The human normal lung fibroblast cell line MRC-5, was purchased from the Shanghai Cell Collection (Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL) at 37°C under a humidified 5% CO₂ atmosphere.

The patient-derived primary cultures of cardiac myxoma cells were obtained from fresh tumor specimens from patients as previously described (12). Briefly, the single-cell suspension was obtained by mechanical manipulation of the cardiac myxoma specimen. The primary culture was maintained in DMEM supplemented with 10% FBS. The established primary myxoma cells were designated as CM001-CM006.

The expression levels of mRNA of the MEF2 family and miR-218 were determined by qPCR. To examine the levels of MEF2A, MEF2C and MEF2D mRNA in cardiac myxoma, fresh tissues (cardiac myxoma samples and the matched non-cancerous tissue, n=10) were obtained with written informed consent from patients following the protocols approved by the Ethics Review Board of Henan University of Science and Technology (China). The patients underwent surgical operation at the Department of Cardiovascular Surgery, The First Affiliated Hospital of Henan University of Science and Technology. Total RNA was extracted with TRIzol solution (Sigma-Aldrich) according to the protocol provided by the manufacturer. The total RNA was transcribed into cDNAs using ReverTra Ace quantitative PCR (qPCR-RT) kit (Toyobo, Japan) according to the manufacturer’s instructions. qPCR was performed using TaqMan^® 2X Universal PCR Master Mix (Applied Biosystems) on a CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA) supplied with analytical software. cDNA was obtained by PCR. The sequences of the used primers were previously described by Ma et al (9).

To detect the expression of miR-218, total RNA was extracted with TRIzol solution according to the protocols provided by the manufacturer. Reverse transcription reaction was carried out using All-in-One™ First-Strand cDNA Synthesis kit (AORT-0020; GeneCopoeia) following the manufacturer’s instructions. qPCR was performed using All-in-One™ miRNA qRT-PCR Detection kit (AOMD-Q020; GeneCopoeia) on a CFX96™ Real-Time PCR Detection System supplied with analytical software. U6 was used as an endogenous reference. The primers and probes for miR-218 were purchased from GeneCopoeia (HmiRQP0327 and HmiRQP0326).

To examine the expression level of the indicated proteins, the lysate containing the total proteins extracted using M-PER^® Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL, USA) was separated by polyacrylamide gel electrophoresis and transferred onto 0.45-μm nitrocellulose membranes. After a 2-h blocking with 5% fat-free dry milk, the membranes were then incubated with primary antibodies for 2 h. The membranes were incubated with the corresponding secondary antibody for 1 h and finally, visualized with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific). The involved antibodies were all purchased from Cell Signaling Technology (Beverly, MA, USA).

The MEF2D-specific siRNA was purchased from the Shanghai GenePharma Co. (Shanghai, China) as well as the control siRNA. The cells were transfected with the indicated siRNA (50 nM) 24 h prior to the subsequent experiments.

Primary cardiac myxoma cells (5×10³) were planted into each well of 96-well plates. Overnight, the cells were treated under the indicated conditions. At the indicated time points, the cells were treated with 10 μl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml) for 4 h. MTT was then removed, and 150 μl DMSO was replaced. The spectrophotometric absorbance was detected on a Model 550 microplate reader at 570 nm with a reference wavelength of 655 nm.

All procedures for the animal experiments were approved by the Committee on the Use and Care on Animals in Henan University of Science and Technology and were performed following institutional guidelines. After the indicated treatments, human cardiac myxoma xenografts were established by subcutaneously inoculating 5×10⁵ cells into both flanks of 5-week-old BALB/c nude mice (n=9). Tumor diameters were periodically measured with calipers. The volumes were calculated following the formula: Volume (mm³) = length (mm) x [width (mm)]²/2. All animals received humane care according to the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy.

The cells were exposed to the indicated treatments, followed by being harvested, fixed in 70% ethanol and stained with propidium podide (PI; 200 mg/ml) for flow cytometric analysis with the Aria II sorter (BD Biosciences). For each group, 10,000 cells were counted to determine the percentages of the G₀/G₁, S and G₂/M populations.

