Down‐regulated HDAC3 elevates microRNA‐495‐3p to restrain epithelial‐mesenchymal transition and oncogenicity of melanoma cells via reducing TRAF5

Abstract MicroRNAs (miRNAs) are emerging biomarkers in biological processes and the role of miR‐495‐3p has been identified in melanoma, while the detailed molecular mechanisms remain to be further explored. We aim to explore the effect of histone deacetylase 3 (HDAC3) and miR‐495‐3p on epithelial‐mesenchymal transition (EMT) and oncogenicity of melanoma cells by regulating tumour necrosis factor receptor‐associated factor 5 (TRAF5). Levels of HDAC3, miR‐495‐3p and TRAF5 in melanoma tissues and pigmented nevus tissues were determined, and the predictive roles of HDAC3 and miR‐495‐3p in prognosis of melanoma patients were measured. The melanoma cells were screened and transfected with relative oligonucleotides and plasmids, and the expression of HDAC3, miR‐495‐3p and TRAF5, and phenotypes of melanoma cells were gauged by a series of assays. The relations between HDAC3 and miR‐495‐3p, and between miR‐495‐3p and TRAF5 were confirmed. HDAC3 and TRAF5 were increased while miR‐495‐3p was decreased in melanoma cells and tissues, and the low expression of miR‐495‐3p as well as high expression of HDAC3 indicated a poor prognosis of melanoma patients. Inhibited HDAC3 elevated miR‐495‐3p to suppress EMT and oncogenicity of melanoma cells by reducing TRAF5. HDAC3 particularly bound to miR‐495‐3p and TRAF5 was the target gene of miR‐495‐3p. Our results revealed that down‐regulated HDAC3 elevates miR‐495‐3p to suppress malignant phenotypes of melanoma cells by inhibiting TRAF5, thereby repressing EMT progression of melanoma cells. This study may provide novel targets for melanoma treatment.


| INTRODUC TI ON
Skin cancer is the commonest form of cancer, and the number of diagnosed cases is increasing every year. Skin cancers are separated into non-melanoma skin cancers and melanoma. 1 Melanoma is the most aggressive skin cancer and accounts for >80% of skin cancer-related deaths. 2 Though the incidences of most cancers are reducing, the prevalence of melanoma has been steadily increased all over the world. 3 Melanocytes are naturally occurring pigmented cells in the epidermis and malignant transformation of melanocytes induces melanoma. Melanocytes are responsible for the production of an endogenous pigment melanin that protects skin against harmful ultraviolet radiation. 4 It is reported that exposure to the highly energetic spectrum of ultraviolet radiation is the main cause of melanoma. 5 Although treatments for melanoma such as targeted and immune therapies have been developed recently, additional effective therapeutic strategies for melanoma are still needed. 2 Histone deacetylases (HDACs) knock out acetyl groups, typically promoting a closed chromatin structure that constrains gene expression. 6 HDACs are separated into four groups based on their structure and function, and HDAC3 belongs to class I. 7 It was previously suggested that the proliferation of melanoma cells was inhibited by reduction of HDAC3, 8 and a recent study revealed that the inhibited HDAC3 contributed to killing melanoma cells. 9 MicroRNAs (miRNAs) are non-coding short RNAs that regulate gene expression and negatively impact the stability and translation of mRNAs by binding to complementary sequences at 3′ untranslated region (3′UTR). 10 It was reported that miR-137 acted as a tumour repressor in malignant melanoma, 11 and miR-337 was demonstrated to be a vital negative regulator in melanoma. 12 As one of the miRNAs, miR-495-3p was involved in the development of melanoma. For instance, it was identified that the elevation of miR-495-3p was capable of inhibiting malignant behaviours of melanoma cells, 13 and miR-495 was verified as a tumour repressor in melanoma. 14 Nevertheless, relation between HDAC3 and miR-495-3p remains unknown. Tumour necrosis factor receptor-associated factor family members (TRAFs) are intracellular adaptors that regulate cellular effects via binding to their cognate cellular receptors. One of the family members, TRAF5, plays an essential role in cell biological processes. 15 It was revealed that suppression of TRAF5 was able to inhibit proliferation while induce apoptosis of melanoma cells, 16 while the target relation between miR-495-3p and TRAF5 is little known.
We aim to explore the impact of HDAC3/miR-495-3p/TRAF5 axis on melanoma, and we supposed that HDAC3 may mediate miR-495-3p to regulate the biological processes of melanoma cells by modulating TRAF5.

