miRNA expression profile of vulvar squamous cell carcinoma and identification of the oncogenic role of miR ‐ 590 ‐ 5 p

MicroRNAs (miRNAs), a class of small non-coding RNA molecules, are associated with a variety of human cancers. Currently, little data are available regarding miRNA expression in vulvar squamous cell carcinoma (VSCC); the mechanism of action of miRNAs in VSCC still requires investigation. The aim of the present study was to investigate the miRNA expression profile in VSCC using a miRCURY™ LNA array. The expression levels of selected miRNAs were quantified by RT-qPCR. The relationship between miR-590-5p expression and clinical pathology was assessed. The expression levels of crucial transforming growth factor-β (TGF-β) and Smad pathway factors were detected. We further investigated the role of miR-590-5p via in vitro studies in the A431 human VSCC cell line. A total of 157 miRNAs showed significantly altered expression in this type of carcinoma. Of particular interest, miR-590-5p, miR-182-5p and miR-183-5p were upregulated, and miR-603, miR-103a-3p and miR-107 were downregulated. A positive relationship was found between miR-590-5p expression and lymph node metastasis. In VSCC, TGFβ1 and TGFβ2 were significantly overexpressed and TGFβRII and Smad4 were significantly underexpressed at both the RNA and protein levels. In A431 cells, overexpression of miR-590-5p promoted proliferation, migration and G1-S phase transition and downregulated TGFβRII. The knockdown of TGFβRII by siRNA promoted malignant behaviours in the A431 cells. In conclusion, we present the miRNA expression profile in VSCC, and our findings suggest that the upregulation of miR-590-5p promotes cellular malignant behaviours via the target gene TGFβRII.


Introduction
Vulvar tumours constitute 4% of all gynaecological malignancies and are critical carcinomas of the female reproductive system (1).According to the National Cancer Institute, the incidence of vulvar cancer has increased in recent years in the US (2).The early diagnosis of vulvar cancer results in a five-year survival greater than 70%.The prognosis of vulvar neoplasia depends on the clinical grade, tumour diameter and status of lymph node metastasis.Standard surgery can cause considerable morbidity and significant disfigurement.Patients require individualised therapy and less radical surgery to reduce the incidence of complications.At present, the majority of vulvar carcinomas are vulvar squamous cell carcinomas (VSCCs) (90%) (3), and recent studies have shown that the overall incidence of VSCC has risen steadily at a rate of 20% over the past 40 years (4).Previous studies have not completely explained the aetiology of this disease.Thus, the identification of new tumour markers for the early detection and prognostic monitoring of VSCC remains urgently needed.
MicroRNAs (miRNAs) are short non-coding RNAs that silence mRNAs at the post-transcriptional levels.The transcription of approximately one-third of mRNAs is regulated by miRNAs, which are considered important regulators in key biological processes, such as cell growth, differentiation, adhesion, angiogenesis and inflammation (5)(6)(7).Changes in miRNA levels can cause different types of cancers.The miRNA expression profiles in cancer have been used to identify early diagnostic markers and therapeutic targets.At present, little data are available regarding miRNA expression in vulvar cancer.de Melo Maia et al (8) identified 79 miRNAs that showed markedly different expression levels in vulvar cancer compared with control samples.Although many miRNAs have been found to be associated with vulvar cancer, the mechanism of action of miRNAs in vulvar tumourigenesis still requires further investigation.
In the present study, we compared the miRNA expression profiles of VSCC and adjacent non-cancerous samples and investigated the mechanism of action of miR-590-5p in the A431 human VSCC cell line.The target of miR-590-5p was identified to elucidate the possible function of miR-590-5p in VSCC.

