Expression of microRNA-122 contributes to apoptosis in H9C2 myocytes

The microRNAs (miRNAs) can post-transcriptionally regulate gene expression and heart development. The Pax-8 gene knockout mice have apparent heart abnormalities. This study investigated the role of miRNAs in regulation of cardiac apoptosis and development in the knockout mice. MicroRNA microarrays demonstrated differential expression of microRNAs between Pax-8−/− and Pax-8+/− mice, confirmed by real-time PCR. The miR-122 was up-regulated by 1.92 folds in Pax-8−/− mice. There were ventricular septum defects in Pax-8−/− mice, and increased numbers of apoptotic cells in the left ventricular wall and interventricular septum in Pax-8−/− mice. In H9C2 myocytes, treatment with miR-122 mimics or miR-122 inhibitor affects the expression of CCK-8 and activity of Caspase-3. The miR-122 is up-regulated in the myocytes of Pax-8−/− mice and may participate in the apoptotic gene expression and pathogenesis of heart development defect.


Introduction
Non-coding microRNAs are small and endogenous RNAs that regulate gene expression [1][2][3]. The microRNAs can repress translation or promote cleavage of mRNA by targeting the 3′ non-coding region (3′-UTR) [4], achieving the post-transcriptional gene silencing effect. The microRNAs function in the regulation of development, cell proliferation, apoptosis and haematopoiesis [5][6][7]. The microRNAs are highly expressed in the heart and regulate heart development [8,9].
Congenital heart disease (CHD) affects about 1% of newborn children in the world and results in high morbidity and mortality, especially in infants under 1 year of age [10]. The CHD represents the largest class of birth defects and accounts for 25% of all human congenital abnormalities [11]. Ventricular septum defect (VSD) is a common CHD, however, it's specific mechanism and risk factors are yet to be clarified.
Paired box gene 8, also known as Pax-8, is a protein which in humans is encoded by the Pax-8 gene. This gene is a member of the paired box (Pax) family of transcription factors. Members of this gene family typically encode proteins which contain a paired box domain, an octapeptide and a paired-type homeodomain. This nuclear protein is involved in thyroid follicular cell development and expression of thyroidspecific genes. Pax-8 gene knockout mice develop various forms of heart abnormalities, particularly those similar to VSD and spheroidal heart with left ventricle enlargement. Apoptosis cells prevail in left ventricular wall and interventricular septum in Pax-8 knockout mice. In addition, myocytes apoptosis increases after down-regulation of Pax-8 gene in vitro [12]. Therefore, we investigated whether miRNAs are involved in the cardiac structural abnormalities of Pax-8 knockout mice.

microRNA microarray analysis
Total RNA was isolated from the mice heart of experimental and control groups with TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). The RNA quality was identified by ultraviolet absorption test and formaldehyde denatured agarose gel electrophoresis. The microRNAs were isolated, purified and labelled with Cy3, then hybridized with the miRNA oligonucleotide microarray (Agilent, Santa Clara, CA, USA). The gel was scanned by an Agilent scanner, and the density was determined by Feature Extraction software (Scan resolution 5 lm, PMT 100% and 5%). Mean value ratio between the two groups more than 2 or less than 0.5 is considered significantly up-regulated or down-regulated.

Real-time PCR analysis
Reverse transcription of miRNAs was performed with the miScription Reverse Transcription Kit (Qiagen, Valencio, CA, USA) according to manufacturer's recommendations. The PCR reaction was performed with the ABI7300. The relative amount of each miRNA was normalized to the U6 RNA using the equation 2 ÀDCT , where DCT = (CT miR-NAÀCTU6RNA ) (CT was indicated to the cycles required by fluorescence signal intensity reaching to the threshold value in PCR amplification process). The miRNA specific real-time quantitative RT-PCR primers ( Table 1, synthesized by Invitrogen) were used in the real-time PCR together with the miScript Universal Primer which was included in the kit. The test was repeated three times.
Morphological detection on the heart of pax-8 knockout mice Hearts were harvested from E15.5 Pax-8 +/À and Pax-8 À/À mice and made for four-chamber heart sections for HE staining. Heart samples were fixed by perfusion with 10% formalin in 19 PBS and then were immersed in the same fixative solution for 72 hrs at room temperature. They were then dehydrated, and embedded in paraffin using standard histological procedures. Sections (4 lm) were treated by conventional HE staining. The hearts from newly born mice of the same groups were cut into thin sections for electron microscopy observation. About one cube millimetre size of the tissue was quickly isolated from the left ventricular wall and interventricular septum, fixed in 2.5% glutaraldehyde for 1 week. The electron microscope samples were post-fixed in 1% osmium tetroxide for 1 hr, and then embedded in epoxy resin for thin section transmission electron microscopy.