| Ethics statement
Written informed consents were acquired from all patients before this study. The protocol of this study was confirmed by the Ethic

| Study subjects
One hundred and fifteen melanoma tissues from patients that had accepted resection in Linyi People's Hospital were collected and confirmed by histopathology. The clinical data of patients, including onset age, gender, tumour classification, tumour, node and metastasis (TNM) stage, lymph node metastasis (LNM) and distant metastasis were collected. Forty control pigmented nevi tissues that obtained from skin biopsy in Linyi People's Hospital were taken as the control tissues.

| Cell grouping
Cells were screened and classified into seven groups and, respec-

| 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide (MTT) assay
Cells after transfection were collected, and the concentration was adjusted to 1 × 10 4 cells/mL. The cell suspension was seeded and incubated for 24 hours, and each well was supplemented with 20 μL MTT solution (5 mg/mL, Sigma-Aldrich Chemical Company) at 24 hours, 48 hours and 72 hours of the incubation, respectively, then continuously incubated for 4 hours. Cells were centrifuged with the supernatant discarded, and each well was appended with 150 μL dimethyl sulfoxide. The blue crystal was completely dissolved after shaken for 10 minutes, and then, the optical density (OD) value at 570 nm of each well was analysed by a microplate reader (FUJIFILM Wako Pure Chemical Corporation). This experiment was repeated for three times, and the results were recorded.

| Colony formation assay
Cells were detached using 0.05% trypsin and seeded into 6-cm culture discs at 1000 cells/well, then cultured for 12-15 d. With the medium removed, the cells were fixed with 10 mL 4% paraformaldehyde (Sigma) for 20 minutes and stained with crystal violet dye solution (Sigma) for 30 minutes. The dried cells were photographed under a microscope, and the colonies were counted.

| Flow cytometry
Cell cycle determination: cell concentration was modulated to 1 × 10 6 cells/mL and the cells were centrifuged at 4°C and 2000 rpm for 10 minutes, then suspended by 1 mL phosphate-buffered saline.
Next, the cells were stained with 10 μL propidium iodide (PI) and Cell apoptosis detection: cell concentration was modulated to 1 × 10 6 cells/mL and the cells were centrifuged at 4°C and 2000 rpm for 10 minutes and then suspended by 300 μL 1 × binding buffer; thereby, the concentration was adjusted to 1 × 10 6 cells/mL. Each well was appended with 5 μL Annexin V-fluorescein isothiocyanate (Thermo Fisher Scientific Inc) for 15-minutes incubation without light exposure at 2-8°C. Cells were filtered by a 400-mesh net, incubated with 10 μL PI at 4°C in dark for 15 minutes and added with 200 μL 1 × binding buffer, and then, the samples were put into the flow cytometer in 1 hour. The apoptosis rate of each group was calculated.

| Hoechst 33342 staining
Detached cells were seeded into 24-well plates at 2 × 10 4 cells/ well until the confluence reached 70%, and then, the medium was replaced by a serum-free one for 12-hours synchronized culture.
With the medium removed, cells in each well were fixed with 1 mL 4% paraformaldehyde at 4°C for 20 minutes and stained by 1 mL

| Scratch test
Cells were seeded onto 24-well plates and cultured for 24 hours under standard conditions, then a straight scratch was made in the middle of the cell monolayer with a sterile pipette tip, and woundhealing status was finally observed under microscopy 24 hours later. The ImageJ 1.35 software was used to analyse the distance of scratches. The wound healing was quantified as the ratio of remaining area without cell and primary wound area.

| Transwell assay
Matrigel (Invitrogen Inc) was fused and put on ice for 1 hour. Apical Five fields of view of each chamber were selected to count the cells.

