Role and mechanism of miR-155/leptin receptor/adenosine phosphate-dependent protein kinase axis in tuberculin-induced osteoclast formation

Zengshun WANG; Angxiu SUONAN; Limin LIU; Jingyuan ZHOU

ABSTRACT

BACKGROUND:

Abnormal activation of osteoclasts plays an important role in the bone destruction due to spinal tuberculosis.During the pathogenesis of osteoporosis,miR-155 knockdown activates adenosine phosphate-dependent protein kinase(AMPK)by increasing the expression of leptin receptors,thereby inhibiting osteoclast differentiation and bone resorption.However,the role of miR-155/leptin receptor(LEPR)/AMPK axis in the bone destruction due to spinal tuberculosis remains unclear.

OBJECTIVE:

To investigate the role and mechanism of miR-155/LEPR/AMPK axis in tuberculin-induced osteoclast formation.

METHODS:

RAW264.7 cells were cultured and treated with different concentrations of purified protein derivative(PPD)(1.0,2.5,5.0,10.0 IU/mL)and transfected with negative control(NC)sequence or miR-155 inhibitor,NC siRNA sequence or LEPR siRNA sequence.Tartrate resistant acid phosphatase staining was used to detect the number of osteoclasts.Fluorescence quantitative PCR was used to detect the expression of miR-155.Western blot was used to detect the expression of LEPR and p-AMPK.Double luciferase reporter gene was used to verify miR-155 targeting LEPR. RESULTS AND

CONCLUSION:

Compared with the control group,the number of osteoclasts and the expression level of miR-155 significantly increased,while the expression level of LEPR and p-AMPK significantly decreased in 2.5,5.0,and 10.0 IU/mL PPD groups(P＜0.05).Compared with NC+5.0 IU/mL PPD group,the number of osteoclasts and the expression level of miR-155 significantly decreased,while the expression level of LEPR and p-AMPK significantly increased in the miR-155 inhibitor+5.0 IU/mL PPD group(P＜0.05).Compared with the NC group,the fluorescence activity of LEPR wild-type double luciferase reporter gene was increased in the miR-155 inhibitor group,and decreased in the miR-155 mimic group(P＜0.05).Compared with si-NC+miR-155 inhibitor+5.0 IU/mL PPD group,the expression level of miR-155 had no significant change,the number of osteoclasts significantly increased,and the expression levels of LEPR and p-AMPL significantly decreased in si-LEPR+miR-155 inhibitor+5.0 IU/mL PPD group(P＜0.05).To conclude,tuberculin can induce osteoclast formation by increasing miR-155 expression and inhibiting downstream LEPR expression and AMPK activation.