Cardiovascular and cerebrovascular ischemic disease is a large class of diseases that is harmful to human health. The primary treatment for the ischemic disease is to recover the blood perfusion and relieve the tissue hypoxia and the shortage of the nutrients in the supply of nutrients. In recent years, investigations found that IGF-1 has a protective effect on cardiovascular disease, especially in myocardial ischemia-reperfusion injury. Investigation into molecular mechanism of ischemia-reperfusion injury may offer potential targets for the development of novel diagnostic strategies. In this study we defined IGF-1 was differentially expressed in the I/R model of the Mus musculus and IGF-1 was the target gene of miR-29a and Let7f. After ischemia-reperfusion, the expression of miR-29a and Let7f increased, while the expression of IGF-1 decreased significantly in the animal model assay. Further studies have found that IGF-1 could inhibit cell apoptosis signaling pathway, thus protecting the reperfusion injury. These results provide new understanding of ischemia-reperfusion injury, with the hope of offering theoretical support for future therapeutic studies.
Cardiovascular and cerebrovascular ischemic disease is a large class of diseases that is harmful to human health [
Insulin-like growth factor-1 (IGF-1) is an important growth factor, which plays an irreplaceable role in the regulation of cardiac structure and function [
MicroRNA (microRNA, miRNA) is a kind of endogenous single chain noncoding small RNA which has the tissue specificity or is expressed in specific developmental stage [
In this study, we predict microRNA regulated IGF-1 by using software analysis. We construct the animal IR model and then explore the effect of microRNA on IGF-1 and apoptosis.
The mRNA and miRNA profiles data were collected from GEO database (
Mouse miRNA sequences were downloaded from the Rfam website (
C57BL/6 mice, weighing 25–30 g, were provided with free access to food and water. Mice were randomly divided into 2 equal groups (10 mice each group): (a) sham (IR−); (b) IR+. Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals. Every effort was made to reduce the suffering of animals.
H9C2 cells were cultured in DMEM supplemented with 10% FBS. Cell were transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Mimic-miR-29a, mimic-Let7f, anti-miR-29a, and anti-Let7f were bought from Life Technologies, Beijing, China.
Mice were anesthetized using 3% pentobarbital sodium and then supine fixation in the surgical table. Removed neck hair was cut 1 cm longitudinally, and the trachea was exposed. Insert tracheal intubation and connected with breathing machine. Removed chest hair was cut longitudinally 2 cm at the left side 0.5 cm of the midline of the chest, the chest was cut open, third to fifth ribs carefully were cut, the heart was exposed, and IR device was placed.
Quantitative real-time polymerase chain reaction (PCR) was performed as previously described by using Takara SYBR Green.
Broken DNA in the nucleus was labeled with TUNEL assay and visualized by fluorescence microscopy. TUNEL assay kit was bought from Roche.
The cells were schizolysised in RIPA buffer containing protease inhibitors and phosphatase inhibitors. Protein concentration was determined colorimetrically by BCA assay. Protein lysates were separated by 12% SDS-PAGE electrophoresis and were transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 5% BSA for 1 h, the membranes were incubated with antibodies at 4°C overnight, followed by incubation with secondary antibodies for 1 h. Then, the blots were visualized using ChemiDocTM XRS + System with Image LabTM Software (Bio-Rad Laboratories, Inc., USA). Experiments were performed 3 times.
Elisa kit was bought from Shanghai Enzyme-linked Biotechnology Co., Ltd. Elisa assay was carried out according to the manufacturer’s instructions.
We explored the expression value of IGF-1 in mouse heart with and without ischemia-reperfusion (I/R) injury and found that IGF-1 was significantly downregulated in mouse heart with I/R injury (Figure
(a) IGF-1 related molecular network in I/R injury mouse. Upregulated genes are marked with red, while downregulated genes are marked with green. (b) TargetScan results of IGF-1.
There has been researches that showed that IGF-1 played a vital role in the inhibition of apoptosis, and IGF-1 could significantly reduce the apoptosis number of myocardial cells after ischemia-reperfusion injury. In this study, IGF-1 protein was quantified by ELISA, and IGF-1 mRNA was quantified by RT-PCR. As shown in Figure
IGF-1 expression decreased after ischemia-reperfusion. (a) IGF-1 protein expression in IR− and IR+ group (
We first identified by the literature search and in silico analysis two microRNAs, miR-29a and Let7f, as candidate regulators of IGF-1. Both published literature and bioinformatics analysis told us that suppressing miR-29a and Let7f could promote IGF-1 function in a variety of biological systems.