| Western blot analysis
Cells or tissues were lysed by 1 × radio-immunoprecipitation assay lysis buffer (Beyotime) on ice for 30 minutes and boiled at 100°C for protein denaturation. The protein fluid of cells or tissues was stored at −80°C. Prepared 10% separation gel was poured into the gel caster and added with water saturated n-butanol, then the gel surface was flattened and the gel was placed for 30 minutes. Afterwards, the water with ionized water and placed for 30 minutes. The gel device was taken out, and the gel was added with 1 × electrophoretic buffer. Each well was added with 30 μL protein fluid and protein maker. After the polyacrylamide gel electrophoresis, proteins were transferred onto membranes, which were blocked with 5% skim milk powder for 1 hour and incubated with primary antibodies HDAC3 (1:1000 and from Abcam Inc, Cambridge), TRAF5 (1:500), glyceraldehyde phosphate dehydrogenase (GAPDH, 1:10 000, both from Proteintech Group Inc), N-cadherin and E-cadherin (both 1:1000 and from Cell Signaling Technologies) for 1 hour and stayed overnight at 4°C. Subsequently, the membranes were incubated with relative secondary antibody for 2 hours and added with horseradish peroxidase enhanced chemiluminescent reagent, then scanned by C-DiGit instrument and analysed by Image Studio software.
GAPDH was the internal reference protein, and the ratio of grey value was used for the quantitative determination of protein analysis.

| Dual luciferase reporter gene assay
The target relation between miR-495-3p and TRAF5 was verified using dual luciferase reporter gene assay. were applied to determine the luciferase activity.

| Chromatin immunoprecipitation (ChIP) assay
The experiment was in strict line with direction of EZ-ChIP kits (Millipore Inc). A375 and SK-MEL-28 cells were incubated with 1% formaldehyde on a shaking table, and the crosslink was stopped by glycine after 10 minutes. Cells were centrifuged at 2000 rpm for 5 minutes and added with sodium dodecyl sulphate lysis buffer for ultrasonication, then centrifuged at 10 000 g and 4°C for 10 minutes.
The product of ultrasonication (100 μL) was added with 900 μL ChIP dilution buffer, 20 μL 50 × pre-initiation-complex and 60 μL Protein A Agarose/SalmonSperm DNA, then mixed at 4°C for 1 hour and placed for 10 minutes. Cells were centrifuged at 700 rpm for 1 minutes, and 20 μL sample was taken as the input. A tube was added with 1 μL HDAC3 antibody and immunoglobulin G antibody, which were not added in the other tube. Tubes were incubated at 4°C overnight, and the samples were washed, eluted and decrosslinked.
DNA sample was recycled and conducted with RT-qPCR. Tumour volume = long diameter × short diameter 2 /2.

| Statistical analysis
All data analyses were conducted using SPSS 21.

| Reduced miR-495-3p and overexpressed HDAC3 indicate a poor prognosis of melanoma patients
We Relation between HDAC3/miR-495-3p expression and pathological features of melanoma patients was analysed. The results revealed that (

| HDAC3 mediates miR-495-3p to regulate TRAF5
ChIP assay was applied to detect whether HDAC3 could bind to the promoter of miR-495-3p (Figure 2A,B). The results showed that HDAC3 was related to promoter of miR-495-3p, while was not related to uncorrelated intergenic region, suggesting that HDAC3 was able to directly regulate the expression of miR-495-3p.