Materials and methods
Tissue collection.We obtained three pairs of freshly frozen VSCC samples and adjacent non-cancerous tissues for our microarray study (Table Ⅰ).For miRNA analysis and target miRNA expression profile of vulvar squamous cell carcinoma and identification of the oncogenic role of miR-590-5p gene validation step, 30 freshly frozen VSCC samples and adjacent non-cancerous tissues were collected.All of the tissues were obtained from the Department of Gynaecology at our hospital at the time of surgery between January 2011 and January 2015, and we snap-froze the samples rapidly for future use.We staged all of the lesions using the new vulvar cancer classification system (9).The pathology of the frozen specimens was analysed.Clinical records were retrospectively reviewed.We enrolled patients who had not undergone chemotherapy or radiation treatment prior to surgery.The ethics Committee of the First Affiliated hospital of China Medical University approved our research and we obtained the informed consent from patients.
miRNA microarray and bioinformatics.Kangchen Bio-tech Inc. (Shanghai, China) extracted the total RNA from the tissue samples and performed our microarray analysis using a miRCURY™ LNA array (v.18.0; exiqon).After carefully reviewing the literature, we chose 25 upregulated and 25 downregulated miRNAs for further study.Three web-based miRNA target prediction programs were used to explore potential target genes: TargetScan (https://www.targetscan.org/vert60/), MicroCosm (http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/) and miRanda (http://www.microrna.org/microrna/home.do).To improve the accuracy of our results, we further examined the putative genes that were identified by the three algorithms for further study.We conducted Gene Ontology (GO) and KEGG pathways analyses on these target genes.
Real-time quantitative PCR of miRNAs.We selected fragments of the differentially expressed miRNAs for further verification in 30 additional VSCC tissues and the corresponding adjacent non-cancerous vulvar samples.Total RNA was isolated from tissues using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions.As previously described (10), we applied stem-loop RT-PCR to detect the miRNA levels.cDNA synthesis was performed using a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA, USA).We performed RT-qPCR in triplicate following standard protocols.We calculated the miRNA expression levels using the 2 -ΔΔCt method with U6 as the internal control.The PCR cycle parameters were the following: 95˚C for 10 min followed by 40 cycles of 95˚C for 10 sec and 60˚C for 60 sec.The miRNA primers are shown in Table Ⅱ.
We detected mRNAs of TGFβ1, TGFβ2, TGFβRⅡ and Smad4 in the tissue samples by RT-qPCR.We quantified the amplified products using the SYBR-green method (Takara Bio, inc., Dalian, China) with gAPDh as the internal control.The fold-changes were quantified using the 2 -ΔΔCt method.The primers were as follows: gAPDh forward, AAggTgAAgg TCGGAGTCAAC and reverse, GGGTCATTGATGGCAAC AATA; TGFβ1 forward, AAGGACCTCGGCTGGAAGTG and reverse, CCCGGGTTATGCTGGTTGTA; TGFβ2 forward, TGCCGCCCTTCTTCCCCTC and reverse, GGAG CACAAGCTGCCCACTGA; TGFβRⅡ forward, AAgATgA CCGCTCTGACATCA and reverse, CTTATAGACCTCAGC AAAGCGAC; and Smad4 forward, CGCTTTTGTTTGGGT CAACT and reverse, CCCAAACATCACCTTCACCT.The RT-qPCR parameters were the following: 95˚C for 10 min followed by 40 cycles of 95˚C for 5 sec and 60˚C for 34 sec.
Cell culture and transfection.The A431 cell line was obtained from ATCC (Manassas, VA, USA) and cultured in RPMi-1640 medium containing 10% fetal bovine serum under standard conditions at 37˚C and 5% Co 2 in a humidified atmosphere.The cells were transfected with Dharmacon miRIDIAN miR-590-5p mimic (miR-590-5p) and the negative control (Thermo Fisher Scientific, Lafayette, Co, USA) at a final concentration of 100 nmol/l.A small interfering RNA targeting TGFβRⅡ (siRNA-TgFβRⅡ) was obtained from Santa Cruz Biotechnology, inc.(Santa Cruz, CA, USA) (sc-36657).The cells were transfected using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer's recommendation.
MTT assay.We performed an MTT assay to evaluate cell proliferation.A431 cells were plated in 96-well sample culture plates at a density of 5x10 4 cells per well with the miR-590-5p mimics or siRNA-TGFβRⅡ and the corresponding negative controls.The cells were cultured for 48 and 96 h, and the optical absorbance was read at 490 nm on a microplate reader.The experiments were conducted in triplicate.
Cell migration.A Transwell assay was performed to examine cell migration.Forty-eight hours after transfection, 5x10 4 cells were placed in the upper chambers of Transwell plates with an untreated membrane.After 24 h of incubation, the chambers were treated with 4% paraformaldehyde and then fixed with hematoxylin and eosin.The cells that passed through the membrane were counted.The migration assays were conducted in triplicate.