Cell culture
The H9C2 myocytes were obtained from the cell bank of the Chinese Academy of Sciences, Shanghai, China. The cells were cultured with DMEM (high glucose; Gibco BRL, Gaithersburg, MD, USA) medium containing 10% foetal bovine serum (FBS) (Gibco BRL) in a humidified atmosphere of 95% O 2 , 5% CO 2 at 37°C in a CO 2 incubator. When the cells were grown to 90% confluence, they were then harvested by a brief exposure to 0.05% trypsin-EDTA (Gibco BRL) and passaged every 3 days.

Transduction of miR-122
Single-cell suspension was prepared 24 hrs before transfection and cultured into six-well plate with the density of 1 9 10 5 /well. The H9C2 (2-1) myocytes were transfected using LipofectamineTM2000 (Invitrogen) according to the manufacturer's instruction. They were divided into three groups, miR-122 group (experimental group), Negative Control (NC) miRNA group and Blank Control group. The miR-122 mimics were transfected into H9C2 (2-1) myocytes as experimental group. The microRNA synthesized randomly was transfected into H9C2 (2-1) myocytes as negative control group. The H9C2 myocytes were offered with routine culture medium with no miRNA as Blank Control group. Forward test was carried out after the cells were cultured at 37°C in an incubator containing 5% CO 2 for 24 or 48 hrs. Total RNAs were extracted through one-step method with Trizol (Invitrogen). The purity of total RNAs was detected by a spectrophotometer analyser (DU800) (Beckman, Miami, FL, USA) and its quality was detected by formaldehyde denaturation gel electrophoresis. Total 60 ng RNAs were obtained. The microRNA Isolation Kit (Ambion) was used to detach small molecular RNA less than 100nt, and mi-Script Reverse Transcription Kit (Qiagen) was adopted to synthesize cDNA through reverse transcription. At last, real-time quantitative PCR detection was carried out with ABI 7500 FAST real-time PCR. The PCR condition: 95°C 5 sec., 60°C 34 sec., 40 cycles. As an internal control, U6 primers were used for RNA template normalization. Fluorescent signals were normalized to an internal reference, and the threshold cycle (CT) was set within the exponential phase of the PCR. Sequence Detection system (SDS) 2.2.2 of Biosystems was used to analyse the data, and △△CT was used to calculate relativeexpression (RQ) of sample aim gene. The relative quantification of gene expression was analysed by the 2 À△△CT method [13] and was presented as fold over the control. The RQ = 2 À△△CT (CT was indicated to the cycles required by fluorescence signal intensity reaching to the threshold value in PCR amplification process.), △CT sample = CT sample ÀCT U6 sample , △CT control = CT control ÀCT U6 control , △△CT = △CT sample À△CT control ). The test was repeated three times.

Inhibition ratio was detected by CCK-8 assay
Single-cell suspension was prepared 24 hrs before transfection and cultured into a 96-well plate with the density of 7000/well.

Analysis of caspase-3 activity
Within 48 hrs, cells of each group were collected and protein was extracted. Two hundred micrograms of protein reacted with Caspase-3 background according to the instruction manual of Caspase-3 spectrophotometry Kit (Nanjing Kaiji Company, Nanjing, JiangSu, China) after protein concentration was detected by BCA assay with protein concentration determination kit (Pierce, Rockford, IL, USA). The absorbance A value at k = 405 nm by ELISA ECX800 (Bio-TEX) was as experimental value. The specific Caspase-3 activity was calculated by the equation, OD (experimental group) -OD (background group): OD (experimental group) indicated the absorbance of interference group, Negative control group or blank control group at k = 405 nm; OD (background) indicated the absorbance of the background containing 29 Reaction buffer 50 ll and Lysis buffer 50 ll at k = 405 nm.

Cell apoptosis ratio was detected by AnnexinV/PI double dyeing with flow cytometry
Single-cell suspension was prepared and cultured into a six-well plate with the density of 1 9 10 5 /hole. Forty-eight hours after transfection, cells of each group were collected, washed with PBS once, centrifugated (discarded supernatant), resuspended with 200 ll binding buffer, added with 5 ll of AnnexinV-FITC, 10 ll of PI and incubated for 15 min. away from light, detected with flow cytometry (BD), and at last, the data were analysed with the software of MULTCYCLE.

