microRNA-802 inhibits epithelial-mesenchymal transition through targeting flotillin-2 in human prostate cancer

miRNAs are a class of non-coding RNAs that exert critical roles in various biological processes. The aim of the present study was to identify the functional roles of miR-802 in regulating epithelial–mesenchymal transition (EMT) in prostate cancer (PCa). miR-802 expression was detected in 73 pairs of PCa samples and PCa cell lines (PC3 and DU145 cells) by qRT-PCR. Cell proliferation was detected using MTT assay, and cell apoptosis was evaluated using flow cytometry. Transwell assay was conducted to investigate cell migration and invasion. Expression analysis of a set of EMT markers was performed to explore whether miR-802 is involved in EMT program. Xenograft model was established to investigate the function of miR-802 in carcinogenesis in vivo. The direct regulation of Flotillin-2 (Flot2) by miR-802 was identified using luciferase reporter assay. miR-802 was remarkably down-regulated in PCa tissues and cell lines. Gain-of-function trails showed that miR-802 serves as an ‘oncosuppressor’ in PCa through inhibiting cell proliferation and promoting cell apoptosis in vitro. Overexpression of miR-802 significantly suppressed in vivo PCa tumor growth. Luciferase reporter analysis identified Flot2 as a direct target of miR-802 in PCa cells. Overexpressed miR-802 significantly suppressed EMT, migration and invasion in PCa cells by regulating Flot2. We identified miR-802 as a novel tumor suppressor in PCa progression and elucidated a novel mechanism of the miR-802/Flot2 axis in the regulation of EMT, which may be a potential therapeutic target.


Introduction
Prostate cancer (PCa) is the most frequent malignant tumor in elderly males worldwide and is the second most common reason for cancer-related mortality in western countries [1]. It is reported that to date, the 5-year survival rate of PCa patients has not been improved remarkably because of late diagnosis and therapeutic limitations, thus exerting a big burden on public health. PCa is also characterized as a leading and increasing health problem in China [2]. A number of factors, including genetic alternations and tumor micro-environment, have been demonstrated to participate in its pathogenesis [3]. Metastasis is often responsible for the recurrence, unsatisfactory prognosis, and high mortality of PCa [4]. Although extensive studies of PCa have been performed, the pathogenesis of tumorigenesis and progression of PCa has not been fully elucidated.
Epithelial-mesenchymal transition (EMT), featured as a highly conserved trans-differentiation process, is frequently implicated in cancer aggressiveness and metastasis. Cells undergoing EMT exhibit a morphological change to a spindle-shaped phenotype and the process involves loss of epithelial cell markers, such as E-cadherin, and overexpression of mesenchymal markers, including N-cadherin and vimentin) [5]. Evidence is increasing that EMT is a critical step associated largely with cancer development and metastasis [6], and many factors, including miRNAs, have been shown to exert important regulatory functions in maintaining the balance between EMT and mesenchymal-epithelial transition (MET) (the reverse process of EMT) [7]. First discovered in 1993, miRNAs are defined as a class of evolutionarily conserved, endogenous, small non-coding RNAs of 18-24 nt in length through complementary binding to the 3 -UTR of specific target mRNAs, causing either degradation or inhibition of translation, thus effectively silencing their mRNA targets [8]. Approximately 50% of human miRNAs are located at fragile sites and genomic regions associated to cancers [9]. In the last decade, a number of miRNAs have been reported to be implicated in PCa pathogenesis, acting as either oncogenes or tumor suppressors according to the roles of their target genes [10].
miR-802, a miRNA located on chromosome 21, is found to be significantly decreased in human breast cancer tissues and inhibit cell proliferation through suppressing Forkhead box protein M1 (FoxM1) expression [11]. Müller et al. [12] speculated that down-regulation of miR-802 might lead to increased Wnt activity in pancreatic ductal adenocarcinoma. To date, however, little is understood about the clinical significance and biological functions of miR-802 in human PCa.
The present article provides evidence, for the first time, that miR-802 expression is down-regulated in human PCa tissues and cells, and miR-802 could suppress EMT of PCa cells through directly inhibiting Flotillin-2 (Flot2), a member from flotillin family that serves an important role in the pathogenesis and progression of human malignancies [13]. Our results revealed that miR-802 might function as a potential therapeutic strategy in metastatic cancers.

