Effects of miR-200c on the migration and invasion abilities of human prostate cancer Du145 cells and the corresponding mechanism

Abstract

microRNAs (miRNAs) have played a key role in human tumorigenesis, tumor progression, and metastasis. On the one hand, miRNAs are aberrantly expressed in many types of human cancer; on the other hand, miRNAs can function as tumor suppressors or oncogenes that target many cancer-related genes. This study aimed to investigate the effects of miRNA-200c (miR-200c) on the biological behavior and mechanism of proliferation, migration, and invasion in the prostate cancer cell line Du145. In this study, Du145 cells were transfected with miR-200c mimics or negative control miR-NC by using an X-tremeGENE siRNA transfection reagent. The relative expression of miR-200c was measured by RT-PCR. The proliferation, migration, and invasion abilities of Du145 cells were detected by CCK8 assays, migration assays and invasion assays, respectively. The expressions of ZEB1, E-cadherin, and vimentin were observed by western blot. Results showed that DU145 cells exhibited a high expression of miR-200c compared with immortalized normal prostate epithelial cell RWPE-1. Du145 cells were then transfected with miR-200c mimics and displayed lower abilities of proliferation, migration, and invasion than those transfected with the negative control. The protein levels of ZEB1 and vimentin were expressed at a low extent in Du145 cells, which were transfected with miR-200c mimics; by contrast, E-cadherin was highly expressed. Hence, miR-200c could significantly inhibit the proliferation of the prostate cancer cell line Du145; likewise, miR-200c could inhibit migration and invasion by epithelial-mesenchymal transition.

Keywords

miR-200c / proliferation / migration / invasion / prostate cancer / Du145 cell / ZEB1

Introduction

Prostate cancer (CaP) is the second leading cause of cancer associated with 193 000 deaths in 2009 in the United States; among affected individuals, approximately 27 000 are noted to succumb to this disease later. Furthermore, one out of six men is possibly at risk of CaP during their lifetime. Prostate tumors are influenced by various genetic and epigenetic factors, such as age, race, heredity, diet, sexual frequency, and physical activity [ 1].

MicroRNAs (miRNAs) are widely observed as small non-coding RNAs with a length of 18 to 24 nucleotides; miRNAs play a major regulatory part in a broad range of biological processes and complex diseases. Since the discovery of miRNAs [ 2], miRNAs have modulated mRNA levels and translation by canonical base pairing between the seed sequences of miRNAs (nucleotides 2 to 8 at the 5′ end) and the complementary seed match sequences of target mRNAs, which are typically located in the 3′ untranslated region (UTR) [ 3]. miRNAs are essential in metastasis possibly because these miRNAs direct cell invasion, motility, and migration in post-transcriptional gene-regulated networks [ 4]. The miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) is known as the main suppressor of epithelial-to-mesenchymal transition (EMT), a reversible embryonic program aberrantly activated in tumor progression and metastasis [ 5]. One of the validated targets of the miR-200 family is TUBB3 (class III β-tubulin) [ 6]. In the post-transcriptional inhibition of TUBB3 gene expression, miR-200c is distributed in Hey ovarian cancer cells and HeC50 endometrial cancer cells. The forced expression of miR-200c in HeC50 cells and in a xenograft model reverses resistance to chemotherapy, suggesting that miR-200c is a factor that protects against tumor aggressiveness and provides chemoresistance [ 7, 8]. The molecular mechanisms by which miR-200c modulates translation remain unclear, although miR-200c regulates numerous genes encoding proteins implicated in carcinogenesis. EMT involves the molecular reprogramming of cells that gradually lose their epithelial characteristics as they acquire mesenchymal attributes; such attributes promote cellular detachment from primary tumors, stimulate adjacent stroma invasion, direct entry into systemic circulation, and induce extravasation [ 9- 14].

In the present study, the in vitro effects of miR-200c upregulation on the proliferation and invasion of CaP cells were investigated to identify a novel relationship between E-cadherin and miR-200c in CaP. Hurteau, Chen, and Wang et al. [ 15- 17] found striking links between miR-200c levels and E-cadherin expression. These data suggested that miR-200c may be a novel marker of CaP prognosis.

