ABSTRACT
Objective:To investigate the role of microRNA-187-5p (miR-187-5p) on osteoporosis (OP) as well as the related molecular mechanism.Methods:The level of miR-187-5p and tartrate-resistant acid phosphatase (TRAP, the osteoclast marker) was quantitatively detected in TRANKL-induced OP group and control (con) group by reverse transcription-quantitative PCR (RT-qPCR) ; Cell counting kit 8 (CCK8) was used to measure the viability of murine macrophage cell line (RAW264.7) ; Western blot was used to detect the expression of p65 and TNFα. Moreover, the dual luciferase reporter assays was applied to detect the interaction between miR-187-5p and p65.Results:The osteoclast proliferation determined by CCK8 and the mRNA level of TRAP detected by RT-qPCR demonstrated that the growth of osteoclast was inhibited in OP group compared with that in con group ( P=0.008; P=0.017) , which reversed by miR-187-5p mimic stimulation ( P=0.023; P=0.037) . The miR-187-5p was lowly expressed in OP group compared with that in con group ( P=0.031) ,which was increased by miR-187-5p mimic treatment compared with miR-187-5p NC treatment ( P=0.041) . Moreover, western blot indicated that the protein level of p65 and TNFα in OP group were up-regulated compared with that in con group ( P=0.034; P=0.024) , which restored by the miR-187-5p mimic co-incubation ( P=0.028; P=0.036) . Moreover, the RT-qPCR indicated that the mRNA level of p65 in OP group was also increased compared with that in con group ( P=0.039) , and the miR-187-5p mimic co-incubation decreased the mRNA level of p65, compared with miR-187-5p NC group ( P=0.025) . The dual luciferase reporter assays indicated that there was an interaction between miR-187-5p and p65. Conclusion:miR-187-5p alleviates OP through inhibiting the activity of osteoclast via RANKL/NFκB signaling
ABSTRACT
Objective To explore the relationship among the expression level of MMP?13, AGG and Col?II in the chon?drocytes caused by nutritional deficiencies in rabbits. Methods 30 New Zealand white rabbits were randomly divided into autolo?gous chondrocyte transplantation group (control group, n=10), nutrition block group (surgery group, n=10), and peripheral nutrition block group (sham surgery group, n=10). 4 weeks after treatment, the rabbits were sacrificed for undergoing the observations on general and histological level;real?time PCR assay was employed for testing the expression level of MMP?13, AGG and Col?II;cel?lular apoptosis percentage was observed through TUNEL stain. The relationship among the apoptosis level, cartilage cells histologi?cal Mankin score as well as the expression level of MMP?13, AGG and Col?II were analyzed. Results Based on the Mankin score, there was a statistic difference between surgery group and control group. On the other side, there were no statistic differenc?es between sham surgery group and control group. 4 weeks after treatment, surgery group presented a higher apoptotic percentage compared with control group;this value between sham surgery group and control showed no significant differences. There was an increased mRNA expression level of MMP?13 and a decreased mRNA expression level of AGG and Col?II in surgery group com?pared with control group;no statistic differences of all these values was found between sham surgery group and control group. His?tological Mankin score and apoptotic percentage presented positive correlation (r=0.922, P<0.001), the regression equation:Y=-0.548+0.404X, R2=0.844 (F=157.735, P<0.001); the mRNA expression level of MMP?13 and apoptotic percentage presented positive correlation (r=0.942, P<0.001), the regression equation:Y=0.951+0.116X, R2=0.883 (F=219.054, P<0.001). There was a nega?tive correlation between the mRNA expression level of MMP?13 and the mRNA expression level of AGG as well as Col?II (r=-0.956,-0.945, P<0.001). Conclusion Damage of cartilage cells causes the up?regulation of the MMP?13 expression which could ex?acerbate the degeneration of cells. It could induce the down?regulation of AGG and Col?II mRNA expression, which will cause the extracellular matrix synthesis disorder.