| Inhibited HDAC3 elevates miR-495-3p to repress growth of melanoma cells by downregulating TRAF5
We further discussed the role of HDAC3/miR-495-3p/TRAF5 in the biological functions of melanoma cells in vivo and in vitro using MTT assay, colony formation assay and subcutaneous tumorigenesis in nude mice. The results indicated that ( Figure 3A-F)  F I G U R E 2 HDAC3 mediates miR-495-3p to regulate TRAF5 in melanoma cells. A, binding enrichment of HDAC3 and promoter of miR-495-3p in A375 and SK-MEL-28 cells was detected by ChIP assay; B, binding enrichment of HDAC3 and uncorrelated intergenic region in A375 and SK-MEL-28 cells was detected by ChIP assay; C, target relation between miR-495-3p and TRAF5 was predicted by a bioinformatic software; D, target relation between miR-495-3p and TRAF5 was confirmed by dual luciferase reporter gene assay; E, expression of HDAC3, miR-495-3p and TRAF5 in A375 cells of each group; F, protein expression of HDAC3 and TRAF5 in A375 cells of each group; G, expression of HDAC3, miR-495-3p and TRAF5 in SK-MEL-28 cells of each group; H, protein expression of HDAC3 and TRAF5 in SK-MEL-28 cells of each group; N = 3, *P < .05, **P < .01; the measurement data were expressed as mean ± standard deviation, the unpaired t test was performed for comparisons between two groups, one-way ANOVA was used for comparisons among multiple groups and Tukey's post hoc test was used for pairwise comparisons after one-way ANOVA TRAF5 siRNA or miR-495-3p mimics suppressed the proliferation, colony formation ability and tumorigenesis of melanoma cells; the impacts of si-HDAC3 were abolished by miR-495-3p inhibitors.
These findings mirrored that HDAC3 mediated miR-495-3p to regulate TRAF5 expression, thus affecting the growth of melanoma cells.

| Down-regulated HDAC3 elevates miR-495-3p to promote G0/G1 phase arrest and apoptosis of melanoma cells via repressing TRAF5
We conducted flow cytometry to measure the cell cycle arrest and apoptosis rate of A375 and SK-MEL-28 cells. Outcomes unravelled that ( Figure  mimics and si-TRAF5 groups performed apparent apoptotic characteristics compared to their NC groups: the nuclei were shrunk and lysed much completely, there were dense massive lumpy or granular fluorescence in nuclei, increased apoptotic cells and decreased F I G U R E 3 Inhibited HDAC3 elevates miR-495-3p to repress growth of melanoma cells by down-regulating TRAF5. A, viability of A375 and SK-MEL-28 cells was determined by MTT assay; B, colony formation ability of A375 and SK-MEL-28 cells was determined by colony formation assay; C, number of colonies in each group; D, xenografts from nude mice that had been injected with A375 and SK-MEL-28 cells; E, volume of xenografts from nude mice that had been injected with A375 and SK-MEL-28 cells; F, weight of xenografts from nude mice that had been injected with A375 and SK-MEL-28 cells; N = 3, *P < .05, **P < .01; the measurement data were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey's post hoc test was used for pairwise comparisons after one-way ANOVA normal cells. Contrasted to si-HDAC3 group, there were evenly distributed blue fluorescence and markedly decreased apoptotic cells in the si-HDAC3 + miR-495-3p inhibitors, si-NC, mimics NC and Control siRNA groups. These results were in accordance with that of flow cytometry.

| Declined HDAC3 increases miR-495-3p to inhibit migration, invasion and epithelialmesenchymal transition (EMT) of melanoma cells through reducing TRAF5
Scratch test and Transwell assay were utilized to determine the mi-  Furthermore, results of cellular experiments in our study indicated that inhibited HDAC3 was able to elevate miR-495-3p to decelerate proliferation, migration and invasion of melanoma cells, and also induced G0/G1 phase arrest and accelerate melanoma cell apoptosis.
In accordance with the results, Shan et al 8  ; the measurement data were expressed as mean ± standard deviation, one-way ANOVA was used for comparisons among multiple groups and Tukey's post hoc test was used for pairwise comparisons after one-way ANOVA have identified that the repression of TRAF5 was accompanied by inhibited proliferation and enhanced apoptosis of melanoma cells.
Except for in vitro experiments, we also conducted in vivo experiment to probe into the roles of HDAC3, miR-495-3p and TRAF5 in melanoma cell growth. Nevertheless, the detailed molecular mechanisms remain to be explored.

ACK N OWLED G EM ENT
We would like to acknowledge the reviewers for their helpful comments on this paper.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.