Materials and methods

Materials

Human CaP Du145 cell line and non-transformed prostate epithelial cell line RWPE-1 were purchased from the Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS), RPMI 1640 media, and K-SFM media were purchased from Gibico Biotech. A reverse transcription kit in a first-strand cDNA synthesis kit was purchased from the Fermentas Company. YBR Premix Ex Taq^TM II was purchased from Takara, Japan. A CCK-8 kit was purchased from Dojindo Co. Trizol RNA extraction reagent was purchased from Invitrogen Corp. GAPDH and ZEB-1 primers were purchased from Yingjun Biotech Company, Wuhan. miR-200c primers, U6 primers, miR-200c mimics, and miR-NC were purchased from Ruibo Biotech Co., Guangzhou, China. X-tremeGENE siRNA transfection reagent was purchased procured from Roche. The primary antibody of GAPDH and secondary antibodies were purchased from Boshide Biotech Company; and the primary antibodies of E-cadherin (#3195), vimentin (#5741), and ZEB-1 (#3396) were purchased from Cell Signaling Technology Co., USA.

Methods

Cell culture

Du145 cells and RWPE-1 cells were grown in RPMI l640 culture medium containing 10% heat-activated fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. These cells were incubated in a humidified incubator at 37 °C with 5% CO₂ and passaged every 2 d to 3 d by digesting with 0.25% trypsin. All of the experiments were performed using the cells in log phase.

Cell transfection

The cells were trypsinized and seeded in 96-well plates (5×10⁴ cells/well) at 37 °C and exposed to 5% CO₂. miR-NC or miR-200c mimics were mixed with transfection reagents when cell density reached 40% confluency in accordance with the manufacturer’s instructions; after 15 min, the resulting mixture was placed at room temperature and mixed in culture supernatant slowly and evenly. This procedure was continuously performed to generate cells used for further experiments.

CCK-8 assay

After the cells were transfected for 12 h, they were trypsinized and seeded in 96-well plates at 5×10⁴ cells/well and incubated for 24, 48, and 72 h. Untreated cells were used as a control sample. Approximately 10 μl of CCK-8 (Dojindo, Tokyo, Japan) solutions in a culture medium were then added to each well. Plates were incubated for another 2 h. The optical density (OD) of each well was determined using a microplate absorbance reader at a wavelength of 450 nm. Cell viability was calculated using the following equation: Cell survival rate (%) = [(A_{treatment group}–A_{blank wells}) / (A_{negative control group}–A_{blank wells})] × 100%. The experiment was repeated thrice.

Real-time fluorescent quantitative polymerase chain reaction (qPCR)

Total RNA extraction and reverse transcription were performed in strict accordance with the manufacturer’s instructions. Reactions were set on a 96-well plate by mixing 2 μl of cDNA, 1 μl of each primer, and 12.5 μl of SYBR mix; ddH₂O was added to obtain a final volume of 20 μl. GAPDH was used as an internal reference. The thermal cycling conditions were set as follows: reaction systems were amplified and detected by 40 thermo cycles at 95 °C for 15 s and 60 °C for 20 s; the reactions were extended at 70 °C for 20 s. The forward and reverse primer sequences of the ZEB1 transcript were 5′-AACGGAAACCAGGATGAAAG-3′ and 5′-TTGTCACACAGGTCACATGC-3′, respectively; the forward and reverse primer sequences of the GAPDH transcript were 5′-CAAGTTCAAGTGCACGGAGT-3′ and 5′-CAAGTTCAAGTGCACGGAGT-3′, respectively. After amplification was performed, standard curves were constructed and used in conjunction with the cycle threshold (Ct) value of each sample to calculate the mRNA expression of the studied genes; these genes were then normalized by dividing each mRNA value by the GAPDH value of the same sample. The experiment was repeated thrice.

Western blot

The cells were transfected for 48 h, mixed with NP-40, and centrifuged at 12 000 × g for 15 min at 4 °C. The supernatants were aliquoted and stored at -80 °C after protein concentration was determined. Protein samples were treated at 100 °C for 5 min, separated in 10% sodium dodecyl sulfate-polyacrylamide gels, and transferred onto polyvinylidene fluoride membranes. These membranes were blocked with Tris-buffered saline (TBS) containing 0.05% Tween 20 and 1% non-fat milk for 2 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: ZEB-1 (1:1000 dilution); E-cadherin (1:1000 dilution); vimentin (1:2000 dilution), and GAPDH (1:500 dilution). Immunoblots were washed with TBS containing 0.05% Tween-20 and incubated with secondary antibodies conjugated with horseradish peroxidase anti-mouse IgG or anti-rabbit IgG (Boshide Biotech Company, Wuhan, China) for 1 h at room temperature. Immunoreactive proteins were visualized using an ECL detection system. The experiment was repeated thrice.

