Recently years, unreasonable daily routines, dietary habits and some other factors give rise to increased incidence of cardiovascular diseases, which seriously threatens human health. Cardiac hypertrophy is a compensatory response of the heart to increase the wall tension by increasing the wall thickness under the stimulation of various pathological factors . Early cardiac hypertrophy is conducive to maintaining the normal function of the heart. However, sustained cardiac hypertrophy will lead to heart failure and sudden death . Therefore, the study on the mechanism of cardiac hypertrophy is of great significance to find the target of prevention and treatment of cardiac hypertrophy .
It is found that there are many kinds of miRNAs in the endothelial cells and smooth muscle cells of vascular walls and myocardial cells, which are involved in many cell biology processes, such as cell proliferation, migration, and differentiation, playing an important role in normal development of the heart and blood vessels . Previous studies also have demonstrated that many miRNAs, such as miR-1 , miR-101b  are both down-regulated in cardiac hypertrophy, and overexpression of miR-1 and miR-101b both play an anti-hypertrophic role by suppressing hypertrophic responses. At the same time, there are also some other miRNAs, such as miR-297 , miR-22  are up-regulated in cardiac hypertrophy and the deletion of theses related miRNAs also has the effect to attenuate hypertrophic responses. Xiao N et al. found that miR-129-5p was in down-regulated in the serum of chronic heart failure (CHF) patients and transfection of miR-129-5p improved heart function and hemodynamic parameters, as well as attenuated oxidative stress and inflammation factors in CHF rats . However, the direct effect and regulation mechanism of miR-129-5p in cardiac hypertrophy has not been explored yet. In our present study, we found that miR-129-5p was down-regulated in Ang-II induced cardiac hypertrophic cells and the overexpression of miR-129-5p successfully suppressed hypertrophic responses, which were exhibited through the immunofluorescence staining, western blot, and the [3H]-leucine incorporation assay.
Oxidative stress is closely related to cardiac hypertrophy and oxidative stress is considered to be a major inducer for the signal transduction in cardiac cells pathological conditions . Thus, a large number of studies have shown that inhibition of oxidative stress can significantly prevent and improve cardiac hypertrophy [29–31]. In our study, we found that overexpression of miR-129-5p largely rescued the activities of oxidative stress related enzymes SOD and CAT and increased the generation of NO in AngII induced cardiac hypertrophic cells. At the same time, the accumulation of MDA was cleared by miR-129-5p mimic, indicating that overexpression of miR-129-5p effectively suppresses AngII-induced oxidative stress. Thus, miR-129-5p mimic may act as an antioxidant to prevent cardiac hypertrophy.
We have learned from previous studies that keap1-Nrf2 signaling pathway is very important in oxidative stress defense mechanism and the keap1-Nrf2 signaling pathway has become a prospective target for the prevention and treatment of oxidative stress-related diseases, including cancer, cardiovascular, inflammatory diseases and so on [32, 33]. Keap1, the negative regulator of Nrf2, acts as an important role in the activation of keap1-Nrf2 signaling pathway . For example, Liao W et al. reported that miR-140-5p attenuated oxidative stress in Cisplatin induced acute kidney injury by activating Keap1-mediated Nrf2/ARE pathway . In our study, we found that the expression of keap-1 was elevated by AngII stimulation and was then suppressed by miR-129-5p mimic. Bioinformatics analysis predicted complementary sequences between miR-129-5p and keap-1 and the luciferase reporter gene further demonstrated the targeting relationship between them. In addition, we found that inhibiting effect of siRNA-keap-1 on AngII-induced hypertrophic responses and oxidative stress was largely neutralized in the presence of siRNA-miR-129-5p. From another point of view, it demonstrated that miR-129-5p mimic suppressed cardiomyocyte hypertrophy and oxidative stress by down-regulating keap-1.
In addition, we found that co-transfection of siRNA-miR-129-5p abolished the activating effect of siRNA-keap-1 on Nrf2 pathway by strongly suppressing nuclear transport of Nrf2, also indicating that miR-129-5p mimic exerts anti-hypertrophic role through activating the keap-1-Nrf2 pathway. Ying Y et al. revealed that Phloretin prevented diabetic cardiomyopathy by dissociating Keap1/Nrf2 complex and inhibiting oxidative stress . Li R et al. also pointed out that Bailcalin protected against diabetic cardiomyopathy through Keap1/Nrf2/AMPK-mediated antioxidative and lipid-lowering effects . However, the specific association between Keap1/Nrf2 and cardio hypertrophy has never been clearly stated. Thus, we for the first find that the activation of Keap1/Nrf2 pathway may have therapeutic effect on cardio hypertrophy, providing a theoretical basis for the application of Nrf2 pathway activator in cardiac hypertrophy.
Taken together, we constructed an in vitro cardiomyocyte hypertrophy model by stimulating the HCM with Ang II. We for the first time revealed that miR-129-5p mimic has the potential as a therapeutic target on cardiac hypertrophy and it may exert anti-hypertrophic role through activating the keap-1-Nrf2 pathway, proving novel research interest preventive and curative procedures of cardiac hypertrophy. However, there are also limitations to our work. For example, whether miR-129-5p overexpression could attenuate cardiac hypertrophy in vivo model still need further exploration. Thus, we will do more related research in our following experiments.