Study on promotion of bone cancer pain via rho associated coiled-coil forming protein kinase2/LIM domain kinase/Cofilin signaling by negative regulation of microRNA in mice

Abstract

Objective To observe microRNA (miR-93) regulation in bone cancer pain(BCP) and explore the underlying mechanisms of miR-93 and Rho associated coiled-coil forming protein kinase (ROCK) signaling affecting the biological behaviors of BCP in mice. Methods Male mice were randomly divided into Control group, Sham group, bone cancer pain (BCP group), BCP+PBS group and BCP+Y-27632(ROCK inhibitor) group(n=13). The paw mechanical threshold and paw thermal latency were used to evaluate the behavioral changes on 1 day before surgery and 1, 3, 5, 7, 10, 14 after surgery. L4-L6 dorsal root ganglions were removed on postoperative day 14, followed by Real-time PCR to measure the expression of miR-93. The ROCK2 promoter activity regulated by miR-93 was evaluated by a dual luciferase reporter assay. Western blotting was used to evaluate the protein expression level of ROCK2, phospho-LIM domain kinase (LIMK) and phospho-Cofilin. Results Following intrafemoral carcinoma inoculation, robust mechanical allodynia[Sham group(6.81±1.04) g, BCP group(5.12±0.47) g]and thermal hyperalgesia[Sham group(11.75±1.46) s,BCP group (7.61±1.07) s]in C57BL/6J mice were respectively developed on day 5 and 7, and the symptoms grow progressively with time (P<0.05). The expression level miR-93 is decreased in a time dependent way in BCP group with progressively increased pain. However, the ROCK2 expression level was increased (P<0.05), and subsequently activated the LIMK/Cofilin pathways. Dual luciferase reporter assay demonstrated that miR-93 directly targeted the 3'-UTR of the ROCK2 gene, resulting in inhibition of the post-transcriptional translation of ROCK2. ROCK inhibitor significantly up-regulated paw withdrawal threshold[BCP+PBS group(3.43±0.89) g, BCP+Y-27632 group (5.74±1.02) g], paw withdrawal latency[BCP+PBS group (5.48±1.03) s, BCP+Y-27632 group (9.82±1.24) s]and reduced the protein expression level of ROCK2, phospho-LIMK and phospho-Cofilin (P<0.05). Conclusions Reduced expression of miR-93 can promote BCP through activating ROCK2/LIMK/cofilin pathway. Therefore, miR-93 or ROCK inhibitor may be novel targets for BCP. Key words: Bone cancer pain; Analgesia; MicroRNA-93; Rho associated coiled-coil forming protein kinase2/LIM domain kinase/Cofilin signaling