Clinical samples and cell lines
Seventy three pairs of human PCa tissues and their matched normal prostate tissues collected by needle biopsy or autopsy were acquired from the patients who were admitted to Ruijin Hospital North (Shanghai, China). All patients did not receive any treatment including chemotherapy, radiation therapy and androgen-deprivation treatment prior to surgery. The tissue samples were frozen in liquid nitrogen after surgery and rapidly stored at −80 • C until further analysis. All patients and/or their guardians provided written informed consent for tissue donation for research purposes. The protocol was approved by the Committees for the Ethical Review of Research at Shanghai Jiao Tong University, and was performed in accordance with the Declaration of Helsinki. Clinicopathological characteristics of PCa patients included in this research, obtained from their medical files, were recorded in Table 1.

RNA extraction and quantitative real-time reverse transcription PCR
Total RNA was extracted from tissue specimens and cell lines using TRIZOL reagent (Invitrogen, Carlsbad, CA, U.S.A.). RNA purity and concentration were detected by Nanodrop 2000 (Thermo Fisher Scientific, Wilmington, DE, U.S.A.). The cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen), and the PCR amplification for the quantification of the miR-802 and U6 was performed using TaqMan Human miRNA Assay Kit (Applied Biosystems, Foster City, CA, U.S.A.). The relative expression of miR-802 was illustrated as fold difference relative to U6. The PCR amplification for the quantification of the Flot2 and GAPDH mRNAs was conducted using an ABI PRISM 7300 Sequence Detection System (Applied Biosystems) and a SYBR R Premix Ex Taq TM ii (Perfect Real Time) Kit (Takara Bio, Shiga, Japan). The relative expression of Flot2 was illustrated as fold difference relative to GAPDH. Data are presented as a relative amount using the calculation of 2 − C t [14,15].
In cell transfection, cells were seeded in six-well plates and cultured until 50-70% confluency was reached in 1 day.

Western blot
Total protein was isolated using radioimmunoprecipitation assay buffer (

MTT assay
The in vitro proliferation of PCa cells were measured by the MTT assay (Sigma, St Louis, MO, U.S.A.) according to the manufacturer's protocol. Briefly, PCa cells were incubated with MTT solution (1 mg/ml) at 37 • C in a 5% CO 2 incubator for 4 h. After the medium was discarded, formazan crystals were dissolved in DMO (Sigma, St Louis, MO, U.S.A.). The absorbance at a wavelength of 570 nm was measured by a microplate reader at 1, 2, 3, 4, and 5 days, and the cell growth curves were analyzed and drawn.

Flow cytometric analysis of apoptosis
After double staining with FITC-Annexin V and propidium iodide (PI) using Annexin V-FITC apoptosis detection kit (BD Biosciences, San Jose, CA, U.S.A.) at room temperature, cells were analyzed with a flow cytometry (BD LSR-Fortessa flow cytometer; BD Biosciences) equipped with CellQuest software (BD Biosciences).

Transwell assay
For the detection of cell migration, 5 × 10 4 cells in serum-free medium were placed into the upper chamber of an insert (8-μm pore size; BD Biosciences). For the detection of cell invasion, 1 × 10 5 cells in serum-free medium were placed into the upper chamber of a Matrigel-coated insert. Medium containing 10% FBS was added to the lower chamber to serve as chemo-attractant. After 48-h incubation, the cells remaining on the upper membrane were removed, and the cells that had migrated or invaded through the membrane were fixed with 4% paraformaldehyde, stained with Crystal Violet, imaged, and counted under a microscope.

Tumor xenograft assay
Six-week-old BALB/c male nude mice were obtained from Shanghai Laboratory Animal Center (Chinese Academy of Sciences, Shanghai, China). Tumor-bearing mice were maintained under specific pathogen-free (SPF) conditions in Experimental Animal Center of Shanghai Jiao Tong University. All animal experiments were performed in accordance with the Guide for Care and Use of Laboratory Animal and all experimental protocols were approved by the Animal Ethics Committee of Shanghai Jiao Tong University. For the subcutaneous xenograft mouse model, DU145 cells (5 × 10 6 ) that were transiently transfected with miR-802 mimics or miR-Con were suspended in 100 μl medium and subcutaneously injected into the left and right backside flanks of nude mice (n=5 mice/group) respectively. The volume of the in vivo tumor xenograft was calculated with the equation, length width 2 /2. Five weeks after initial injection, mice were sacrificed and prostate cancer xenograft was weighed.

Statistical analysis
Data are presented as mean + − S.D. and analyzed using GraphPad Prism 5 (GraphPad Software Inc., San Diego, U.S.A.) and SPSS 21.0 software (SPSS Inc., Chicago, IL, U.S.A.). Statistical significance was investigated through a two-tailed, unpaired Student's t test or a chi-square test as appropriate. Statistics with P-value <0.05 was regarded as statistical significance.

