miR ‐ 320 a mediates doxorubicin ‐ induced cardiotoxicity by targeting

Background: Vascular homeostasis abnormalities may involve in doxorubicin induced cardiotoxicity. Methods: Enhanced cardiac miR‐320a expression, reduced cardiac microvessel density and impaired cardiac function were observed in mice treated by anthracycline doxorubicin. To further explore the role of miR‐320a in doxorubicin induced cardiotoxicity, microRNA mimics/inhibitor in vitro and rAAV administration in vivo were employed in mice. Results: Knockdown of miR‐320a not only resulted in enhanced proliferation and inhibited apoptosis in cultured endothelial cells, but also attenuated cardiac abnormalities induced by doxorubicin. On the contrary, overexpression of miR‐320a enhanced apoptosis in vitro, and aggravated vessel abnormalities in heart and subsequent cardiac dysfunction in mice. Furthermore, Western blot assays showed that VEGF‐A was a potential target of miR‐320a, which was verified by anti‐Ago2 co‐immunoprecipitation. Moreover, as same as miR‐320a, siRNA against VEGF‐A reinforced doxorubicin induced endothelial cells injury. Finally, the negative effects of miR‐320a on vascular homeostasis and cardiac function were alleviated by VEGF‐A re‐expression in doxorubicin treated mice. Conclusion: Our observations demonstrate that miR‐320a play important roles in doxorubicin induced cardiotoxicity via vessel homeostasis in heart and thus, inhibition of miR‐320a may be applied to the treatment of cardiac dysfunction induced by anthracycline. www.impactaging. com 192 AGING, January 2016, Vol. 8 No.1 endothelium is a cell monolayer lining the luminal surface of blood vessels, acting as a gatekeeper of vascular homeostasis via sensing and responding to stimuli and activating various vasoactive systems via secreting cytokines. Endothelial injury appears to be crucial element in disturbance of vascular homeostasis [7]. Disturbance of vascular homeostasis may results in multiple cardiovascular diseases, such as hypertension, atherosclerosis, and heart failure [7]. Emerging evidences suggest that the disturbance of vascular homeostasis in heart may involve in the development of doxorubicin-induced cardiotoxicity [8-10]. However, whether and how doxorubicin disturbs cardiac vascular homeostasis remains unclear. VEGF family consists of five subtypes: VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PlGF). VEGF-A is encoded by a single gene, and five isoforms of VEGF-A have been identified due to alternative mRNA splicing[11]. Five isoforms of VEGF-A are VEGF-A121, VEGF-A145, VEGF-A165 (the predominant isoform), VEGF-A189, and VEGF-A206 [11]. VEGF-A plays crucial roles in vascular homeostasis via regulating new vessel formation (including angiogenesis, arteriogenesis, and vasculogenesis), stabilization of newly formed vessels, vasodilatation, increased vascular permeability, neuromodulation, recruitment and homing of stem cells and so on [11]. In heart, VEGF-A is secreted from various types of cells, including endothelial cells and mature cardiomyocytes. Cardiomyocyte-specific knockout of VEGF-A results in fewer coronary microvessels, thinned ventricular walls, depressed basal contractile function, and an abnormal response to βadrenergic stimulation [12]. However, the use of VEGF inhibitors in the clinical treatments of cancer has a potential to cause cardiac toxicity and decompensated heart failure. The recombinant human monoclonal antibody bevacizumab is a novel antineoplastic agent that binds human VEGF, and approximately 2%-4% of patients treated with bevacizumab will develop CHF [13]. Moreover, combination of doxorubicin and bevacizumab resulted in an unacceptably higher exacerbation of cardiotoxicity [14]. These prompted a hypothesis that doxorubicin may cause cardiac dysfunction by destroying vascular homeostasis via VEGF signal pathway. MicroRNAs (miRNAs) are a class of small non-coding RNAs which regulate gene expression at the posttranscriptional level by binding to their mRNA targets. Mounting evidences demonstrated that miRNAs play key roles in diverse physiological/pathological processes. It was speculated that a single miRNA can modulate a variety of processes by regulating multiple targets [15]. Some miRNAs are tissue enriched or even tissue restricted, which can control gene expression in certain organs or tissues [16]. More importantly, miRNAs may be promising therapeutic targets for diseases. However, the reports about the roles of miRNAs in doxorubicin induced cardiotoxicity are numbered. Up to now, only miR-532-3p, miR-216b, miR-34c and miR-146a were identified as potential regulators of cardiac complications of doxorubicin [17]. Our previous publication demonstrate that miR-320a contributes to atherogenesis by augmenting multiple risk factors and down-regulating SRF [18]. Interestingly, we found that the circulating miR-320a was decreased in patients treated with anthracycline combined chemotherapy. In the current study, we observed that miR-320a involved in doxorubicin induced cardiotoxicity through regulating vascular homeostasis via VEGF-A.