Objective To investigate the expression of long non-coding RNA NEF (LncRNA NEF) in osteoporosis rats and its effect on the differentiation of osteoblasts and osteoclasts.
Methods Osteoporotic rats were prepared as the experimental group (n=6) by the tail suspension method, and another normal control group (n=6) was set up.The bone mass and structure of rat femur in two groups were measured by dual energy X-ray absorptiometry and microcomputer tomography; the quantitative real-time polymerase chain reaction (qRT-PCR) method was used to detect the changes of LncRNA NEF, ALP, tartrate-resistant acid phosphatase (TRAP), CTSK levels in the femoral tissues of the two groups of rats.The MC3T3-E1 cells of the rat pre-osteoblast cell line and RAW264.7 cells of the macrophage cell line were cultured and induced differentiation in vitro. The ALP activity detection kit was used to determine the ALP activity of osteoblasts. Alizarin red staining and TRAP staining were used to observe the effects of osteogenic differentiation and osteoclast differentiation, respectively. The effects of silencing LncRNA NEF expression on ALP and LncRNA NEF expression and osteogenic effects in MC3T3-E1 cells, as well as CTSK mRNA and its protein expression and osteoclast effect in RAW264.7 cells were explored.
Results The bone mineral density, bone volume fraction (BV/TV), number of trabeculae(Tb.N) and thickness of trabeculae (Tb.Th) in the experimental group were significantly lower, and the trabecular spacing (Tb.Sp) was significantly higher than that in the control group (P < 0.05).The levels of LncRNA NEF and ALP in the femur of rats in the experimental group were significantly, while the levels of TRAP and CTSK were significantly higher than those in the control group (P < 0.05). Pearson correlation analysis showed that LncRNA NEF was positively correlated with ALP expression (r=0.532, P < 0.05), and negatively correlated with TRAP and CTSK expression in osteoporosis rats (r=-0.624, -0.573, P < 0.05). During osteogenic differentiation of MC3T3-E1 cells, the level of LncRNA NEF and ALP activity were significantly up-regulated on day 15 compared with day 0 (P < 0.05); during osteoclast differentiation of RAW264.7 cells, the level of LncRNA NEF was significantly down-regulated, and the CTSK mRNA and its protein levels were significantly up-regulated on day 6 compared with day 0 (P < 0.05). Knockdown of the expression of LncRNA NEF could significantly inhibit the osteogenic differentiation of MC3T3-E1 cells, and promote the osteoclast differentiation of RAW264.7 cells.
Conclusion LncRNA NEF expression is abnormally low during osteogenic differentiation and abnormally high during osteoclast differentiation.LncRNA NEF silencing may induce osteoporosis by blocking osteoblast differentiation and promoting osteoclast differentiation.
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