Analysis of cell cycle distribution.
Cells transfected with the miR-590-5p mimics or siRNA-TGFβRⅡ for 48 h were collected and placed in ethanol (70%) for 24 h.The cells were then treated with propidium iodide (40 µg/ml) for 30 min.Flow cytometry was used for the analysis, and the experiments were performed in triplicate.

Statistical analysis.
The data are presented as the means ± SD, and SPSS 15.0 software (SPSS, inc., Chicago, iL, USA) was used for all of the data analyses.The significance of differences in the mean values was analysed using the Student's t-test.A two-sided Fisher's exact test was used to determine the relationship between miR-590-5p expression and clinicopathological data.P<0.05 was considered to indicate a statistically significant difference.

miRNA expression profile in VSCC.
After confirming the quality of the RNA extracted from the tissue samples, we determined the miRNA expression profile of VSCC.We selected the miRNAs that were overexpressed or underexpressed by more than 2-fold for the bioinformatics analysis and identified 90 upregulated and 67 downregulated miRNAs in the cancer samples (Fig. 1, Tables Ⅲ and Ⅳ).
Bioinformatics.In total, 1,350 genes were predicted.GO analysis and KEGG annotation showed that the transforming growth factor-β (TGF-β) pathway was an enriched pathway in VSCC (Fig. 2).
Relationship between miR-590-5p and clinical pathology of vulvar cancer.We set the 75th percentile of the 2 -ΔΔCt values as the cut-off value for samples with high or low levels of miR-590-5p (12).The miR-590-5p level was not correlated with patient age, tumour differentiation, vascular invasion, FIGO stage, tumour size and depth of invasion but was positively correlated with lymph node metastasis (P=0.009)(Table V).
The results indicated that increased expression of miR-590-5p aids the distant spread of VSCC.
Putative target genes.According to the target prediction programs, TGFβ1 and TGFβ2 are both putative target genes for miR-590-5p (Table Ⅴi), TgFβRⅡ is a target of miR-590-5p in human hepatocellular carcinoma (13), and miR-182-5p acts as an oncogene by knocking down Smad4 in bladder cancer (14).Since the results showed that miR-182-5p and miR-590-5p were upregulated in VSCC (Fig. 3), we detected the mRNA and protein levels of TGFβ1, TGFβ2, TGFβRⅡ and Smad4.The RNA levels of TGFβ1 and TGFβ2 were significantly overexpressed in VSCC compared with control tissues by 2.95-fold and 3.25-fold, respectively (Fig. 4A).The protein levels of TGFβ1 and TGFβ2 were found to be markedly increased in VSCC (Fig. 4B and C).The RNA levels of TGFβRⅡ and Smad4 were significantly underexpressed in VSCC compared with control tissues by 0.21-fold and 0.25-fold, respectively (Fig. 4A), and the protein levels of TGFβRⅡ and Smad4 were markedly decreased in VSCC (Fig. 4B and C).Based on our results and the literature (13), we hypothesized that TGFβRⅡ is a target gene of miR-590-5p in VSCC.
miR-590-5p promotes the proliferation, migration and the G1-to-S transition in A431 cells.A431 cells were transiently transfected with the miR-590-5p mimic at a concentration of 100 nM.At 96 h, the proliferation rate of the A431 cells transfected with the miR-590-5p mimic was markedly higher (P=0.001)than that of the cells transfected with the miRNA negative control (Fig. 5D).The miR-590-5p mimic significantly promoted A431 cell migration.The migration rate of the cells transfected with the miR-590-5p mimics was 40% higher than that of the cells transfected with the negative control (P=0.001) (Fig. 6A).An analysis of the cell cycle distribution by flow cytometry showed that the percentage of A431 cells in the G1 phase was significantly decreased from 58.89±1.69%before transfection to 47.73±1.23%at 48 h after transfection (P=0.001) and that the percentage of cells in the S phase was significantly increased from 27.80±1.95%before transfection to 43.41±1.22%at 48 h after transfection (P=2.998x10 - ) (Fig. 7A).