Synthesis of Pax-8 siRNA and miR-122 inhibitor
As previously mentioned, transduction of miR-122 resulted in the lower rates of cell proliferation, increased Caspase-3 activity, and higher apoptosis in primary cultures of H9C2 myocytes. To further study the function of miR-122 in the pathogenesis of congenital heart disease, especially in the mechanism of VSD, miR-122 inhibitor was transfected 24 hrs before Pax-8 siRNA was transfected into H9C2 cells to induce apoptosis. Then we observed whether miR-122 inhibitor could protect H9C2 cells from apoptosis. The positive-sense strand of Pax-8 siRNA (Sigma-Aldrich) was as follows: 5′-GGAAGUGAGUAUU CUGGCATT-3′, and its antisense strand: 5′-UGCCAGAAUACUCACUUCC TG-3′, The sequence of miR-122 inhibitor (Sigma-Aldrich) was as follows: 5′-CAAACACCAUUGUCACACUCCA-3′, no homology with genome of rats.

Transduction of Pax-8 siRNA and miR-122 inhibitor
Single-cell suspension was prepared 24 hrs before transfection and cultured into a six-well plate with the density of 1 9 10 5 /well. The H9C2 (2-1) myocytes were transfected using LipofectamineTM2000 (Invitrogen) according to the manufacturer's instruction. It was divided into four groups, Pax-8 siRNA + miR-122 inhibitor group (experimental group), Pax-8 siRNA group (apoptosis induction group), Negative Control (NC) miRNA group and Blank Control group. Pax-8 siRNA was transfected into H9C2 (2-1) myocytes as apoptosis induction group. The miR-122 inhibitor was transfected 24 hrs before Pax-8 siRNA was transfected into H9C2 (2-1) myocytes. The miRNA, synthesized randomly, was transfected into H9C2 (2-1) myocytes as negative control group. The H9C2 myocytes were offered with routine culture medium with no miRNA as Blank Control group. Forward test was carried out after these cells were cultured at 37°C in incubator containing 5% CO 2 for 24 or 48 hrs.

Statistics
The data were analysed with the software of SPSS 17.0. All experiments were repeated three times and presented as mean ± SE. The multiple groups were analysed by one-way ANOVA followed by Tukey's post hoc comparisons. Differences were considered statistically significant at P < 0.05.

Developmental defects occurring in the hearts of Pax-8 KO mice
Ventricular septum defect (VSD) was found by H&E staining in the hearts of Pax-8 À/À mice (Fig. 2). By electron microscopy, we observed that Pax-8 À/À mice had fairly organized structures of the myofilament and sarcomere in the left ventricular wall and interven-tricular septum. However, there were a large number of myocardial apoptosis cells with cell shrink, cytoplasmic vacuoles, marked increases of the number of mitochondria and decreases in volume, chromatin concentration and side shifts and formations of apoptotic bodies. The H&E staining of Pax-8 +/À mice hearts showed no significant change in the structure. The electron microscopy observation of Pax-8 +/À mice hearts showed no significant expansion of sarcoplasmic reticulum, the moderate myocardial apoptosis cells, and no obvious abnormalities in size and amount of the mitochondria was found (Fig. 2, more details were showed at Int J Cardiol. 2012; 154: 43-51).

Increased apoptosis of H9c2 myocytes by transduction of miR122
To further study the function of miR-122 in the pathogenesis of congenital heart disease, especially in the mechanism of VSD, H9C2 cells were transfected with the appropriate miR-122 mimics. Transfection efficiency was detected by flow cytometry 24 hrs after different doses of FAM-NC miRNA/LipofectamineTM2000 were transfected into H9C2 (2-1) myocytes. Transfection efficiencies of different groups were shown. (A) Blank Control was 0.1%; (B) 2.5/2.5 ll group was 81.0%; (C) 5/5 ll group was 96.6%; (D) 7.5/7.5 ll group was 98.7%. Considering transfection efficiency and toxicity derived from high concentration of miRNA/LipofectamineTM2000, miRNA 5 ll/Lipofectamine TM2000 was chosen as a transfection condition. High concentration of miRNA/LipofectamineTM2000 (C and D group) could cause cell necrosis which could interfere with experimental results (Fig. 3).
The role of the miR-122 in the mechanism of VSD was determined by examining its apoptosis and proliferation abilities. The inhibition index was higher in the cells of the miR-122 group (35.64 ± 3.82%) than that of the negative control miRNA group (16.77 ± 5.06%) (P < 0.01) (Fig. 4-2). Pairwise comparison among three groups, caspase-3 activity of miR-122 group (0.361 ± 0.051) was obviously higher than that of negative control miRNA group (0.112 ± 0.014) and blank control group (0.099 ± 0.012) (P < 0.01), while the latter two groups had no significant statistical difference (P > 0.05) (Fig. 4-3).