Down-regulation of miR-802 correlates with PCa metastasis
To investigate the expression pattern and clinicopathologic significance of miR-802 in PCa, we first detected the expression and clinical values of miR-802 in 73 cases of paired PCa tissue samples. miR-802 expression was remarkably decreased in PCa tissues than in corresponding adjacent non-tumor mucosa ( Figure 1A, P<0.05). In addition, miR-802 expression was evaluated in two types of PCa cell lines (PC3 and DU145 cells) and normal prostate cells (RWPE-1). As illustrated in Figure 1B, after normalization to U6, the mRNA levels of miR-802 were evidently reduced in two types of PCa cell lines (PC3 and DU145 cells) than that in normal prostate cells (RWPE-1). DU145 cell line exhibited the lowest miR-802 level and was thus selected for further trials.
To analyze the clinicopathological significance of miR-802, the mean fold change of miR-802 expression (0.27) was considered as a cut-off value to categorize all 73 PCa patients into two groups: an miR-802 low expression group  (n=27) and an miR-802 high expression group (n=46). The associations between miR-802 expression and the clinical features of PCa patients were documented in Table 1. miR-802 expression was observed to be correlated closely with Gleason score (P=0.004), distant metastasis (P=0.002) and pathological stage (P=0.02).

miR-802 suppresses cell proliferation and promotes apoptosis in PCa cells in vitro
Function assays were performed to ascertain whether miR-802 could regulate cell proliferation and apoptosis in PCa. We transfected DU145 PCa cells with miR-802 mimics or control miRNA, and then explored the proliferation and apoptosis of cell lines. Overexpression of miR-802 significantly inhibited cell proliferation in DU145 cells compared with the control groups, as demonstrated by MTT assay (P<0.05, Figure 2A). Moreover, up-regulation of miR-802 greatly increased the number of apoptotic PCa cells than control miR-transfected cells, as shown by flow cytometry (P<0.05, Figure 2B).

miR-802 inhibits in vivo PCa tumor growth
To further explore the function of miR-802 on tumor growth in vivo, DU145 cells with overexpressed miR-802 were generated and injected subcutaneously into the dorsal flank of nude mice to establish DU145 tumor xenografts. Tumor growth was closely monitored for 5 weeks. The tumor growth was markedly slower in mice injected with DU145 cells overexpressing miR-802 compared with those in control mice (P<0.05, Figure 3A). Similarly, the average tumor weight was remarkably reduced by miR-802 overexpression (P<0.05, Figure 3B,C), further indicating that miR-802 might suppress tumor growth in vivo.

miR-802 directly targets Flot2 in PCa cells
It is widely acknowledged that miRNAs exert their functions through modulation of target mRNAs. Through computer-based sequence analysis using TargetScan website (http://www.targetscan.org) [16], we have identified that Flot2 is a potential target gene of miR-802. The binding sites of miR-802 were predicted at positions 82-89 in the 3 -UTR of Flot2 mRNA, as demonstrated in Figure 4A. To investigate whether miR-802 directly interacts with the predicted 3 -UTR of Flot2, the 3 -UTR of human Flot2 was cloned downstream the firefly luciferase coding sequence and co-transfected with miR-802 mimics into DU145 cells. Indeed, normalized firefly luciferase activity decreased by approximately 40% in comparison with the transfected control (P<0.05). In addition, site-directed mutagenesis of the seed region dramatically reversed the inhibitory function of miR-802 on firefly luciferase activity ( Figure 4B). To illustrate that the endogenous miR-802 can regulate the expression of Flot2, miR-802 mimics was transfected into DU145 cells. As shown in Figure 4C,D, Flot2 expression was significantly reduced in miR-802 mimics-transfected DU145 cells whatever the mRNA or protein levels (all P<0.05), and down-regulation of miR-802 had the opposite effects in RWPE-1 cells (all P<0.05). These results suggested that miR-802 directly regulated Flot2 expression by targeting 3 -UTR of its mRNA.

miR-802 suppresses EMT in PCa cells by regulating Flot2
In order to verify whether miR-802 can modulate EMT in PCa cells, we investigated several common EMT markers through Western blot analysis. As demonstrated in Figure 5, reduced protein levels of mesenchymal markers (vimentin and N-cadherin) were found in miR-802 mimics-transfected DU145 cells compared with that in control cells (all P<0.05), and epithelial marker (E-cadherin) protein expression was elevated in DU145 cells with overexpressed miR-802 (P<0.05). Besides, the repressive effects of miR-802 on EMT were obviously restored by Flot2 overexpression. Taken together, our data revealed that miR-802 suppresses EMT in PCa cells in vitro.

miR-802 suppresses cell migration and invasion in PCa cells in vitro by regulating Flot2
The effect of miR-802 on the migration and invasion of PCa cells was determined through transwell assay. The results demonstrated that compared with control cells, up-regulation of miR-802 could significantly suppress DU145 cell migration and invasion (Figure 6), and these effects were obviously restored by Flot2 overexpression, clearly indicating that miR-802 can inhibit the migratory and invasive phenotype of PCa cells by regulating Flot2.