Discussion
miRNAs are highly conserved small RNA molecules (18-24 bp in length) that block translation or promote mRNA degradation.
Researchers use miRNA expression profiles to identify new early diagnostic markers and key pathways that are relevant to cancer and to predict outcomes (15).however, few miRNA studies have focused on VSCC.In the present study, we determined the miRNA expression profile of VSCC using a miRCURY™ LNA array.To confirm the reproducibility of the experiment, we determined the expression levels of 6 miRNAs in 30 pairs of tissues by RT-qPCR and found that the results of the two assays were consistent.This investigation of the miRNA profile of VSCC provides valuable information regarding the aetiology of VSCC.Computational approaches, such as GO and KEGG, provide high statistical power for confirming the biological relevance of the experimental data.Using bioinformatics, we  The RNA and protein levels of key factors (TgFβ1, TGFβ2, TGFβRⅡ and Smad4) were measured in tissue samples, and TGFβRⅡ was selected for further research.We observed the upregulation of miR-590-5p in VSCC.miR-590-5p has been reported to increase cardiac muscle cell growth in mammals (16) and has frequently been shown to be upregulated in tumours, which suggests that miR-590-5p promotes tumour progression.miR-590-5p has been shown to be overexpressed in patients with high-risk types of Wilms' carcinoma (17).The target mRNAs of miR-590-5p have been identified in certain studies.For example, miR-590-5p has been shown to exert oncogenic activity through targeting of the ChL1 gene in cervical carcinoma (18). in addition, upregulation of miR-590-5p has been shown to be induced by the downregulation of PBRM1 in renal cancer cell lines (19).Another research group showed that miR-590 is upregulated by TGFβRⅡ in cardiosphere-derived cells during differentiation (20).however, Shan et al reported that miR-590-5p is downregulated in liver cancer and that miR-590-5p decreases the proliferation of carcinoma cells by targeting S100A100 (21).Our study showed that miR-590-5p is overexpressed in vulvar cancer tissues (Fig. 3).Thus, miR-590-5p may be a novel oncogenic miRNA in VSCC.
Our research also showed that TGFβRⅡ is negatively regulated by miR-590-5p in VSCC.The TGF-β/Smad signalling pathway functions as an important regulator of the pathogenesis of many cancers, including colon cancer, oesophageal squamous cell carcinoma, breast cancer and lung cancer (22)(23)(24)(25).Many studies have investigated the effect of TGF-β/Smad signalling on epithelial cell proliferation and carcinoma.In humans, the three TGFβ isoforms, TGFβ1, TGFβ2 and TGFβ3, exhibit high similarity and homology.The TGF-β pathway transfers signals to the inside of cells via TGFβRⅡ has been shown to play roles in tumour invasion and metastasis in several types of cancers.Ganapathy et al showed that TGFβRⅡ is downregulated in haCaT cells and found that TGFβRⅡ inhibits metastasis and progression of late-stage tumour cells (26).The overexpression of TGFβRⅡ inhibits cell growth in cancer (27).Additionally, TGFβRⅡ has also been found to be progressively lost in oral and skin SCC (28).however, the role of TgFβRⅡ in VSCC is unknown, and our research showed that TGFβRⅡ is underexpressed in vulvar carcinoma.
We conducted functional studies in A431 cells, which have previously been used for miRNA research.Alanazi et al found that some miRNAs (miR-663, miR-499-5p, miR-494, miR-602, miR-2861, miR-675 and miR-3185) may have crucial functions in A431 cell apoptosis through a process involving epidermal growth factor (EGF) involvement (29).In addition, miR-21 has been shown to promote the proliferation of A431 cells (30).In the present study, we transfected   the miR-590-5p mimic into A431 cells, and after transfection, the endogenous level of miR-590-5p was upregulated.
The MTT assay revealed that cell proliferation was markedly increased (Fig. 5D) and that TgFβRⅡ was downregulated (Fig. 5A-C).The Transwell assay results suggested that miR-590-5p induced cell migration (Fig. 6A).We also explored changes in cell cycle progression, which is an essential step in carcinogenesis.The FCM results indicated that the upregulation of miR-590-5p promoted the G1-to-S cell cycle transition in the A431 cells (Fig. 7A).Our results provide novel information regarding the roles of miR-590-5p in tumourigenesis.Moreover, we used siRNA to successfully block TGFβRⅡ expression and obtained results that were similar to those obtained after the overexpression of miR-590-5p.We found that miR-590-5p exerted an oncogenic function in vulvar cancer.Based on our results, we hypothesize that  The present study has some limitations.First, vulvar cancer is a rare disease; thus, the number of clinical samples in our study was relatively low, and more microarray studies on vulvar cancer are needed to obtain larger data sets.We did not conduct a dual luciferase reporter assay in our study since it was performed previously (13).We found that miR-590-5p, TGFβ1 and TGFβ2 were all upregulated in VSCC tissues, which suggests that miRNAs other than miR-590-5p may be  responsible for the upregulation of TGFβ1 and TGFβ2 because miRNAs typically decrease the expression of their target mRNAs.however, some miRNAs have also been reported to increase the expression of target genes.For example, miR-373 increases the expression of its target gene, E-cadherin (31).Therefore, further assays need to be performed to determine the targets of miR-590-5p.The relationship between miR-182-5p and Smad4 in VSCC remains to be investigated.
In conclusion, we determined the miRNA expression profile in VSCC and evaluated the expression of a subset of miRNAs by microarray.Target prediction and functional analysis suggested that miR-590-5p is involved in the tumourigenesis of VSCC as the upregulation of miR-590-5p was found to be associated with lymphatic metastasis.Furthermore, the present investigation showed that miR-590-5p plays an oncogenic role in VSCC by promoting cell proliferation and  migration as well as the G1-S transition through the manipulation of TGFβRⅡ expression.These findings suggest that miR-590-5p may be a critical therapeutic target in VSCC.