Migration and invasion assay in vitro

Migration assay was performed using Transwell inserts containing 6.5 mm polycarbonate membranes with 8.0 µm pores. Matrigel invasion assay was conducted using membranes coated with Matrigel matrix. Homogeneous single cell suspensions (2×10⁵ cells/well) were added to the upper chambers and allowed to invade for 24 h at 37 °C in a CO₂ incubator. Migrated or invaded cells were stained with 0.1% crystal violet for 15 min at room temperature and examined by ordinary optical light microscope. Migrated or invaded cells were quantified according to the following criteria: the small membrane at the bottom of the chamber was artificially divided into nine equal parts by using an ordinary optical microscope and four edge frames and one center frame were selected to count the number of cells under each view; the average was then obtained. The experiment was repeated thrice.

Bioinformatics prediction

We used the bioinformatics software of TargetScan, Miranda, and PicTar to predict the target genes of miR-200c. The selection criteria of the candidate target genes were listed as follows: (1) genes exhibit species conservatism and (2) the second to eighth nucleotide sequences (“seed region”) of the 5′ end of the miR-200c sequence and the 3′ UTR of a candidate target gene show fully complementary pairing and the free energy between the two sequences was relatively low.

Statistical analysis

At least five independent experiments were completed for each analysis described in this article. Data were shown as mean ± standard deviation (SD). Paired analysis and multiple group comparison were performed by conducting Student’s t-test and one-way ANOVA, respectively, using SPSS 18.0. P < 0.05 was considered statistically significant.

Results

Detection of miR-200c expression in the cell lines

Compared with the human non-transformed prostate epithelial cell line RWPE-1, the CaP cell line Du145 exhibited a significantly reduced miR-200c expression (Fig. 1). This result suggested that miR-200c may play a very important role in CaP.

Detection of expression in transfected cells

miR-200c and miR-NC (negative vector) were transfected in the Du145 cells; after 48 h, total RNA was extracted. The expressions of miR-200c and ZEB1 in the cells were detected by qPCR. U6 and GAPDH were used as references of miR-200c and ZEB1, respectively. The results showed that the relative expression of the miR-200c group was significantly higher than that of the negative group (12.74398 ± 2.4387, P < 0.05). The relative expression of ZEB1 was 0.24 ± 0.094 (P < 0.05; Fig. 2). These results indicated that miR-200c may regulate the expression of ZEB1 on a transcriptional level.

miR-200c suppressed the proliferation ability of CaP Du145 cells

At the indicated time points, the ODs of the miR-200c transfected group were significantly lower than those of the miR-NC (negative vector) group (Fig. 3A). This result indicated that miR-200c could significantly suppress the proliferation of CaP Du145 cells.

miR-200c attenuated the in vitro migration and invasion abilities of CaP Du145 cells

In the Transwell migration assay, CaP Du145 cells stably transfected with the precursor of miR-200c mimics (34.345 ± 6.238, P < 0.05) presented an impaired migration ability compared with those transfected with miR-NC (Fig.4). In the Matrigel invasion assay, miR-200c mimics (51.279 ± 11.338, P < 0.05) attenuated the invasiveness of Du145 cells (Fig. 3B). The results of in vitro migration and invasion assays showed that miR-200c could significantly suppress the migration and invasion abilities of CaP Du145 cells.

EMT analysis

miR-200c and miR-NC (negative vector) were transfected in Du145 cells; total protein was extracted after 48 h. Western blot result indicated that the expressions of ZEB1 and vimentin were reduced when miR-200c was transfected in the cells; on the contrary, the expression of E-cadherin was increased. This experiment confirmed that miR-200c functioned as a tumor suppressor gene of CaP and participated in the regulatory mechanisms of proliferation, migration, and invasion of CaP cells (Fig. 4).

Prediction of the target genes

We predicted the target genes of miR-200c by using TargetScan, Miranda, and PicTar. We found that locus 5 of the 3′ UTR in ZEB1 could be combined with miR-200c (Fig. 5). A previous study also revealed that ZEB1 is a direct target gene of miR-200c in breast cancer and colon cancer [ 18, 19].