Figure 6. miR-802 suppresses cell migration and invasion in PCa cells in vitro by regulating Flot2
Transwell assay was performed to investigate the migratory and invasive capacities of DU145 cells. Data are presented as mean + − S.D. from at least three independent experiments. Statistical significance was determined by a two-tailed Student's t test: *P<0.05 in comparison with miR-Con, # P<0.05 in comparison with miR-802.

Discussion
Despite outstanding advances in our understanding of the underlying mechanisms of PCa, the prognostic outcomes of PCa patients remain unfavorable. Distant metastasis, featured as the leading cause of cancer-associated mortality in the vast majority of cases of human cancers, is a complex process that requires multiple molecular and cellular events [17,18]. Accordingly, it is of vital importance to identify the molecular switch contributing to this malignant phenotype and clarify the key mechanisms involved in the metastatic evolution of PCa.
During the last two decades, a great number of miRNAs have been confirmed as oncogenes or tumor suppressor in various human cancers [19][20][21][22]. Our data suggested that aberrant miR-802 expression is significantly correlated to distant metastasis in PCa patients. Intriguingly, the regulatory function of miR-802 in carcinogenesis remains controversial. The article of Li et al. [23] indicated that decreased miR-802 expression might play a critical role in the carcinogenesis and metastasis of pulmonary cells induced by long-term exposure to PM 2.5. In osteosarcoma, however, miR-802 expression was found to be remarkably up-regulated in tumor tissues [24]. Accordingly, it is necessary to further clarify the contribution of miR-802 in multiple malignancies in the near future.
EMT, a co-ordinated, organized program which facilitates epithelial cells to acquire invasive mesenchymal phenotype, is attracting increasing attention of cancer researchers as an important mechanism for the initial step of metastasis of cancer cells [25], and EMT might be an important therapeutic target in PCa [26]. Up to recent years, it has become increasingly apparent that miRNAs are critical regulators of a wide variety of biological processes, including EMT and tumorigenesis. It has been reported that miR-10b and miR-203 can promote or suppress cancer metastasis and invasion by modulating EMT [27,28]. The present study revealed that mesenchymal markers, such as N-cadherin and vimentin, were down-regulated, but epithelial cell markers, such as E-cadherin were up-regulated in PCa cells overexpressing miR-802, indicating that miR-802 suppresses EMT. Dysregulated activation of EMT in cancer might promote cancer cell migration, invasion, and ultimately lead to metastasis [29]. Consistently, our results demonstrated that up-regulation of miR-802 in PCa cells significantly inhibits cell proliferation, migration and invasion.
With bioinformatics prediction, it was identified that Flot2 is the theoretical target of miR-802. In turn, experimental validation demonstrated that Flot2 is a bona fide target of miR-802 in PCa cells. Flot2, a highly conserved 47-kDa protein located in a region on human chromosome 17q11.2, is originally characterized as an obbligato part of lipid raft domains that tether growth factor receptors linked to signal transduction pathways [30,31]. Recently, emerging evidence has demonstrated that Flot2, a downstream gene of p53 family members [32], might be involved in the progression of metastasis in several kinds of solid tumors, including gastric carcinoma [33], breast cancer [34], renal cell carcinoma [35] and lung adenocarcinoma [36]. Zhao et al. [37] recently suggested that in nasopharyngeal carcinoma, Flot2 is an indispensable member for transforming growth factor-β (TGF-β) family signaling, which epithelial cells receive from their micro-environment to drive EMT during cancer progression [38][39][40]. Therefore, we considered that decreased Flot2 expression induced by overexpressed miR-802 might be critical in suppressing PCa metastasis associated with TGF-β-mediated EMT.
To draw a conclusion, this might be the first study to identify miR-802 as a novel potential oncogene in PCa through induction of an EMT program. Our findings revealed that overexpression of miR-802 in PCa cells dramatically inhibits cell proliferation, migration and invasion in vitro, which raises the possibility for the development of miR-802 as a potent target for novel, promising therapies for the highly aggressive and malignant PCa in clinical application.