背景:癌症恶病质诱导的骨骼肌纤维化(SMF)损害肌肉再生,改变肌肉结构和功能,降低抗癌药物的功效,降低了患者的生活质量,缩短了总生存期。RUNX家族转录因子2(Runx2),转录因子,和I型胶原蛋白α1链(COL1A1),SMF的主要组成部分,以前已经联系过了,与Runx2显示直接调节COL1A1mRNA水平。左旋肉碱,癌症恶病质的标志,可以减轻肝和肾模型的纤维化;然而,其在癌症恶病质相关纤维化中的作用以及Runx2在该过程中的参与仍有待探索。
方法:雌性C57小鼠(48周龄)皮下接种MC38细胞,建立癌性恶病质模型。通过口服管饲法给予5mg/kg剂量的左旋肉碱或等效体积的水14天,然后评估肌肉功能(握力)和纤维化。为了阐明DeltexE3泛素连接酶3L(DTX3L)/Runx2/COL1A1轴与转化生长因子β1刺激的NIH/3T3细胞纤维化之间的相互作用,一套分子技术,包括定量实时PCR,蛋白质印迹分析,免疫共沉淀,分子对接,免疫荧光和Duolink分析,被使用。还在体内模型中探索了腓肠肌中DTX3L/Runx2/COL1A1轴的相关性。
结果:补充l-肉碱可减少癌症恶病质引起的握力下降(>88.2%,P<0.05)和腓肠肌内胶原纤维面积(>57.9%,P<0.05)。在5mg/kg剂量下,L-肉碱还抑制COL1A1和α-平滑肌肌动蛋白(α-SMA)蛋白表达,它们是SMF和肌成纤维细胞的标志物。对TRRUST数据库的分析表明,Runx2同时调节COL1A1和COL1A2。体外,左旋肉碱降低Runx2蛋白水平并促进其泛素化。Runx2的过表达消除了左旋肉碱对COL1A1和α-SMA的影响。免疫共沉淀,分子对接,免疫荧光和Duolink分析证实了DTX3L和Runx2之间的相互作用,l-肉碱增强了这种相互作用以促进Runx2的泛素化。补充左旋肉碱可将DTX3L水平恢复到非恶病质条件下观察到的水平,在体外和体内。敲除DTX3L消除了左旋肉碱在体外对Runx2,COL1A1和α-SMA的影响。在未经处理的NIH/3T3细胞中,DTX3L的表达与Runx2和COL1A1的水平呈负相关。
结论:这项研究揭示了SMF中Runx2和DTX3L之间以前未被识别的联系,并证明l-肉碱对癌症恶病质相关的SMF具有显著的治疗作用,可能通过上调DTX3L。
BACKGROUND: Cancer cachexia-induced skeletal muscle fibrosis (SMF) impairs muscle regeneration, alters the muscle structure and function, reduces the efficacy of anticancer drugs, diminishes the patient\'s quality of life and shortens overall survival. RUNX family transcription factor 2 (Runx2), a transcription factor, and collagen type I alpha 1 chain (COL1A1), the principal constituent of SMF, have been linked previously, with Runx2 shown to directly modulate COL1A1 mRNA levels. l-Carnitine, a marker of cancer cachexia, can alleviate fibrosis in liver and kidney models; however, its role in cancer cachexia-associated fibrosis and the involvement of Runx2 in the process remain unexplored.
METHODS: Female C57 mice (48 weeks old) were inoculated subcutaneously with MC38 cells to establish a cancer cachexia model. A 5 mg/kg dose of l-carnitine or an equivalent volume of water was administered for 14 days via oral gavage, followed by assessments of muscle function (grip strength) and fibrosis. To elucidate the interplay between the deltex E3 ubiquitin ligase 3L(DTX3L)/Runx2/COL1A1 axis and fibrosis in transforming growth factor beta 1-stimulated NIH/3T3 cells, a suite of molecular techniques, including quantitative real-time PCR, western blot analysis, co-immunoprecipitation, molecular docking, immunofluorescence and Duolink assays, were used. The relevance of the DTX3L/Runx2/COL1A1 axis in the gastrocnemius was also explored in the in vivo model.
RESULTS: l-Carnitine supplementation reduced cancer cachexia-induced declines in grip strength (>88.2%, P < 0.05) and the collagen fibre area within the gastrocnemius (>57.9%, P < 0.05). At the 5 mg/kg dose, l-carnitine also suppressed COL1A1 and alpha-smooth muscle actin (α-SMA) protein expression, which are markers of SMF and myofibroblasts. Analyses of the TRRUST database indicated that Runx2 regulates both COL1A1 and COL1A2. In vitro, l-carnitine diminished Runx2 protein levels and promoted its ubiquitination. Overexpression of Runx2 abolished the effects of l-carnitine on COL1A1 and α-SMA. Co-immunoprecipitation, molecular docking, immunofluorescence and Duolink assays confirmed an interaction between DTX3L and Runx2, with l-carnitine enhancing this interaction to promote Runx2 ubiquitination. l-Carnitine supplementation restored DTX3L levels to those observed under non-cachectic conditions, both in vitro and in vivo. Knockdown of DTX3L abolished the effects of l-carnitine on Runx2, COL1A1 and α-SMA in vitro. The expression of DTX3L was negatively correlated with the levels of Runx2 and COL1A1 in untreated NIH/3T3 cells.
CONCLUSIONS: This study revealed a previously unrecognized link between Runx2 and DTX3L in SMF and demonstrated that l-carnitine exerted a significant therapeutic impact on cancer cachexia-associated SMF, potentially through the upregulation of DTX3L.