Figure 1 .
Figure 1.miRNA expression in VSCC as determined by microarray.Each row represents an miRNA and each column represents a sample.Green indicates low relative expression and red indicates high relative expression.

Figure 2 .
Figure 2. Relationship of the TGF-β signalling pathway to VSCC.The orange nodes indicate putative target genes of the miRNAs that were found to be differentially expressed in VSCC, and the green nodes indicate genes in the TGF-β pathway.

Figure 4 .
Figure 4. Expression of TGFβ1, TGFβ2, TGFβRⅡ and Smad4 in VSCC samples.(A) mRNA expression levels of TGFβ1, TGFβ2, TGFβRⅡ and Smad4 in 30 pairs of VSCC and corresponding normal tissue samples.(B and C) Western blot analysis was conducted with the indicated antibodies.gAPDh was used as the internal control.* P<0.05.

Figure 8 .
Figure 8. TGFβRⅡ regulates A431 cell proliferation.siRNA targeting TgFβRⅡ suppressed the mRNA (A) and protein (B and C) expression levels of TgFβRⅡ.(D) Knockdown of TgFβRⅡ by siRNA increased the cell proliferation rate at 96 h after transfection.* P<0.05.

Table I .
Characteristics of the patients in the microarray study.

Table Ⅱ .
Primers used in reverse transcription and real-time PCR for the miRNAs.

Table Ⅲ .
miRNAs with a 2-fold increased change in VSCC.
found that the TGFβ signalling pathway is important in VSCC.

Table Ⅳ .
miRNAs with a 2-fold decreased change in VSCC.-specific receptors including TGFβRⅡ, which plays a role in cell growth and carcinogenesis.Smad4 is a major transducer of intracellular signals of the TGF-β pathway.