Background
Ewing’s sarcoma (ES) is the second most frequent primary malignant bone tumor in children and adolescents. ES is characterized by specific translocations resulting in the fusing the EWS gene with different members of the ETS transcription family, the most frequent is being the EWS-FLI-1 fusion [
1]. Over the past two decades, there has been great efforts have been made to elucidate the underlying mechanisms of ES development over the past two decades, and to identify novel therapeutic targets for ES patients [
2]. However, survival estimates remain miserable for patients who present with overt metastatic disease or who relapse following initial therapy, survival estimates remain miserable.
MicroRNAs (miRNAs) are a large family of non-coding single-stranded RNAs of 18–25 nucleotides that play a role in post-transcriptional regulation. miRNAs have been shown to be involved in many cellular processes, and are implicated including in cancer as either tumor suppressors or oncogenes [
3]. By binding of the miRNA to a partially homologous sequence mostly usually located within the 3′untranslated region (UTR) of the target mRNA a transcript, it can either block its translation of the target mRNA translation or lead to its degradation. And miRNAs play important roles in the regulation of gene expression both in normal tissues and as well as in disease pathogenesis [
4‐
6].
FAS, also known as (also termed APO-1, CD95, tumor necrosis factor receptor superfamily member 6, or TNFRSF6) is a death domain-containing member of the TNF receptor superfamily [
7]. Binding to Fas by its physiological ligand, FasL, triggers receptor trimerization, followed by formation of the death-inducing signaling complex (DISC), and subsequent apoptosis [
8]. Several reports have demonstrated that FAS is involved in cell apoptosis in human cancer [
9,
10].
In the present study, we conducted explored genome wide whole genome array expression analysis of both miRNAs and mRNAs in human mesenchymal stem cells (hMSCs) and five human ES cell lines. The results showed that the expression of miR-181c was increased in all five ES cell lines, whereas that of FAS was repressed in all five ES cell lines. We hypothesized that the effect of FAS in ES cells might be mediated, at least in part, via miR-181c, directly or indirectly. The aim of our study is to evaluate whether the expression of FAS is repressed by miR-181c and the pathway could play a role in malignancy in ES cells.
Methods and materials
Cell lines and reagents
Human MSC (hMSCs) was obtained from Takara Biotechnology. Human Ewing sarcoma cells (SK-N-MC, RD-ES, SK-ES-1 and SCCH) were purchased from Japanese Collection of Research Bioresources (Tokyo, Japan). WE-68, a human ES cell line, was generously provided by Prof. Frans van Valen (Westfalische-Wilhelms University, Munster, Germany). High glucose-Dulbecco’s modified eagle medium (DMEM), RPMI 1640 medium, minimal essential medium (MEM), and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). Mesenchymal Stem Cell (MSC) Basal Medium, Chemically Defined (MSCBM-CD) and MSCGM-CD SingleQuats were obtained from Takara Biotechnology (Otsu, Japan). RNeasy kit and miRNeasy Mini kit were obtained from Qiagen, (Valencia, CA, USA), and TRizol reagent was from Invitrogen. The microRNAs, miR-181c-5p mimic (5′-AACAUUCAACCUGUCGGUGAGU-3′), miR-181c-5p mutant (5′-AUGUAAGUACCUGUCGGUGAGU-3′), hsa-miR-181c inhibitor or negative control (NC) miRNAs were purchased from Invitrogen. A transfection reagent Lipofectamine 2000 and antibiotics-free OptiMEM were also obtained from Invitrogen. Actinomycin D was from Sigma-Aldrich (Tokyo, Japan). FAS expression plasmid (SC321759) was obtained from OriGene Tech. Inc (Iowa, USA). Antibodies produced in rabbits for FAS (#8023), caspase 3 (#9662), cleaved caspase 3 (#9661), caspase 7 (#9492), cleaved caspase 7 (#9491), caspase 8 (#4790), cleaved caspase 8 (#9496), PAR/poly (ADP-ribose) polymerase (PARP) (#9542), cleaved PARP (#9541) and β-Actin (#4970) were purchased from Cell Signaling Technology (Tokyo, Japan). Horseradish peroxidase-conjugated anti-rabbit immunoglobulin G antibodies and ECL Prime system were obtained from GE Healthcare (Tokyo, Japan). Cycletest Plus DNA reagent kit and Annexin V-FITC apoptosis detection kit was obtained from BD Biosciences (Tokyo, Japan). BALB/c nu/nu nude mice were purchase from Kudo (Tosu, Japan). All protocols for animal experiments in the present study were approved by the Ethics Review Committee for Animal Experimentation of Oita University.