Discussion
Many miRNAs have been found in vertebrates. Their functions in the ontogenetic process, especially in the process of cell development are deeply investigated [14,15]. The microRNAs also play an important role in the process of myocardial injury and apoptosis. Pax belongs to an important gene family related to development, and Pax protein is an important transcription regulator that plays an important role in the differentiation of tissues and organs during embryonic development. Pax-8 is one transcription factor of the family of mammals Pax protein. Pax-8 gene expression has tissue specificity. Pax-8 knockout mice develop thyroid dysplasia and thyroxine descent [16]. In current studies, significant structural abnormalities of the heart of Pax-8 knockout mice including ventricular septum defect are found. A large 57 ± 1.88%) was obviously higher than that of negative control miRNA (6.70 ± 1.23%) and blank control cells (4.33 ± 1.00%) (P < 0.01) while the latter two groups had no significant statistical difference (P > 0.05) and the index of apoptosis of Pax-8 siRNA + miR-122 inhibitor transduced H9c2 cells (16.10 ± 0.70%) was obviously lower than that of Pax-8 siRNA group (25.57 ± 1.88%) (P < 0.01).
number of myocardial apoptosis cells in the left ventricular wall and interventricular septum are further confirmed by electron microscopy. The volume of the mitochondria within cells is reduced and the quantity is increased.
In microarray studies, 10 miRNAs were differentially expressed in significance between pax-8 +/À and pax-8 À/À mice hearts. The level of expression of eight miRNAs was significantly up-regulated in pax-8 À/À by 1.21-to 8.98-fold (miR-451, 8.98-fold, miR-142-3p, 7.77-fold, miR-144, 2.79-fold, miR-7a, 1.98-fold, miR-122, 1.92-fold, miR-142-5p, 1.85-fold, miR-148, 1.59-fold, miR-486, 1.21-fold). The level of expression of miR-125-3p was reduced by 0.56-fold. The miR-218-2 was not detected by RT-PCR. And miR-142-3p was found probably closely related to the haemopoietic system disease [17], miR-451 is being revealed to participate in the late stages of erythropoiesis [18] and closely related to the resistance of cancer cell to a broad range of chemotherapeutics [19]. The role of both in the heart development has not yet been reported. In this study miR-122 mimics were transfected into H9C2 (2-1) myocardial cells of rat to upregulate miR-122 expression. In up-regulated miR-122 group, myocardial apoptosis was increased while proliferation inhibited, indicating that miR-122 might promote myocardial cell apoptosis. As miR-122 may promote myocardial cell apoptosis, then what's the role of miR-122 inhibitor in myocardial cell apoptosis. Therefore, in the following experiment, the H9C2 (2-1) myocardial cells transfected with Pax-8 siRNA were as an apoptotic model. Then miR-122 inhibitor was transfected into H9C2 (2-1) myocardial cells 24 hrs before Pax-8 siRNA was transfected. At last, we observed whether miR-122 inhibitor had protective role in cell apoptosis. We found that the expression of Pax-8 in H9C2 (2-1) myocardia cells trasfected with Pax-8 siRNA was down-regulated, which caused increased myocardial apoptosis and inhibited proliferation. However, when H9C2 (2-1) myocardia cells were transfected with miR-122 inhibitor 24 hrs before Pax-8 siRNA, we found cell apoptosis decreasing and proliferation increasing compared with those only transfected with Pax-8 siRNA. So we infer an individual miRNA such as miR-122 may be a new target for the therapy of VSD. Modulating the expression of a single microRNA can, in principle, influence an entire gene network and thereby modify complex disease phenotypes. It has been reported that a kind of miR-NA antagonist rectifying fault signal transduction pathway had the potency of target prevention and therapy.
However, in our research there also exists defects. In Pax-8 knockout mice, Pax-8 gene was silent completely, and we found 10 miRNAs were differentially expressed, among which miR-122 was up-regulated 1.92 fold. However, RNA interference was often used to partly silence gene expression. When the H9C2 (2-1) myocardial cells transfected with Pax-8siRNA were as apoptotic model to study whether miR-122 inhibitor had protective role in cell apoptosis, Pax-8 gene was silent partly. Besides, the fold that miR-122 up-regulated in Pax-8 knockout mice was only 1.92, so when Pax-8 was down-regulated with siRNA, the level of the miR-122 expression was not increased significantly.
