微小RNA-362通过调控组蛋白去乙酰化酶6抑制大鼠骨癌痛

Analgesic effects of miR-362 on bone cancer pain process via targeting HDAC6 in rats

目的探讨骨癌痛(BCP)大鼠模型中微小RNA(miR)-362和组蛋白去乙酰化酶6(HDAC6)的表达变化,以及上调miR-362产生的镇痛效应和潜在作用机制。方法大鼠骨髓腔内注射Walker 256乳腺癌细胞建立BCP模型。用质粒转染方法调控miR-362和HDAC6表达,用Van Frey纤维丝和热痛刺激仪检测大鼠机械缩足阈值(PWT)和热缩组潜伏期(PWL)变化,用qRT-PCR检测脊髓miR-362和HDAC6 mRNA表达,Western blotting检测HDAC6和核因子κB p65(NF-κB p65)蛋白表达,用ELISA检测炎性因子白细胞介素(IL)-1β、IL-6和肿瘤坏死因子α(TNF-α)蛋白水平,最后用荧光素酶活性实验明确miR-362与HDAC6的上下游关系。结果与sham组相比,BCP组大鼠PWT和PWL显著下降,脊髓miR-362表达显著减少,HDAC6 mRNA和蛋白显著增加(均P<0.05)。与BCP组相比,BCP+LV-miR-362组大鼠出现PWT和PWL显著上升,脊髓HDAC6 mRNA和蛋白显著减少(均P<0.05)。与BCP+LV-miR-362组比较,BCP+LV-miR-362+LV-HDAC6组大鼠PWT和PWL显著下降(均P<0.05)。此外,与BCP组相比,BCP+LV-HDAC6 siRNA组大鼠脊髓NF-κB p65、IL-1β、IL-6和TNF-α蛋白显著减少(均P<0.05)。最后,与mimic miR-362+HDAC63’UTR^(MUT)组相比,mimic miR-362+HDAC63’UTR^(WT)组荧光素酶活性明显降低(P<0.05)。结论在大鼠BCP模型中,miR-362可通过“HDAC6-NF-κB p65”通路抑制BCP,提示miR-362有可能成为BCP分子治疗的新靶点。

Objective To detect the expressions of microRNA(miR)-362 and histone deacetylase 6(HDAC6)in bone cancer pain(BCP)rats and investigate the analgesic effect of miR-362 and its potential analgesic mechanism.Methods The BCP model was developed by injecting Walker 256 mammary gland carcinoma cells into bone marrow cavity.Plasmid transfection was used to regulate the expressions of miR-362 and HDAC6.The Van Frey filaments and radiant heat instrument were used to detect the paw withdrawal threshold(PWT)and paw withdrawal latency(PWL).qRT-PCR was used to detect mRNA expression levels of miR-362 and HDAC6,and Western blotting was used to detect protein expression of HDAC6 and nuclear factor kappa-B p65(NF-κB p65).ELISA assay was used to detect the protein levels of interleukin(IL)-1β,IL-6 and tumor necrosis factorα(TNF-α).Luciferase activity assay was used to determine the relationship between miR-362 and HDAC6.Results Compared to sham group,the significant decrease of PWT and PWL,decrease of miR-362 and the increase of HDAC6 mRNA and protein in the spinal were detected in BCP group(P<0.05).Compared to BCP group,the significantly increase of PWT and PWL and decrease of HDAC6 mRNA and protein in the spinal were detected in BCP+LV-miR-362 group(P<0.05).Compared to BCP+LV-miR-362 group,PWT and PWL significantly decreased in BCP+LV-miR-362+LV-HDAC6 group(P<0.05).In addition,compared to BCP group,significant decreases of NF-κB p65,IL-1β,IL-6 and TNF-αin spinal were detected in BCP+LV-HDAC6 siRNA group(P<0.05).Moreover,compared to mimic miR-362+HDAC63’UTR^(MUT) group,the luciferase activity significantly decreased in mimic miR-362+HDAC63’UTR^(WT) group(P<0.05).Conclusion As a key factor regulating the mechanism of BCP through“HDAC6-NF-κB p65”signal pathway in rats,targeting miR-362 may be a novel therapeutic method for BCP.