Cell culture
SK-N-MC and RD-ES cells were cultured in DMEM high-glucose medium and SK-ES1 and WE-68 cells were cultured in RPMI 1640 medium. The media were supplemented with 10% FBS and 1% penicillin/streptomycin. SCCH cells were cultivated in MEM medium supplemented with 10% FBS and 0.1 mM non-essential amino acids. hMSCs were maintained in MSCBM-CD supplemented with SingleQuats. The cells were incubated at 37 °C in the incubator chamber supplemented with 5% CO2 and passaged when the cells were grown to approximately 70% confluent.
miRNA and mRNA expression analysis using microarray
miRNA and total RNA were extracted by miRNeasy and RNeasy kits, respectively, from the cells according to the manufacturer’s recommendation. The quality of RNA was confirmed using Bioanalyzer 2100 (Agilent, Santa Clara, CA). An aliquot (1 μg) of small RNA fraction including miRNAs from each of five ES cells and hMSCs was biotin-labeled by FlashTag Biotin HSR Kit (Genisphere, Hatfield, PA) and subjected to miRNA expression array analyses with GeneChip miRNA 3.0 array (Affymetrix, Santa Clara, CA). The array data were quantile normalized, log2-transformed using miRNA QC software (Affymetrix), and analyzed using GeneSpring GX 11.0 (Agilent). For mRNA expression analysis, 1 ng total RNA from each of five ES cells and hMSCs was used to generate double stranded cDNA by reverse transcription, and then used to generate biotinylated cRNA by in vitro transcription using the 3′IVT Express Kit (Affymetrix). The cRNA probes were subjected to hybridization to GeneChip Genome HG U133 Plus 2.0 Array (Affymetrix). GeneSpring GX 11.0 software was used for the array analyses including normalization and filtering (20.0–100.0th‰). The whole experiments were repeated twice. The variant analyses were carried out to determine the significant difference between two groups. Genes indicating twofold or more significant increase or reduction in the expression were listed up. KEGG pathway database (
http://www.genome.jp/kegg/pathway.html) was used for the pathway analyses.
Target prediction of miRNAs
Transfection and cell proliferation analysis
Cells (1 × 105) were cultured in 2 ml medium without antibiotics in 6-well plates. miR-181c-5p mimic, miR-181c-5p mutant, anti-miR-181c inhibitor, negative control miRNAs, FAS expression vector and Mock vector were transfected using Lipofectamine 2000. 10 μg/ml of Actinomycin D was used to inhibit the de-novo RNA transcription. The transfected cells were incubated for 48 h and subjected to further analyses. The number of viable cells was counted by TC10 Automated Cell Counter (BioRad). The cell cycle distribution was monitored by propidium iodide (PI) staining using a fluorescence activated cell sorting (FACS) analyzer FACSVerse (BD Biosciences). The number of cells in the cell cycle phases of G0/G1, S, and G2/M were analysed. All experiments were performed in triplicate manner.
Quantitative RT-PCR
The transfected cells were harvested and lysed using TRizol reagent for the extraction of total RNA. Then cDNA was generated and quantitative real-time PCR (qRT-PCR) was carried out with Light Cycler 480 (Roche). The relative expressions of FAS and GAPDH were calculated by 2-(ΔΔCt) method. The following primers were used; FAS-forward 5′-TGCAGAAGATGTAGATTGTGTGATGA-3′, FAS-reverse 5′-GGGTCCGGGTGCAGTTTATT-3′; GAPDH-forward 5′-CCTCTATGCCAACACAGTGC-3′, GAPDH-reverse 5′-GTACTCCTGCTTGCTGATCC-3′.
Western blotting
The cellular protein was extracted and an aliquot (15 μg) was applied onto 10% Tris–HCl Criterion precast gel (Biorad). The proteins on the gel were transferred onto PVDF membrane, and reacted with anti-FAS (#8023), caspase 3 (#9662), cleaved caspase 3 (#9661), caspase 7 (#9492), cleaved caspase 7 (#9491), caspase 8 (#4790), cleaved caspase 8 (#9496), PAR/poly (ADP-ribose) polymerase (PARP) (#9542), cleaved PARP (#9541) and β-Actin (#4970) were purchased from Cell Signaling Technology (Tokyo, Japan). The blots were treated with anti-rabbit IgG antibodies and signals were detected using ECL Prime system (GE Healthcare). The quantification of the protein expression was carried out using ImageQuant TL software (GE Healthcare). All experiments were done in triplicate.
Detection of apoptosis
The cells underwent apoptosis were detected by FACS analysis and western blotting. SK-ES-1 (1 × 106 cells) were cultured and transfected with anti-miR-181c miRNA or FAS expression vector. Forty-eight hours after the transfection, the cells were subjected to analysis using FACSVerse for detection of cell death by the expression of Annexin V. As the positive control of apoptosis, the cells were treated with low dose (5 μg/ml) of doxorubicin. The expression of apoptosis-related proteins, including PARP and cleaved PARP were also analyzed by western blot.
In vivo experiments using nude mice
SK-ES-1 cells (2 × 106) transfected with anti-miR-181c were suspended in 100 μl normal saline and injected into the gluteal region of BALB/c nu/nu mouse. Total of 20 mice were divided into four groups (5 mice each): (1) untreated control, (2) transfected with control-miRNA, (3) transfected with anti-miR-181c, and (4) transfected with FAS expression vector. The changes in weight of the treated mice and volume of the tumors were monitored for 6 weeks. The volume of the tumor was calculated using the formula of V = (length × width2)/2. Then mice were sacrificed and xenografted tumors were removed and subjected to immunohistochemistry. The resected tumors were fixed with 4% formaldehyde, paraffin embedded, sectioned using microtome, and reacted with anti-FAS and cleaved caspase 3 (#9661) antibodies were purchased from Cell Signaling Technology (Tokyo, Japan). The expression of proteins in the section was visualized using DAB and EnVision System (Dako).
