Study On Correlation Between MiR-672-5p、miR-146a-5p And Steroid-induced Avascular Necrosis Of Femoral Head

Objective1.To investigate the differential expression of miR-672-5p in rats’osteoblasts in rats after intervention by different concentrations of glucocorticoid.2.To investigate the differential expression of miR-672-5p in osteoblasts in rats after intervention by long-acting,middle-acting and short-acting hormone.3.To investigate the differential expression of miR-146a-5p in different kind of patient with steroid-induced osteonecrosis of the femoral head（SONFH）.Methods1.The primary osteoblasts of SD rats were cultured.The third generation rat osteoblasts were added into dulbecco’s modified eagle medium（DMEM）high glucose medium;then divide into steroid group and blank group.The concentration of steroid group was 10^-33 mol/L,10^-44 mol/L,10^-55 mol/L,10^-66 mol/L,respectively.All groups were conducted by total RNA extraction,reverse transcription reaction,quantitative PCR reaction;and calculated 2^（-△△Ct）value to evaluate miR-672-5p expression.2.The primary osteoblasts of SD rats were cultured.The third generation rat osteoblasts were added into DMEM high glucose medium;then into the final concentration was 10^-5mol/L to methylprednisolone,hydrocortisone and Dexamethasone and blank control group;four groups were conducted by total RNA extraction,reverse transcription reaction,quantitative PCR reaction;and calculated 2^（-△△Ct）value to evaluate miR-672-5p expression.3.18 cases of osteonecrosis of the femoral head patients who were selected in hospitalized in the orthopedic department of the first affiliated hospital of Guangzhou University of Chinese Medicine from November,2016 to March,2017.These patients were included 6 cases of SONFH patients,6 cases of alcohol-induced osteonecrosis of the femoral head（AONFH）patients and 6 cases of trauma-induced osteonecrosis of the femoral head（TONFH）patients.At the same time,6 healthy volunteers were also included in this study.Collected their serum and all these serum samples were conducted by total RNA extraction,reverse transcription reaction,quantitative PCR reaction;and calculated2^（-△△Ct）value to evaluate miR-146a-5p expression.Results1.Compared with the blank group,the expression quantity of miR-672-5p in four different concentrations of glucocorticoid groups were increased 6.16times、2.56 times、2.35 times and 1.26 times,respectively.Moreover,the expression quantity of miR-672-5p was significantly increased in Group 10^-3mol/L.2.Compared with the blank group,the expression of miR-672-5p in the osteoblasts of the three groups was up-regulated,and the expression of miR-672-5p was 2.35 in the methylprednisolone group,indicating the highest expression.There was significant difference between the methylprednisolone group and the dexamethasone group or the dexamethasone group,but there was no significant difference between the hydrocortisone group（1.50）and the dexamethasone group（1.53）.3.Compared with the healthy volunteers group（6.68±3.61）,the expression of miR-146a-5p in patients with osteonecrosis of the femoral head groups were down-regulation.the expression of miR-146a-5p in patients with SONFH group was 3.12±2.56,in patients with AONFH group was 4.48±3.93 and in patients with TONFH group was 3.65±1.94.However,the variance analysis showed that there were no statistical significance between these groups（F=1.71,p=0.1965>0.05）.Conclusion1.The expression quantity of miR-672-5p was vary from groups,and increase with the increase concentrations of glucocorticoid.Moreover,the expression quantity of was significantly increased in Group 10^-3 mol/L.We therefore thought 10^-3 mol/L concentrations of glucocorticoid might be a critical value,which may increase the risk of steroid induced osteonecrosis of the femoral head.Further research will focus attention on the target RNA and signaling pathway of miR-672-5p.2.The expression of miR-672-5p in rat osteoblasts by middle-acting hormone is the highest,which suggests that methylprednisolone may play an important role in steroid-induced femoral head necrosis.3.The expression of miR-146a-5p was different between patients with osteonecrosis of the femoral head groups and healthy volunteers group.However,there no statistical significance between these groups.Therefore,miR-146a-5p cannot be a specific expression gene in patients with SONFH.