A primary culture of human nucleus pulposus (NP) cells was established and identified by immunofluorescence. An in vitro IDD model was constructed by compressing human NP cells, and the MTT assay was used to measure cell viability. Changes in the ncRNA group were analysed by RNA-seq. The expression levels of hsa_circ_7042, CDH2, and miR-369-3p were detected by qPCR. Cell apoptosis, senescence, and extracellular matrix (ECM) metabolism were detected by flow cytometry, β-galactosidase staining, and Western blotting. hsa_circ_7042, miR-369-3p, and bone morphogenetic protein 2 (BMP2) were verified by luciferase and RNA immunoprecipitation (RIP) analyses. The PI3K/Akt pathway was validated by transfection of BMP2 siRNA. Furthermore, a mouse model of lumbar spine instability was constructed. circ_7042 adenovirus was packaged and injected into the intervertebral discs of mice, and the influence of circ_7042 overexpression on intervertebral disc degeneration was determined.
Western blotting, qPCR, and flow cytometry analyses confirmed that overexpression of circ_7042 could downregulate miR-369-3p and upregulate the expression of CDH2 and BMP2 in IDD cell and animal models. Additionally, the levels of apoptotic and senescent cells decreased, and ECM degradation decreased. The PI3K/Akt pathway was significantly activated after circ_7042 was overexpressed. The injection of circ_7042-overexpressing adenovirus effectively reduced ECM degradation and the level of apoptosis in NP tissue.
circ_7042 could upregulate the expression of CDH2 and BMP2 by absorbing miR-369-3p, and the increased BMP2 activated the PI3K/Akt pathway, thus improving IDD.
建立了人髓核(NP)细胞的原代培养物,并通过免疫荧光进行了鉴定。通过压缩人NP细胞构建体外IDD模型,MTT法检测细胞活力。通过RNA-seq分析ncRNA组中的变化。qPCR检测hsa_circ_7042、CDH2和miR-369-3p的表达水平。细胞凋亡,衰老,用流式细胞仪检测细胞外基质(ECM)代谢,β-半乳糖苷酶染色,和西方印迹。hsa_circ_7042,miR-369-3p,通过荧光素酶和RNA免疫沉淀(RIP)分析验证和骨形态发生蛋白2(BMP2)。通过转染BMP2siRNA验证PI3K/Akt途径。此外,构建了腰椎不稳定的小鼠模型。将circ_7042腺病毒包装并注射到小鼠的椎间盘中,并确定了circ_7042过表达对椎间盘退变的影响。
蛋白质印迹,qPCR,和流式细胞术分析证实circ_7042的过表达可以下调miR-369-3p,并上调IDD细胞和动物模型中CDH2和BMP2的表达。此外,凋亡和衰老细胞的水平下降,ECM降解减少。PI3K/Akt通路在circ_7042过表达后显著激活。注射过表达circ_7042的腺病毒可有效降低NP组织中ECM的降解和凋亡水平。
circ_7042可以通过吸收miR-369-3p上调CDH2和BMP2的表达,增加的BMP2激活了PI3K/Akt通路,从而改善IDD。