Abstract:

Objective To discuss the effect of microRNA-7 （miR-7） on the steroid-induced avascular nectosis of femoral head （SANFH） cell model in vitro，and to clarify its mechanism. Methods The lentivirus miR-7 mimic（miR-7 mimic） and miR negative control（miR NC） were transfected into the osteoblast MC3T3-E1 cells，and control group was set up， real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the transfection of MC3T3-E1 cells in various groups.The MC3T3-E1 cells were divided into control group， SANFH group， miR-7 mimic group， and miR-7 mimic+tumicamy （TM） group.CCK- 8 method was used to detect the proliferation activities of cells in various groups； flow cytometry was used to detect the apoptotic rates of cells in various groups； alkaline phosphatase （ALP） staining method was used to detect the ALP staining of cells in various groups；colorimetric method was used to detect the levels of ALP in cells in various groups； Alizarin red S staining was used to observe the formation of mineralization nodule in the cells in various groups； RT-qPCR method and Western blotting method were used to detect the expression levels of runt-related transcription factor 2 （Runx2）， osteocalcin （OCN）， osteopontin （OPN） mRNA and proteins in the cells in various groups； Western blotting method was used to detect the expression levels of glucose regulated protein 78 （GRP78）， CCAAT/enhancer-binding protein（CHOP）， cysteinyl aspartate specific proteinase（Caspase）-12， Caspase-9， and Caspase-3 proteins in the cells in various groups； immunofluorescence double-labeling staining was used to detect the fluorescence intensities of expressions of GRP78 and Caspase-3 proteins in the cells in various groups. Results Compared with control group and miR NC group， the expression level of miR-7 in the MC3T3-E1 cells in miR-7 mimic group was increased （P<0.05）. Compared with control group， the proliferation activity of the MC3T3-E1 cells in SANFH group was decreased （P<0.05）， the apoptotic rate and TUNEL positive rate were increased （P<0.05）， the number of ALP staining cells was less and the ALP activity was decreased （P<0.05）， the number of formation of mineralized nodules in the cells was decreased， the expression levels of Runx2， OCN， and OPN mRNA and proteins in the cells were decreased （P<0.05）， the expressions levels of GRP78， CHOP， Caspase-12， Caspase-9， and Caspase-3 proteins in the cells were increased （P<0.05）， and the fluorescence intensities of expressions of GRP78 and Caspase-3 proteins were increased （P<0.05）； compared with SANFH group， the proliferation activity of the MC3T3-E1 cells in miR-7 mimic group was increased （P<0.05）， the apoptotic rate and TUNEL positive rate were decreased （P<0.05）， the number of ALP staining cells was increased， the ALP activity was increased （P<0.05）， the number of formation of mineralized nodules in the cells was significantly increased， the expressions levels of Runx2， OCN， and OPN mRNA and proteins in the cells were increased （P<0.05）， while the expression levels of GRP78， CHOP， Caspase-12， Caspase-9， and Caspase-3 proteins in the cells were decreased （P<0.05），and the fluorescence intensities of expressions of GRP78 and Caspase-3 proteins in the cells were decreased （P<0.05）；compared with miR-7 mimic group， the proliferation activity of the MC3T3-E1 cells in miR-7 mimic+TM group was decreased （P<0.05）， the apoptotic rate and TUNEL positive rate were increased （P<0.05）， the number of ALP staining cells was less and the ALP activity was decreased （P<0.05）， the number of formation of mineralized nodules in the cells was decreased， the expression levels of Runx2， OCN， and OPN mRNA and proteins in the cells were decreased （P<0.05）， the expression levels of GRP78， CHOP， Caspase-12， Caspase-9， and Caspase-3 proteins in the cells were increased （P<0.05）， and the fluorescence intensities of expressions of GRP78 and Caspase-3 proteins were increased （P<0.05）. Conclusion miR-7 can promote the osteogenesis of the MC3T3-E1 cells in SANFH state， and the mechanism may be related to the inhibition of endoplasmic reticulum stress-mediated apoptosis pathway.

Key words: Steroid-induced avascular necrosis of femoral head, MicroRNA-7, Endoplasmic reticulum stress, Apoptosis, Osteogenesis