Effects Of Different Mechanical Loadings On The Gene Expression Profiles Of MG-63 Cells

Objective:Osteoblasts play important roles in the balance of bone formation and resorption. Osteoblasts can sense mechanical signals, transform these mechanical stimuli into biochemical signals, integrate these signals into cellular responses of osteoblasts and osteoclasts, then lead to appropriate changes in the architecture of bone. Bone loss in astronauts is primarily related to microgravity environment. Previous works have highlighted the important role of mechanical loading in skeletal development and the maintenance of skeletal architectural integrity. While much attention has been paid to the certain molecules which had been confirmed to play important roles in the regulation of osteoblasts, the change of whole gene profiles under simulated microgravity was rarely studied. Microarray(Gene chip) is such a technology that all the nucleotide probes are arrayed on special supports and then hybridized with the target samples labeled with fluorescent dye, scanned with laser co-focus in order to measure the target DNA. Therefore, it’s necessary to observe the changes of genetic expression under different mechanical loadings in osteoblasts by using microarray.By detecting the gene expression of MG-63 cells under fluid shear stress and simulated microgravity, the study aimed to find out the alteration of gene expression profiles of MG-63 cells under different mechanical loadings. Further more, as the expression of candidate gene ESM1 was significantly increased under simulated microgravity, the effect of ESM1 on osteoblasts’ proliferation, differentiation and mineralization was verified. Methods: 1. Microarray study on the gene expression profile of MG-63 cells under fluid shear stress.The MG-63 cells were seeded on the glass slides(2.55×2.15 cm) at 1×105 cells/slide, then cultured in the six-well plate for 72 h. The slides were divided into fluid shear stress group(FSS) and control group(CON) randomly. The FSS group was subjected to fluid shear stress(1.5 Pa, 60 min).The CON group was cultured in identical conditions but without the shear stress. Then, total RNAs of two groups were isolated and the gene expression profiles of both groups were detected by using microarray.As compare with CON group, in the gene expression profile of FSS group, the expression of 6223 genes were changed. 2554 genes were up-regulated and 3669 genes were down-regulated. Their main molecular functions were focused on the regulation of transcription, intracellular signaling cascade, intracellular signaling cascade, regulation of apoptosis and regulation of cell proliferation. And they were mainly involved in MAPK pathway, TGF-β pathway, P53 pathway, focal adhesion pathway and Wnt pathway.2. Microarray study on the gene expression profile of MG-63 cells under simulated microgravity. The MG-63 cells were seeded on the glass slides(2.55×2.15 cm) at 1×105 cells/slide, then cultured in the six-well plate for 72 h. The slides were randomly divided into simulated microgravity group(MG) and control group(CON). Clinostat was used to simulate microgravity with 24 rpm for up to 48 h. The CON group was cultured in identical conditions but without rotating. Then, total RNAs of two groups were isolated and the gene expression profiles of both groups were detected by using microarray.As compare with CON group, in the gene expression profile of MG group, the expression of 197 genes was changed. 151 genes were up-regulated and 46 genes were down-regulated. The main molecular functions of up-regulated genes were focused on the regulation of transcription, negative regulation of biosynthetic process and cell death. The main molecular functions of down-regulated genes were focused on the negative regulation of macromolecule metabolic process, negative regulation of gene expression, cytoskeletal protein binding and actin binding. 3. Effect of ESM1 on the biological functions of MG-63 cells.Microarray results showed that the expression of ESM1 was significantly increased under simulated microgravity environment. So a validation study was conducted to verify the fuction of ESM1 on MG-63 cells. We used real time quantitative RT-PCR to observe the expression of ESM1. In addition, in order to observe the effect of ESM1 on osteoblasts’ proliferation, differentiation and mineralization, we inhibited the expression of ESM1 by transfecting si RNA into MG-63 cells. Various tests, including CCK-8, quantitative RT-PCR and alizarin red staining, were conducted to evaluate the functions of MG-63 cells.The results verified the significant increase of ESM1 gene. Compared with control group, the number of MG-63 cells increased in si RNA group, gene expression of ALP and RUNX2 significantly decreased, and the number of mineralized nodules increased remarkably. We get a preliminary conclusion that ESM1 inhibits MG-63 proliferation, mineralization and promotes differentiation. Conclusion:The gene expression profiles of MG-63 cells were altered under fluid shear stress and simulated microgravity. We can draw the conclusion that different mechanical loadings can regulate osteoblasts’ biological functions by changing gene expression. Additionally, the functions of many altered genes in the regulation of osteoblasts’ biological functions are still unknown. It is important to explore and elucidate the roles of these altered genes in the regulation of osteoblasts under different mechanical loadings. More interesting is to explore the roles of these altered genes in the countermeasures of bone loss induced by microgravity.