The potential evolutionarily conserved miR-218 targets were predicted using the algorithm TargetScan (http://www.targetscan.org/). Luciferase assay was applied to further confirm whether MEF2D is an authentic target of miR-218. To generate the luciferase reporter vectors, a 251-bp fully synthesized DNA construct identical to the region of the MEF2D 3′UTR containing the predicted miR-218 binding site (AAGCACA) was inserted into the SpeI and HindIII sites of the pMIR-REPORT vector (Applied Biosystems), generating pMIR-MEF2D-WT. To construct a control vector (pMIR-MEF2D-MUT), a mutation in the miR-218 seed region was introduced into the Luc-m 3′UTR (AAGCTGA). The mimics of miR-218 were purchased from GenePharma Co. Plasmids for luciferase assays and mimics were cotransfected by Lipofectamine 2000 (Invitrogen) according to the protocols provided by the manufacturer. Subsequently, detection of luciferase activity was performed with the Dual-Luciferase^® Reporter Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

Each experiment was performed for at least three times. All values are reported as means ± SD, and compared at a given time point by the unpaired, two-tailed Student’s test. Data were considered to indicate a statistically significant result at P<0.05 and P<0.01.

The expression profile of MEF2A, MEF2C and MEF2D mRNA was investigated in fresh cardiac myxoma specimens and their matched non-cancerous tissues (n=10). No significant difference was detected in the mRNA level of MEF2A and MEF2C between the cancer and normal tissues (Fig. 1A). In contrast, MEF2D mRNA was found to be overexpressed in the cardiac myxoma specimens (Fig. 1A). Furthermore, immunoblot assay confirmed the differential expression of MEF2D between the cancer and normal tissues (Fig. 1B).

Given the role of MEF2D in the proliferation of hepatocellular carcinoma, we subsequently investigated whether aberrant expression of MEF2D is also associated with the proliferation of cardiac myxoma cells. MTT assays were employed to determine the proliferation rate of the primary myxoma cells. The data revealed that the levels of MEF2D protein were inversely correlated with the doubling time of the myxoma cells (R=−0.943, P=0.005) (Fig. 2A and B), revealing that cancer cells with higher MEF2D expression proliferated more rapidly.

We used small interfering RNA (siRNA) to specifically silence MEF2D expression, followed by investigation of the proliferation rate in myxoma cells. Our data showed that MEF2D siRNA selectively inhibited MEF2D expression, yet not MEF2A and MEF2C (Fig. 2C). We found that MEF2D downregulation inhibited the growth of the tested primary cardiac myxoma cells (Fig. 2D). Collectively, MEF2D overexpression promoted the proliferation of myxoma cells.

The effect of MEF2D overexpression on the tumorigenicity of myxoma cells was further studied in a mouse model. The tumors from the MEF2D siRNA-treated CM001 and CM002 cells were found to grow slower than the control groups by 71 and 65%, respectively (Fig. 3A and B).

To elucidate the mechanisms underlying the promotion of proliferation and tumorigenesis of myxoma cells by MEF2D, we examined the expression of known MEF2D targets, RPRM, GADD45A, GADD45B and CDKN1A, following MEF2D siRNA treatment. Immunoblot analysis revealed that the expression levels of the above mRNAs were all increased when MEF2D expression was downregulated in the myxoma cells (Fig. 4A). Consistently, G2/M cell cycle arrest was also detected in both the CM001 and CM002 cells treated with MEF2D siRNA, evidenced by flow cytometric analysis (Fig. 4B). These results indicated that MEF2D contributed to the proliferation of myxoma cells by preventing G2/M cell cycle arrest.

The bioinformatic analysis showed that MEF2D is a predictive target of miR-218 using an algorithm available online (http://www.targetscan.org/) (Fig. 5A). Therefore, we employed luciferase assays to confirm whether miR-218 negatively regulates the expression of MEF2D by binding its putative recognition sites within 3′UTR of MEF2D mRNA. Luciferase expression by pMIR-MEF2D-WT, but not pMIR-MEF2D-MUT, was shown to be greatly reduced in the myxoma cells treated with the miR-218 mimics (Fig. 5B). Furthermore, miR-218 overexpression was able to suppress the levels of endogenous MEF2D proteins in the CM001 and CM002 cells (Fig. 5C), while miR-218 suppression restored the expression of MEF2D in normal fibroblast MRC-5 cells (Fig. 5D). The above data indicated that MEF2D is an authentic miR-218 target.

In the same tumor specimens, miR-218 was found to be underexpressed (Fig. 6A). There was an inverse association between MEF2D mRNA and miR-218 levels (R=−0.881, P=0001) (Fig. 6B). MTT assays also demonstrated that miR-218 restoration was able to reduce the proliferation of the myxoma cells (Fig. 6C). These data indicate that downregulation of miR-218 could be responsible for the elevated expression of MEF2D and increased proliferation rates in the myxoma cells.