背景:在我们之前的研究中发现侵袭性垂体腺瘤(PA)存在骨侵袭,提示PA细胞可能参与破骨细胞的形成过程。据报道,miR-19a(作为miR-17-92簇的关键成员)可激活核因子κB(NF-κB)途径并促进炎症,这可能参与了垂体腺瘤的骨侵袭过程。
方法:在这项工作中,FISH用于检测PA患者组织中的miR-19a分布。建立PA骨侵袭模型,用miR-19a模拟物转染GH3细胞,HE染色检测破骨细胞的分级。进行qPCR以确定在RANKL诱导的破骨细胞生成的整个过程中miR-19a的表达。用miR-19a模拟物转染后,BMM用RANKL治疗指定时间,并通过qPCR和WesternBlot检测破骨细胞标记基因。通过凹坑形成和F-肌动蛋白环测定来评估破骨细胞的功能。TargetScan数据库和GSEA用于发现miR-19a的潜在下游,这是由Co-IP验证,西方印迹,和EMSA。
结果:这里,我们发现miR-19a的表达水平与PA的骨侵袭显著相关,在临床样本和动物模型中。在骨吸收之前的破骨细胞形成被miR-19显着增强,这是由减少的圆柱瘤(CYLD)表达介导的,增加肿瘤坏死因子受体相关因子6(TRAF6)的K63泛素化。因此,miR-19a通过激活下游NF-κB和丝裂原活化蛋白激酶(MAPK)途径促进破骨细胞生成。
结论:总结一下,这项研究的结果表明PA来源的miR-19a通过抑制CYLD表达和增强NF-κB和MAPK通路的激活来促进破骨细胞的生成.
BACKGROUND: The presence of bone invasion in aggressive pituitary adenoma (PA) was found in our previous study, suggesting that PA cells may be involved in the process of osteoclastogenesis. miR-19a (as a key member of the miR-17-92 cluster) has been reported to activate the nuclear factor-кB (NF-кB) pathway and promote inflammation, which could be involved in the process of the bone invasion of pituitary adenoma.
METHODS: In this work, FISH was applied to detect miR-19a distribution in tissues from patients with PA. A model of bone invasion in PA was established, GH3 cells were transfected with miR-19a mimic, and the grade of osteoclastosis was detected by HE staining. qPCR was performed to determine the expression of miR-19a throughout the course of RANKL-induced osteoclastogenesis. After transfected with a miR-19a mimic, BMMs were treated with RANKL for the indicated time, and the osteoclast marker genes were detected by qPCR and Western Blot. Pit formation and F-actin ring assay were used to evaluate the function of osteoclast. The TargetScan database and GSEA were used to find the potential downstream of miR-19a, which was verified by Co-IP, Western Blot, and EMSA.
RESULTS: Here, we found that miR-19a expression levels were significantly correlated with the bone invasion of PA, both in clinical samples and animal models. The osteoclast formation prior to bone resorption was dramatically enhanced by miR-19, which was mediated by decreased
cylindromatosis (CYLD) expression, increasing the K63 ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6). Consequently, miR-19a promotes osteoclastogenesis by the activation of the downstream NF-кB and mitogen-activated protein kinase (MAPK) pathways.
CONCLUSIONS: To summarize, the results of this study indicate that PA-derived miR-19a promotes osteoclastogenesis by inhibiting CYLD expression and enhancing the activation of the NF-кB and MAPK pathways.