Bevacizumab (BVZ) is the first recombinant humanized monoclonal antibody against vascular endothelial growth factor (VEGFA) approved by the FDA for the treatment of different kinds of cancers, especially colorectal cancer. Although the anti-tumor effects have been verified, the side effects of BVZ are also noteworthy, among which, cardiotoxicity may be the most serious side effect of BVZ. However, the exact mechanisms of cardiotoxicity induced by BVZ have been little explored. This study was conducted in vitro in a human cardiac myocyte (HCM) model. MTT assay was conducted to determine BVZ-stimulated cell viability. For testing the function and mechanism, the cells were transfected with miR-140-5p mimics, miR-140-5p inhibitor and/or VEGFA small interfering RNA (si-VEGFA). Then, apoptosis of the cells was detected via annexin V/propidium iodide (AV-PI) staining followed by flow cytometry. qRT-PCR and western blot assays were applied to measure gene expression (i.e. mRNA) and protein levels, respectively. The CK, LDH, SOD, CAT and GSH-Px activities and MDA level were determined using commercial kits. ROS levels were determined by DCFH-DA assay. Mitochondrial membrane potential was measured by JC-1 assay. Dual-luciferase reporter assay was used to detect the interaction between miR-140-5p and VEGFA. BVZ could inhibit HCM proliferation and induce apoptosis. miR-140-5p was upregulated in response to BVZ treatment and miR-140-5p restraint could alleviate HCM damage caused by BVZ treatment. In contrast, VEGFA and 14-3-3γ expressions were down-regulated by BVZ, and miR-140-5p could inhibit the expression of 14-3-3γ by directly targeting VEGFA. Moreover, VEGFA suppression enhanced HCM injury stimulated by BVZ and partially reversed the functional role of the miR-140-5p inhibitor in BVZ-treated cells. Taken together, miR-140-5p promoted BVZ-treated cardiomyocyte toxicity by targeting the VEGFA/14-3-3γ signal pathway. Collectively, miR-140-5p mediated the BVZ-induced cytotoxicity to cardiomyocytes by targeting the VEGFA/14-3-3γ signal pathway, indicating that miR-140-5p may be a novel target for treating BVZ-induced cardiotoxicity.

中文翻译：

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miR-140-5p通过靶向VEGFA /14-3-3γ信号通路介导贝伐单抗对心肌细胞的细胞毒性。

贝伐单抗（BVZ）是第一种针对血管内皮生长因子（VEGFA）的重组人源化单克隆抗体，已获得FDA批准，可用于治疗各种癌症，尤其是结直肠癌。尽管已经证实了抗肿瘤作用，但BVZ的副作用也很值得注意，其中，心脏毒性可能是BVZ最严重的副作用。但是，由BVZ引起的心脏毒性的确切机制尚未探索。这项研究是在人心肌细胞（HCM）模型中进行的。进行MTT测定以确定BVZ刺激的细胞活力。为了测试功能和机制，将细胞用miR-140-5p模拟物，miR-140-5p抑制剂和/或VEGFA小干扰RNA（si- VEGFA）转染。）。然后，通过膜联蛋白V /碘化丙啶（AV-PI）染色，然后通过流式细胞仪检测细胞的凋亡。使用qRT-PCR和Western blot分析分别测量基因表达（即mRNA）和蛋白质水平。使用商业试剂盒测定了CK，LDH，SOD，CAT和GSH-Px活性以及MDA水平。ROS水平通过DCFH-DA测定法确定。线粒体膜电位通过JC-1测定法测量。使用双重荧光素酶报告基因检测来检测miR-140-5p与VEGFA之间的相互作用。BVZ可以抑制HCM增殖并诱导细胞凋亡。miR-140-5p响应BVZ治疗而上调，而miR-140-5p的抑制作用可以减轻BVZ治疗引起的HCM损伤。相比之下，VEGFABVZ下调了14-3-3γ和14-3-3γ的表达，miR-140-5p可以通过直接靶向VEGFA抑制14-3-3γ的表达。此外，VEGFA抑制增强了BVZ刺激的HCM损伤，并部分逆转了miR-140-5p抑制剂在BVZ处理的细胞中的功能作用。总之，miR-140-5p通过靶向VEGFA /14-3-3γ信号通路来促进BVZ治疗的心肌细胞毒性。miR-140-5p通过靶向VEGFA /14-3-3γ信号通路共同介导BVZ诱导的对心肌细胞的细胞毒性，表明miR-140-5p可能是治疗BVZ诱导的心脏毒性的新靶标。