Discussion

miRNAs, small non-coding RNAs of 18 to 24 nucleotides in length, play an important role in gene regulation in animals, plants, and viruses [ 20, 21]. miRNAs regulate gene expression by targeting mRNAs for either cleavage or translational repression through base pairing with them at partially or fully complementary sites [ 22]. Studies have found that miRNAs contribute to the regulation of oncogenes and tumor suppressor genes, as well as tumorigenesis and human cancer development [ 23]. miR-200c is a member of the miRNA-200 family, which also comprises miR-200a, miR-200b, miR-141, and miR-429; miR-200c is located on human chromosome 12 [ 24]. Studies have demonstrated that miR-200c performs an oncogenic or tumor suppressor role in different kinds of cancers, including prostate cancer. For instance, Duk-Soo Bae et al. [ 25] found that miR-200c expression is increased in endometrial cancer and promotes cancer by transcriptionally inhibiting the BRD7 gene. Yuichiro Doki et al. [ 26] further demonstrated that patients with esophageal cancer manifested by high miR-200c expression are resistant to chemotherapy. By contrast, miR-200c functions as a tumor suppressor by inhibiting EMT in colon cancer, bladder cancer, breast cancer, and other tumors [ 18, 27, 28]. Banyard and Kim [ 29, 30] found that miR-200c functions as a tumor suppressor in prostate cancer; however, ZEB1 as a direct target of miR-200c in prostate cancer has not been indicated. Hurteau et al. [ 31] indicated that the overexpression of miR-200c can lead to an increase in E-cadherin expression in estrogen receptor-positive breast cancer but not in prostate cancer. Kong et al. [ 32] found that the family of miR-200, particularly miR-200b, can regulate EMT, adhesion, and invasion of CaP cells.

The E-box binding protein (ZEB) family comprises zinc finger transcription factors that are essential for embryonic development [ 33]. The ZEB family of transcription factors plays an important role in tumorigenesis. Among the members of this family, ZEB1 interacts with the E2 box [CACCT(G)] of the E-cadherin promoter, resulting in the transcriptional inhibition of E-cadherin and induction of EMT; as such, cell invasion and tumor metastasis are likely enhanced [ 34]. Target prediction software has shown that miR-200c interacts with several locations on the 3′ UTR of ZEB1, thereby inhibiting EMT by post-transcriptionally inhibiting the expression of ZEB1 [ 35, 36].

In this study, the expression of miR-200c in the prostate cancer cell line Du145 was significantly reduced compared with that in the non-transformed prostate epithelial cell line RWPE-1. Thus, miR-200c may be a key factor involved in tumor suppression. Indeed, the proliferation of cells transfected with miR-200c mimics was significantly reduced compared with that of the negative control Du145 cells, which were transfected with miR-NC. Likewise, transwell experiments demonstrated that the migration and invasion abilities of Du145 cells were evidently reduced after the cells were transfected with miR-200c mimics. This result is consistent with the significantly increased expression of E-cadherin; by contrast, the expressions of mesenchymal markers, namely, ZEB1 and vimentin, were significantly reduced. Therefore, miR-200c functioned as a tumor suppressor of CaP. Indeed, miR-200c participated in the negative regulation of proliferation, migration, and invasion of CaP cells. Preliminary results also indicated that miR-200c-mediated anticancer activities in CaP cell line occurred by post-transcriptionally inhibiting ZEB1, upregulating cell adhesion molecule expression, restraining EMT, and subsequently impeding cell migration and invasion.

In summary, miRNAs act as oncogenes in some tumors; in other cases, miRNAs function as tumor suppressors. Therefore, miRNAs could be effectively applied in anticancer therapy. Thus far, studies have demonstrated that specific miRNAs can be injected in animal models to reduce tumor burden and inhibit metastasis [ 37]. In this study, miR-200c elicited an inhibitory effect on CaP cell line Du145. Thus, a potential measure was developed to treat CaP; indeed, miR-200c could be used to develop new biotherapies to treat CaP.

Compliance with ethics guidelines

Runlin Shi, Haibing Xiao, Tao Yang, Lei Chang, Yuanfeng Tian, Bolin Wu, and Hua Xu declare that they have no conflicts of interest. This article does not contain experiments with human or animal subjects performed by any of the authors.

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