Role Of Micro Rna In The Effect Of Oxidative Stress On Osteoblast Function And Frax(?) Assessment Of Fracture Probability In Postmenopausal Women With Hyperglycemic Stress

Objective: The study is to explore the role of micro RNA in the effect of oxidative stress on osteoblast function, FRAX? assessment of fracture probability in postmenopausal women with hyperglycemic stress.Methods: 1. To stimulate MC3T3-E1 cells in variable durations with H2O2 at different concentrations, to detect cell damage, oxidative stress, osteogenic differentiation genes and micro RNA by Cell Counting Kit-8(CCK-8), Real-time PCR. 2. As previously reported, we intervened the osteogenic differentiation of MC3T3-E1 with 0.3m M H2O2. The level of osteogenic differentiation was identified by alkaline phosphatase(ALP) activity measurement and staining; the expression level of osteogenesis marker gene m RNA was detected by osteogenesis marker gene m RNA expression level in order to observe the effects of H2O2 on osteogenic differentiation. 3. Intervening the level of mi R-141 or mi R-200 a in MC3T3-E1, and stimulating it with H2O2, detecting osteogenesis marker gene m RNA expression level by Real- time PCR in order to identify the potential role of mi R-141 or mi R-200 a on osteogenic differentiation ability. 4. A Chinese-specific FRAX? model was used in calculating the 10-year probability of hip fracture(HF) and a major osteoporotic fracture(MF) of postmenopausal women with hyperglycemic stress.Results: 1.Under variable durations with H2O2 at different concentrations, the mortality of MC3T3- E1 cells appeared significant difference, and it dependents on concentration and time. RT-PCR analysis revealed that the m RNA levels of runt-related transcription factor 2(Runx2), Collagenase I(Col-I) and ALP were significantly decreased, the m RNA levels of Superoxide Dismutase(SOD), Growth arrest and DNA damage 45(Gadd45) were significantly increased, and the levels of mi R-200 a or mi R-141 were significantly increased. 2. Culturing MC3T3-E1 in osteogenic medium. Compared with normal control, the resultshow that ALP staining was shallow in experimental group, and ALP activity was significantly decreased. At day 3 and 7, RT-PCR analysis revealed that the m RNA levels of Runx2, Col-I and ALP in experimental group were significantly decreased. These results show that H2O2 can reduce the osteogenic differentiation ability of MC3T3-E1 cell. 3. Compared with normal control, the m RNA levels of Runx2, Col-I and ALP in mi R-141 or mi R-200 a overpression group were decreased, the m RNA levels of Runx2, Col-I and ALP in mi R-141 or mi R-200 a knockdown group were increased. These results suggested that reducing the expression of mi R-141 or mi R-200 a can relieve the damage of oxidative stress on MC3T3-E1 functions. 4. Bone mineral densities(BMDs) at the femoral neck(FN) and lumber spine 2-4(L2-4) was significantly higher in the type 2 diabetes mellitus(T2DM) cohort as compared with the healthy controls. There is no significant difference with probability of HF and MF between the women with T2 DM and healthy controls.Conclusions: 1. Oxidative stress can cause MC3T3 E1 cell damage, and osteogenesis function were decreased; the levels of mi R-200 a or mi R-141 were significantly increased. 2. Reducing the expression of mi R-141 or mi R-200 a can relieve the damage of oxidative stress on MC3T3-E1 functions. 3. We need to put hyperglycemic stress as an independent risk factor into the future FRAX? algorithm.