To further study the effect of miR-29a and Let7f on the function of IGF-1, we transfected H9C2 cells with scrambled miRs, mimic-miR-29a, mimic-Let7f, anti-miR-29a, and anti-Let7f, respectively. The ELISA and RT-PCR results showed that after transfection, IGF-1 protein and mRNA level increased several times compared with the scrambled miRs transfection group (Figures
After transfection with anti-miR-29a and anti-Let7f, IGF-1 protein (a and c) and mRNA (d) level increased significantly. However, we found IGF-1 protein (b and e) and mRNA (f) level decreased after transfection with mimic-miR-29a and mimic-Let7f.
Currently, PI3K/Akt signaling pathway is considered as the classical pathway of IGF-1 inhibition of apoptosis [
Caspases is the abbreviation for cysteine aspartate-specific protease. Among the 14 known caspases, the relationship of caspase-3, caspase-8, and caspase-9 with apoptosis was most closely. By PI3K-Akt pathway, IGF-1 activated Akt, and then Ser196 of Akt was phosphorylated, directly prevented caspase-3 activation, and inhibited apoptosis [
In our study, after H9C2 cells were treated 48 hours with serum-free culture, we transfected H9C2 cells with scrambled miRs, mimic-miR-29a, mimic-Let7f, anti-miR-29a, and anti-Let7f, respectively. From TUNEL assay results (Figure
MiR-29a and Let7f influenced IGF-1 downstream related apoptosis pathway. (a) H9C2 cell apoptosis was detected by TUNEL assay. (b) We transfected cell with mimic-miR-29a and mimic-Let7f, then found the protein level of p-Akt and Bcl-2 decreased while caspase-3 and Bax increased. (c) After transfection with anti-miR-29a and anti-Let7f, Western blot results showed that p-Akt and Bcl-2 significantly increased, while caspase-3 and Bax protein significantly decreased.
Cardiovascular and cerebrovascular disease is a major disease that endangers human health and life. The primary treatment for the ischemic disease is to recover the blood perfusion and relieve the tissue hypoxia and the shortage of the nutrients in the supply of nutrients. Prevention and treatment of myocardial I/R injury have been studied for many years; control of reperfusion conditions, application of cell protective agent, and mobilizing the endogenous protection mechanism have achieved certain results [
The existing studies have indicated that apoptosis is the essential component of the life activity of multicellular organisms, and it is the need of survival and exists throughout the total life cycle of the organism [
IGF-1 can regulate cell metabolism and promote its growth and development, in recent years, as a key regulator of cardiovascular disease [
In this study, applied bioinformatics analysis method, we found that IGF-1 was differentially expressed in I/R model of the Mus musculus. Using Targetscan, mirSVR, and PicTar methods, there exist the binding sites of miR-29a and Let7f in the sequence of IGF-1. Therefore we predicted that IGF-1 is target gene regulated by the miR-29a and Let7f.
To confirm the ideas above we then implemented the animal model assay and found that, after I/R, the expression of miR-29a and Let7f increased, while the expression of IGF-1 decreased significantly.
As an inhibitor of apoptosis, IGF-1 can interact with multiple signaling pathways to play a role in inhibiting apoptosis. We used serum starvation method to induce apoptosis, and then we transfected the cell with anti-miR-29a and anti-Let7f, respectively. Western blot results showed the level of p-Akt decreased and cleaved caspase-3 was upregulated, whereas the anti-miR-29a and anti-Let7f transfection group was just the opposite and thus inhibited the apoptosis and protected from the I/R injury.
Although regulation mechanism of IGF-1 on apoptosis inhibition has not been fully elucidated and there may still exist many unknown signaling pathways and regulatory microRNA played a role, IGF-1 may be seen as a target which provides a new way for the treatment of related diseases.
The authors declare that there is no conflict of interests.
This study was supported by the Science and Technology Project of Xi’an Municipal Health Bureau (no. 2013028) and the Provincial Natural Science Basic Research Foundation of Shaanxi (no. 2014JM4152).