微小核糖核酸-1205沉默Cullin-RING泛素E3连接酶4A激活AMPK信号传导保护人成骨细胞免受地塞米松损伤的研究

Silencing Cullin-RING ubiquitin E3 ligase 4A by microRNA-1205 to activate AMPK signaling and protect human osteoblasts from dexamethasone

目的:腺苷酸活化蛋白激酶(AMPK)激活可以抑制地塞米松(Dex)诱导的成骨细胞损伤。Cullin-RING泛素E3连接酶4A(CUL4A)决定了AMPKα泛素化和蛋白酶体降解。本研究鉴定了一种新型的靶向CUL4A的微小核糖核酸-1205(miR-1205)。方法:为了研究miR-1205对Dex诱导人类成骨细胞的氧化损伤和细胞死亡的影响,对hFOB1.19成骨细胞和原代人成骨细胞进行培养和分化,提取第3~10代的原始人类成骨细胞总细胞RNA,并通过反转录获得c DNA。利用qPCR、2ΔΔCt方法进行数据定量,分析miR-1205的表达。将生成表达premiR-1205的慢病毒(lv-premiR-1205)或miR-1205反义慢病毒(lv-antagomiR-1205),进行过滤并添加至h FOB1.19细胞或人成骨细胞。将细胞接种到六孔板中,通过miR模拟物转染,进行遗传修饰。检查CUL4A 3’-UTR荧光素酶的活性,并对结果进行量化。在对hFOB1.19细胞使用Dex处理后,对细胞功能包括细胞生存力测定,细胞死亡检测,以及通过核TUNEL染色和caspase-3活性测定及Western印迹测定进行细胞凋亡检查。结果:将CUL4A七个靶向miRNA模拟物中的每一个都分别转染到hFOB1.19成骨细胞中。其中,miR-1205模拟物导致最显著的CUL4A mRNA降低。miR-1205的亚细胞分布表明,内源性miR-1205的大部分位于细胞质中。生物素化的miR-1205与hFOB1.19细胞中的CUL4A m RNA直接关联并使其沉默,从而导致AMPK级联激活,并显著减弱h FOB1.19细胞中Dex诱导的细胞毒性。此外,miR-1205的过表达显著抑制了hFOB1.19细胞中Dex诱导的凋亡激活。结论:在h FOB1.19细胞和人类成骨细胞中,通过miR-1205沉默CUL4A,激活了AMPK信号传导,并调节其下游靶标发挥有效的抗氧化活性,极大减弱Dex诱导的细胞毒性,从而显著保护成骨细胞。

Objective: Adenosine 5’-monophosphate-activated protein kinase(AMPK) activation can inhibit dexamethasone(Dex)-induced osteoblast injury. Cullin-RING ubiquitin E3 ligase 4 A(CUL4 A) dictates AMPKα ubiquitination and proteasomal degradation. To identified a novel CUL4 A-targeted microRNA-1205(miR-1205). Methods: In order to study the effect of miR-1205 on Dex-induced oxidative damage and cell death of human osteoblasts, hFOB1.19 osteoblasts and protogenic human osteoblasts were cultured and differentiated, the total cell RNA of primitive human osteoblasts in the 3 rd and 10 th generation was extracted, and cDNA was obtained through reverse transcription. The expression of miR-1205 was analyzed by qPCR and 2ΔΔCt methods for data quantification. Chronic viruses(lv-premiR-1205) or miR-1205 antonymous chronic viruses(lvantagomiR-1205) which expressed premiR-1205 were generated, and the virus were filtered and added to hFOB1.19 cells or human osteoblasts. The cells were inoculated into a six-hole plate and transfected by miR simulator for genetic modification.Check the activity of CUL4 A 3’-UTR fluoresceinase and quantify the results. After Dex treatment of hFOB1.19 cells, cell functions including cell viability were measured, cell death was detected, and apoptosis was tested through nuclear TUNEL staining, caspase-3 activity determination and Western blot. Results: Each of the seven targeted miRNA simulators of CUL4 A was transfected into hFOB1.19 osteoblasts respectively. Among them, the miR-1205 analogue led to the most significant reduction in CUL4 A mRNA. The subcellular distribution of miR-1205 indicated that most of the endogenous miR-1205 was located in the cytoplasm. Biotinylated miR-1205 was directly associated with CUL4 A mRNA in hFOB1.19 cells and silenced it, resulting in AMPK cascade activation and significantly reducing Dex-induced cytotoxicity in hFOB1.19 cells. In addition, the overexpression of miR-1205 significantly inhibited Dex-induced apoptosis activation in hFOB1.19 cells. Conclusions: In hFOB1.19 cells and human osteoblasts, AMPK signal transmission is activated through miR-1205 silencing CUL4 A, and its downstream target is regulated to play an effective antioxidant activity, greatly reducing Dex-induced cytotoxicity, thus significantly protecting osteoblasts.