microgravity conditions,The results showed that the alkaline phosphatase（ALP）,osteocalcin（OCN）and typeⅠcollagen（CoL-I）mRNA and protein level were dramatically decreased under the simulated microgravity.Meanwhile,the IκB-α（NF-κB inhibitor α）protein level was decreased and the expressions of phosphorylation of IκB-α（p-IκB-α）,p65,p-p65（phosphorylation of p65）was significantly up-regulated,in addition,the IL-6 content was increased compared to CON.These results indicated that simulated microgravity could activate the NF-κB pathway to regulate ME3T3-E1 cells differentiation.（5）.We finally built the heterozygotes CKIP-1 knockout cell line,RT-PCR results showed that the expression of CKIP-1 was significantly decrease.Conclusion: In the present study,we demonstrated that the process of bone loss due to disuse osteoporosis in healthy young mice and the MC3T3-E1 osteoblast cell proliferation and differentiation was markedly inhibited in the micro-gravity condition.Moreover,a large of number of LncRNA,miRNA,CircRNA and mRNA have been changed,which means non-coding RNA participated in the progress of osteoblast cell proliferation and differentiation.NONMMUT114186.1-let-7c-5p-Il6ra、circ₀14154-let-7c-5p-Il6 ra may be an important progress in regulation of osteogenic differentiation.In micro-gravity condition,the differentiation of osteoblast was inhibited by increasing IL-6 expression and activating the NF-κB pathway.In MC3T3-E1 cell,crisper cas 9 could not construct the perfect CKIP-1 knockout cell line,we finally obtained the heterozygotes cell line.