环状 RNA CDR1as 通过靶向 miR-135b/FIH-1 轴抑制骨微血管内皮细胞活性和血管生成。
CircCDR1as Suppresses Bone Microvascular Endothelial Cell Activity and Angiogenesis Through Targeting miR-135b/ FIH-1 Axis.
机构信息
Department of Rehabilitation, The third Affiliated Hospital, Southern Medical University, Guangzhou, China.
Home for the Aged Guangzhou, Guangzhou, China.
出版信息
Orthop Surg. 2021 Apr;13(2):573-582. doi: 10.1111/os.12883. Epub 2021 Feb 22.
OBJECTIVE
The current study investigated the role of CircCDR1as on angiogenesis of bone microvascular endothelial cells (BMECs) isolated from non-traumatic ONFH.
METHODS
Forty corticosteroid-induced ONFH patients received THA were enrolled in our study. Expressions of CircCDR1as, miR-135b, and FIH-1 were detected by qRT-PCR in affected necrosis tissue and non-affected normal tissue. Bone microvascular endothelial cells (BMEC) were isolated from six patients and treated with 0.1 mg/mL hydrocortisone to establish a GC-damaged model of BMECs. Circ CDR1as plasmid and miR-135b mimic were transfected into BMECs. BMEC proliferation was assessed using MTT assays. The migration ability of cells was detected by scratch-wound assays. Matrigel assay was performed to detect angiogenesis in vitro. Western blot assay was used to detect HIF-1α, VEGF, and FIH-1 expressions. FISH, RNA pull down, RIP, and luciferase assay were carried out to determine the interaction of CircCDR1as, miR-135b, and FIH-1.
RESULTS
CircCDR1as was upregulated(2.02 ± 0.30 vs. 1.00 ± 0.10,P < 0.001) whereas miR-135b was downregulated (0.55 ± 0.12 vs. 1.00 ± 0.10,P < 0.001) in affected tissues than in non-affected tissues. Expression of CircCDR1as and FIH-1 were negatively associated with miR-135b in affected tissues (CircCDR1as with miR-135b: r = -0.506, P < 0.001; FIH-1 with miR-135b r = -0.510, P < 0.001). Total blood tubule density was increased when CircCDR1as was silenced compared with NC (P < 0.01 vs. NC). The number of migrated BMECs were significantly increased in CircCDR1as silencing group compared with NC group (P < 0.05 vs. NC). In addition, CircCDR1as plasmids transfection increased the protein expressions of FIH-1 (P < 0.05 vs. NC) and reduced the HIF-1α as well as VEGF expression compared with NC group (P < 0.05 vs. NC). FISH, RNA pull down, RIP, and luciferase assay identified that FIH-1 was a target of miR-135b and could be modulated by CircCDR1as.
CONCLUSION
CircCDR1as decreases angiogenesis and proliferation of BMECs by sponging miR-135b and upregulate FIH-1.
目的
本研究旨在探讨 CircCDR1as 在非创伤性股骨头坏死（ONFH）患者骨髓微血管内皮细胞（BMEC）血管生成中的作用。
方法
纳入 40 例接受 THA 的皮质类固醇诱导性 ONFH 患者作为研究对象。通过 qRT-PCR 检测病变组织和非病变正常组织中 CircCDR1as、miR-135b 和 FIH-1 的表达。从 6 例患者中分离骨髓微血管内皮细胞（BMEC），并使用 0.1mg/mL 氢化可的松处理以建立 BMEC 的 GC 损伤模型。将 Circ CDR1as 质粒和 miR-135b 模拟物转染到 BMEC 中。使用 MTT 法评估 BMEC 增殖。通过划痕实验检测细胞迁移能力。进行体外血管生成实验。采用 Western blot 法检测 HIF-1α、VEGF 和 FIH-1 的表达。通过 FISH、RNA 下拉、RIP 和荧光素酶测定来确定 CircCDR1as、miR-135b 和 FIH-1 的相互作用。
结果
与非病变组织相比，病变组织中 CircCDR1as（2.02±0.30 比 1.00±0.10，P<0.001）上调，而 miR-135b（0.55±0.12 比 1.00±0.10，P<0.001）下调。病变组织中 CircCDR1as 和 FIH-1 的表达与 miR-135b 呈负相关（CircCDR1as 与 miR-135b：r=-0.506，P<0.001；FIH-1 与 miR-135b：r=-0.510，P<0.001）。与 NC 相比，沉默 CircCDR1as 后总血管管密度增加（P<0.01 比 NC）。与 NC 组相比，沉默 CircCDR1as 组 BMEC 迁移数明显增加（P<0.05 比 NC）。此外，与 NC 组相比，CircCDR1as 质粒转染组 FIH-1 蛋白表达升高（P<0.05 比 NC），HIF-1α 和 VEGF 表达降低（P<0.05 比 NC）。FISH、RNA 下拉、RIP 和荧光素酶测定鉴定出 FIH-1 是 miR-135b 的靶基因，并且可以被 CircCDR1as 调节。
结论
CircCDR1as 通过海绵吸附 miR-135b 降低 BMEC 的血管生成和增殖，并上调 FIH-1。
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