Our research showed that the apoptosis in the Pax-8 siR-NA + miR122 inhibitor transduced H9C2 cells was still high (Fig. 8): 16% (in D) versus 6.7% (in B). This suggests that miR-122 alone is not sufficient to ablate the apoptotic events induced by down-regula-tion of Pax-8. That is to say that other genes may participate in apoptotic events induced by down-regulation of Pax-8. We have used gene chip to identify down-stream genes of Pax-8.
As to the potential target genes of the 10 miRNAs, we have used miRNAs target prediction software (pictar, targetscan, miRBase) to screen the target genes. HAND2 which is the potential target gene of miR-122 is a basic helix-loop-helix (bHLH) transcription factor that plays an essential role in cardiac morphogenesis. In HAND2 mutant embryos, the right ventricle (RV) forms but apoptosis undergoes [20], suggesting that Hand2 may be required for survival of the cardiomyocytes. Loss of HAND2 in the natural cytotoxic lineage leads to misalignment of the outflow tract and aortic arch arteries. HAND2 related defects include pulmonary stenosis, interrupted aortic artery, retro-oesophageal right subclavian artery and ventricular septum defect [21]. Disruption of HAND2 severely impairs development of the right ventricle, outflow tract and aortic arch arteries, indicating that HAND2 is indispensable for the formation of the superior elements of the developing heart [22]. In patients with right ventricular hypertrophy (e.g. tetralogy of Fallot), the level of HAND2 mRNA expression is increased [23]. The investigation of HAND2 mutations in 131 Chinese congenial heart disease (CHD) children indicates that HAND2 would be a potential candidate gene of some kinds of CHD [24]. How miR-122 regulates HAND2 gene expression needs further study.
The potential target genes of miR-144 include Apaf-1, Caspase-3, MAP3K4, LGR4 and so on. Apoptotic protease activating factor-1 (Apaf-1) binds to cytochrome c (Cyt c) released from cells to form complexes called apoptosomes, and then bind to and cleave caspase-9 precursors, which creates an expanding cascade of proteolytic activity and causes apoptosis. This is one of the classic pathways of apoptosis. However, Dmitriy Ovcharenko et al. [25] predicted that miR-144 directly targeted caspase-3 to achieve anti-apoptosis effect. The mitogen-activated protein kinase kinase kinase 4 (MAP3K4) has been identified as a crucial anti-apoptotic element required for normal development of mouse embryo by regulation of the MKK3/p38/MAP-KAPK-2 pathway [26], hence miR-144 may also target MAP3K4 to induce apoptosis. On account of these two aspects, we could presume that miR-144 plays different roles at different time points: it may be mainly to induce apoptosis at the stage of embryonic development and inversely mainly inhibit apoptosis in the adult. One of the potential target genes LGR4 is widely distributed in the embryo and adult, Mazerbourg et al. [27] indicated that LGR4 was closely related to the process of embryonic development in LGR4 knockout mouse model. On the other hand, after the prediction of the target genes of miR-125-3p, the LBH gene related to heart development has aroused our interest. Briegel et al. [28] have used transgenic technology to make LBH excessive expression, which leads to severe defects in morphology and function of heart, such as left ventricular hypertrophy, ventricular septum defect and so on, and this finding is similar to our study on Pax-8 knockout mouse, hence we can presume miR-125-3p could be involved in the abnormal heart development of pax-8 knockout mouse embryo by targeting LBH.
In summary, to assess the involvement of miRNA in the pathogenesis of congenital heart disease, especially in the mechanism of VSD development, we analysed differential miRNA expression in Pax-8 gene knockout mice. We found that miR-122 was up-regulated sigª 2012 The Authors nificantly. The role of miR-122 in apoptosis of myocardial cells was confirmed by using miR-122 mimics and miR-122 inhibitor transfected into H9C2 (2-1) myocytes respectively. Transduction of miR-122 mimics resulted in the lower rates of cell proliferation, increased Caspase-3 activity and higher apoptosis in primary cultures of H9C2 myocytes, and transduction of Pax-8 siRNA into H9C2 (2-1) myocytes resulted lower expression of Pax-8, correspondingly causing increased caspase-3 activity, higher apoptosis and decreased cell proliferation. However, when H9C2 (2-1) myocardia cells were transfected with miR-122 inhibitor 24 hrs before Pax-8 siRNA, we found cell apoptosis decreasing and proliferation increasing compared with those only transfected with Pax-8 siRNA.