XIST/miR-376c-5p/OPN axis modulates the influence of proinflammatory M1 macrophages on osteoarthritis chondrocyte apoptosis

The inflammatory microenvironment in the joints is one of the critical issues during osteoarthritis (OA) and also the main factor that may aggravate symptoms. Under inflammatory microenvironment, M1 macrophages are activated and produce large numbers of proinflammatory mediators, leading to the production of degradative enzymes, the disturbance of chondrocyte apoptosis and cartilage catabolic processes, and finally the deterioration of OA. In the present study, we reveal that the overexpression of osteopontin (OPN), a cytokine, and a matrix protein involved in arthritis and chondrocyte apoptosis in OA, could exacerbate the inflammatory microenvironment in OA via promoting the production of proinflammation cytokines and the levels of degradative enzymes in M1 macrophages, therefore, enhancing the cytotoxicity of M1 macrophage on chondrocytes. XIST expression significantly increases in OA tissue specimens. XIST serves as a competing endogenous RNA for miR-376c-5p to compete with OPN for miR-376c-5p binding, thus counteracting miR-376c-5p-mediated OPN suppression. XIST knockdown could improve the inflammatory microenvironment in OA via acting on M1 macrophages, subsequently affecting the apoptosis of cocultured chondrocytes. miR-376c-5p inhibition exerts an opposing effect on M1 macrophages and cocultured chondrocytes, as well as significantly reverses the effect of XIST knockdown. As a further confirmation, XIST and OPN mRNA expression significantly increased in OA tissues and was positively correlated in tissue samples. In summary, we provide a novel mechanism of macrophages and the inflammatory microenvironment affecting chondrocyte apoptosis. XIST and OPN might be potential targets for OA treatment, which needs further in vivo experimental confirmation.     

从异常核型人胚胎干细胞中筛选不同X染色体失活状态的亚系

目的:从异常核型人胚胎干细胞系中分离两种不同X染色体失活(XCI)状态的细胞,建立亚系,并进行对其XCI状态特征和多能性标记进行鉴定。方法:G显带鉴定人胚胎干细胞系ch HESC-3早晚期代数细胞的核型,H3K27me3免疫荧光染色鉴定早晚期ch HESC-3表观遗传差异,RT-PCR检测早晚期ch HESC-3中XIST基因的表达。利用单细胞克隆的培养分选亚系,H3K27me3、RNA polymeraseⅡ以及DAPI三种标记的共染后每种表观标记各选两株进行RT-PCR,检测两种亚系中XIST基因的表达。并对这四株细胞进行干细胞标记鉴定。结果:G显带结果证明早期ch HESC-3为正常核型,晚期代数核型为异常核型,牵涉到8条染色体的复杂结构变异。H3K27me3免疫荧光染色证明异常核型ch HESC-3中有部分细胞出现了H3K27me3凝集点,而正常核型细胞中未发现。正常核型细胞(ch HESC-3N)没有XIST基因表达,异常核型细胞(ch HESC-3C)中有表达。在RNA polymeraseⅡ着色缺口中发现H3K27me3凝集点的细胞亚株XIST基因表达阳性,polymeraseⅡ着色缺口中未发现H3K27me3凝集点的细胞亚株XIST基因表达阴性,XIST阳性和阴性细胞各选两株进行多能性标记免疫荧光染色均为阳性。结论:成功从异常核型人胚胎干细胞系中分离两种不同XCI状态的细胞并建立亚系,两种表观类型的亚系均保持多能性标记并能在长期培养中保持各自特性。     

Downregulation of XIST ameliorates acute kidney injury by sponging miR-142-5p and targeting PDCD4

Acute kidney injury (AKI) is a common kidney disease that markedly affects public health. To date, the roles of long noncoding RNA XIST in AKI are poorly understood. Here, we investigated the biological functions of XIST in AKI. We observed that XIST expression increased in patients with AKI and HK-2 cells stimulated by CoCl₂. In addition, a rat AKI model induced by ischemia–reperfusion was established. Tumor necrosis factor-α, interleukin-6, and cyclooxygenase-2 messenger RNA expression were induced in vivo; moreover, XIST expression was upregulated. Knockdown of XIST significantly repressed CoCl₂-triggered injury in HK-2 cells. However, microRNA (miR)-142-5p, a downstream target of XIST, was downregulated in AKI. miR-142-5p was repressed by XIST and miR-142-5p could inhibit CoCl₂-induced injury in HK-2 cells. Moreover, PDCD4 expression was significantly increased in AKI. PDCD4 was predicted to be the target of miR-142-5p. Subsequently, loss of PDCD4 was able to retard injury in HK-2 cells exposed to CoCl_2. Thus, we suggest that XIST regulates miR-142-5p and PDCD4, and it has the potential to function as a biomarker in therapeutic strategies for AKI.