Therapeutic targets of broad relevance are likely located in pathogenic pathways common to disorders of various etiologies. Screening for targets of this type revealed CCN genes to be consistently upregulated in multiple cardiomyopathies. We developed RNA interference (RNAi) to silence CCN2 and found this single-target approach to block multiple proinflammatory and profibrotic pathways in activated primary cardiac fibroblasts (PCFBs). The RNAi-strategy was developed in murine PCFBs and then investigated in "individual" human PCFBs grown from human endomyocardial biopsies (EMBs). Screening of short hairpin RNA (shRNA) sequences for high silencing efficacy and specificity yielded RNAi adenovectors silencing CCN2 in murine or human PCFBs, respectively. Comparison of RNAi with CCN2-modulating microRNA (miR) vectors expressing miR-30c or miR-133b showed higher efficacy of RNAi. In murine PCFBs, CCN2 silencing resulted in strongly reduced expression of stretch-induced chemokines (Ccl2, Ccl7, Ccl8), matrix metalloproteinases (MMP2, MMP9), extracellular matrix (Col3a1), and a cell-to-cell contact protein (Cx43), suggesting multiple signal pathways to be linked to CCN2. Immune cell chemotaxis towards CCN2-depleted PCFBs was significantly reduced. We demonstrate here that this RNAi strategy is technically applicable to "individual" human PCFBs, too, but that these display individually strikingly different responses to CCN2 depletion. Either genomically encoded factors or stable epigenetic modification may explain different responses between individual PCFBs. The new RNAi approach addresses a key regulator protein induced in cardiomyopathies. Investigation of this and other molecular therapies in individual human PCBFs may help to dissect differential pathogenic processes between otherwise similar disease entities and individuals.

中文翻译：

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单靶RNA干扰可阻断心脏成纤维细胞中多种相互作用的促炎和促纤维化途径。

具有广泛相关性的治疗目标可能位于各种病因疾病共有的致病途径中。筛选这种类型的靶标显示，在多种心肌病中，CCN基因始终被上调。我们开发了RNA干扰（RNAi）使CCN2沉默，并发现这种单靶标方法可以阻断激活的原发性心脏成纤维细胞（PCFBs）中的多个促炎和促纤维化途径。RNAi策略是在鼠类PCFBs中开发的，然后在从人心内膜活检组织（EMBs）生长的“个体”人PCFBs中进行研究。筛选具有高沉默效力和特异性的短发夹RNA（shRNA）序列可分别在鼠或人PCFB中产生沉默CCN2的RNAi腺载体。RNAi与表达miR-30c或miR-133b的CCN2调节microRNA（miR）载体的比较显示出更高的RNAi功效。在鼠类PCFB中，CCN2沉默导致伸展诱导的趋化因子（Ccl2，Ccl7，Ccl8），基质金属蛋白酶（MMP2，MMP9），细胞外基质（Col3a1）和细胞间接触蛋白（Cx43）的表达大大降低。 ，表明有多个信号通路与CCN2相关联。对CCN2耗尽的PCFBs的免疫细胞趋化性显着降低。在这里，我们证明了这种RNAi策略在技术上也适用于“个体”人PCFB，但是这些分别显示出对CCN2耗竭的明显不同的响应。基因组编码因子或稳定的表观遗传修饰可能解释了各个PCFB之间的不同响应。新的RNAi方法解决了心肌病中诱导的关键调节蛋白。对单个人PCBFs中这种和其他分子疗法的研究可能有助于剖析其他相似疾病个体与个体之间的不同致病过程。