Statistical analysis
Two-tailed Student’s t test was carried out for continuous variables. The differences among more than 3 groups were analyzed using ANOVA and Scheffe test. The results were expressed as the mean ± standard deviation (SD), the differences were considered significant when p value were less than 0.05. All statistical analyses were done using SPSS 23.0 software (IBM, Tokyo, Japan).
Discussion
MiRNAs are a group of small, noncoding RNAs that regulate the protein coding genes [
14,
15]. To recognize consequential miRNA–mRNA relationship in ES, we carried out genome-wide miRNA array and cDNA array. In the present study, miRNA array analysis showed that the expression of miR-181c was upregulated in all of the five tested ES cell lines. Several studies have shown that miR-181c is involved in various biological and pathological processes, including development, differentiation, inflammation, apoptosis, and cancer [
5,
16]. Up-regulation of miR-181c is reported to be involved in tumor cell growth [
4] and chemotherapy resistance [
17] in other malignant tumors.
The data from cDNA array analysis showed that FAS mRNA expression is decreased in all five ES cell lines compared with hMSCs. Furthermore, sequence analysis suggested possible association of miR-181c with 3′UTR of FAS. Numerous reports suggest a key role for the transcriptional regulation by FAS, in the complex signaling network of apoptosis. Malignant tumor cells tend to downregulate FAS expression to avoid FAS-mediated apoptosis signaling [
16,
18]. Our data in ES cells is consistent with previous studies reporting that the downregulation of FAS may contribute to malignant phenotypes.
Although miR-181c might influence the expression of many genes, we focused on FAS as the target of miR-181c in ES cells. Our cDNA array analysis demonstrated that FAS was the only miR-181c-target gene whose expression was uniformly upregulated in all five ES cell lines, whereas the expression of other candidate genes was different among the ES cells. Analysis using several algorithms, such as BLAST and TargetScan, further suggested that FAS was a putative target of miR-181c. Among these abnormalities, from a cDNA array, we first focused on FAS as a gene generally considered strongly affect the biology of cancer cells. Also, when we examined whether microRNA that binds to the FAS mRNA 3′-UTR is elevated in a microRNA array, we found that miR-181c was elevated in all five Ewing sarcoma cell lines. Therefore, we hypothesized that an elevation in miR-181c results in inhibition of FAS mRNA expression, and we investigated our hypothesis. Therefore, we analyzed the possibility that miR-181c might play an anti-cancer regulatory role in ES cells by targeting FAS. However, FAS mRNA degradation was promoted even after the termination of de novo mRNA transcription. This effect was not observed in the mutant miR-treated cells. Therefore, miR-181c may have affected FAS mRNA directly at least in part.
We examined the functions of miR-181c in the regulation of its possible target gene, FAS, and the changes in the biological characteristics of ES cell lines. Forced expression of miR-181c resulted in the repression of FAS protein, indicating that miR-181c might function as an oncogene in ES cells. Our results are the first evidence that suggest that the same miR-181c mediated regulatory mechanism of FAS expression might exist in ES cells.
Our data shows that miR-181c promotes the proliferation of ES cells via induction of anti-apoptosis mechanisms, and not by affecting the cell cycle pausing at G1/G0 phase. Transfection of anti-miR-181c or FAS expression vector into SKES1 cells resulted in the increase of sub G1 fraction but did not influence the proportion between G1/G0, S and G2/M phases. Thus, we can infer that the upregulation of FAS, resulting from transfection of anti-miR-181c or FAS expression vector, induced apoptosis of ES cells. This indicates that the repression of ES cell growth with FAS restoration was a result of apoptosis rather than the cell cycle retardation.
Considering that FAS acts as a major regulator of apoptosis related pathways via caspase and PARP cleavage, the repression of FAS by 181c-miR might play an important role in apoptosis of other types of cells as well. The receptor ligation followed by binding with Fas-associated death domain protein leads to the recruitment of caspase-8, resulting in the cleavage and activation of caspase-8 and downstream caspases [
19]. Caspase-8 subsequently activates caspase-3 [
20]. Our results show that transfection with anti-miR-181c and FAS expression vector enhances caspase-8, and caspase 3/7 activity, and increases cleaved PARP expression levels.
Furthermore, the repression of miR-181c results in the inhibition of ES tumor growth in vivo. Consistent with the data from in vitro experiments, xenograft ES models also indicated that miR-181c repression is capable of inhibiting ES tumor development in vivo following restoration of FAS expression and translation.
Authors’ contributions
MK, KT designed and performed experiments, wrote the manuscript. MK, KT and TI performed experiments. KT and II gave suggestion on study design, discussed and interpreted the data. HT designed and supervised study, discussed and interpreted the data. All authors read and approved the final manuscript.