MicroRNA-19b promotes the migration and invasion of ovarian cancer cells by inhibiting the PTEN/AKT signaling pathway

Abstract

Local and systemic metastasis is the main reason for the poor survival rate of patients with ovarian cancer (OC). MicroRNAs (miRNAs/miRs) are short non-coding RNAs that serve critical roles in the initiation and progression of OC. The present study demonstrated that expression of miR-19b was significantly increased in OC tissues and cell lines. Analysis of clinicopathological features revealed that the increased expression of miR-19b was associated with advanced International Federation of Gynecology and Obstetrics stage and lymphatic metastasis of OC patients. Loss-of-function experiments demonstrated that the silencing of miR-19b reduced the migration and invasion of OVCAR-3 cells; contrarily, the overexpression of miR-19b facilitated the migration and invasion of CAOV-3 cells. Furthermore, miR-19b regulated the expression of phosphatase and tensin homolog (PTEN) and the activity of the PTEN/RAC serine/threonine-protein kinase pathway in vitro. Notably, the results of dual-luciferase reporter assays indicated that PTEN was a direct downstream target of miR-19b in OC. Taken together, the results of the current study demonstrated that miR-19b serves an oncogenic role in the progression of OC, and could potentially act as a biomarker and therapeutic target for OC patients.

Introduction

Ovarian cancer (OC), which is the one of the most fetal gynecologic cancer, is responsible for the highest percentage of incidences of cancer-associated mortality for women with gynecological cancer (1). In early-stage OC, no evident clinical symptoms can be readily observed in patients (2,3). There are also a lack of effective biomarkers for the early diagnosis of OC (4). Therefore, the majority of patients with OC already have late-stage disease when they are first diagnosed (5). The majority of patients with advanced-stage disease exhibit local and systemic metastases, which is behind the poor prognosis of patients with OC (5). Therefore, finding effective biomarkers for the early diagnosis of OC patients and the clarification of the molecular mechanisms driving the metastasis of OC is urgently required.

MicroRNAs (miRNAs/miRs) are short non-coding RNAs that post-transcriptionally modulate gene expression; they have been widely recognized as critical regulators of tissue- and disease-specific gene expression (6). Numerous studies into human cancer demonstrated that the aberrant expression and function of miRNAs served fundamental roles in the pathogenesis of malignant diseases (7,8). miRNAs were also found to be actively involved in the development and progression of OC (9,10), and could serve as diagnostic and prognostic markers, and attractive therapeutic targets of OC (11–13).

Of the numerous cancer-associated miRNAs, miR-19b was found to be an active participator in the pathogenesis of human cancer. miR-19b is overexpressed and has oncogenic roles in lung cancer (14,15), melanoma (16), breast cancer (17) and osteosarcoma (18). However, studies into gastric cancer found that miR-19b was downregulated, indicating that miR-19b may have a tumor-suppressive role in gastric cancer (19). Therefore, the role of miR-19b in human cancer seems to be cancer-type specific. However, to the best of our knowledge, the expression and biological function of miR-19b is yet to be clarified in patients with OC.

The present study demonstrated that the miR-19b expression level was significantly elevated in the clinical tissues of patients with OC and in OC cell lines. The elevated expression of miR-19b was associated with adverse clinical features in patients with OC. Functionally, miR-19b promoted the migration and invasion of OC cells. miR-19b could also directly regulate the expression of PTEN by interacting with the 3′-untranslated region (3′-UTR) of phosphatase and tensin homolog (PTEN).

Materials and methods

Clinical samples and cell culture

A total of 50 OC tissues and adjacent non-tumor tissues were obtained from patients who underwent surgical resection between January 2004 and December 2008 in the Department of Gynecology, Cangzhou Central Hospital (Cangzhou, China). The age range was 18–75 years with a median age of 50.6 years. Stage was retrospectively assessed for every patient based on a modified International Federation of Gynecology and Obstetrics (FIGO) staging system (20). No patients received chemotherapy before surgery. After written informed consent was obtained from every patient, the clinical samples were used for this study and were stored in liquid nitrogen. The protocol involving patients' samples in this study was approved by the Institutional Research Ethics Committee of Cangzhou Central Hospital. The clinicopathological characteristics of all enrolled patients were presented in Table I.

Table I.

Association between the clinicopathological characteristics and miR-19b expression in patients with ovarian cancer (n=50).

		miR-19b expression
Characteristics	Patients, n	High, n	Low, n	P-value
Pathological type
Mucinous	20	8	12	0.248
Serous	30	17	13
FIGO stage
I–II	32	12	20	0.018a
III–IV	18	13	5
Differentiation
Well/moderate	33	14	19	0.136
Poor	17	11	6
Lymphatic metastasis
No	38	16	22	0.047a
Yes	12	9	3

^aP<0.05. miR-19b, microRNA-19b; OC, ovarian cancer; FIGO, International Federation of Gynecology and Obstetrics.

The present study also used 5 human OC cell lines (CAOV3, SKOV-3, ES-2, HO-8910 and OVCAR3) and the immortalized human fallopian tube FTE187 epithelial cell line cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The OC cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.), 100 mg/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). FTE187 cells were kept in Medium 199 and MCDB105 medium (Sigma-Aldrich; Merck KGaA) containing 10% FBS and 10 ng/ml epidermal growth factor (Sigma-Aldrich; Merck KGaA). All cells were maintained at 5% CO₂ in a humidified atmosphere of 37°C.

Cell transfection

The expression vector of miR-19b (pCMV-miR-19b) and the corresponding control (pCMV-MIR), synthetic oligonucleotide against miR-19b (miR-19b inhibitors) and control oligonucleotide (negative control) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA). miR-19b expression vectors (400 ng) and miR-19b inhibitors (100 nM) were transfected into SKOV-3 and OVCAR-3 cells, respectively, using Lipofectamine 2000 following the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequent experiments were performed 48 h after transfection.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis

Isolation of the miRNA fraction from OC tissues and cells was performed using the Isolation of Small RNA kit (Macherey-Nagel GmbH, Düren, Germany). For miRNA analysis, RNA was reverse-transcribed using a TaqMan™ MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed using the Kapa Probe Fast qPCR Master Mix (Kapa Biosystems, Inc., Wilmington, MA, USA). The thermocycling conditions were as follows: Incubation at 95°C for 60 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec, as previously described (21). The primers used were as follows: Has-miR-19b (RT primer: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGTT-3′; forward, 5′-TGTGCAAATCCATGCAAAACTGA-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′), U6 forward, 5′-TCGGCAGCACATATACTAA-3′ and reverse, 5′-ATGGAACGCTTCACGAAT−3′. The relative expression of miR-19b was shown as fold difference relative to U6 using the 2^−ΔΔCq method (22).

Western blot analysis

Radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) was used to obtain cellular proteins, followed by quantification with a Bradford Protein assay kit (Beyotime Institute of Biotechnology). In total, 20–40 µg protein per lane was subjected to 10% SDS-PAGE and were transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% non-fat milk for 1 h at room temperature, then were incubated with primary antibodies against the following proteins: PTEN (cat no. sc-7974; 1:1,500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), RAC serine/threonine-protein kinase (AKT; cat no. 9272; 1:1,500), phosphorylated (p)-AKT (cat no. 4060; 1:500; both Cell Signaling Technologies, Inc., Danvers, MA, USA) and GAPDH (cat no. sc-47724; 1:1,500; Santa Cruz Biotechnology, Inc.) overnight at 4°C, and were then incubated with horseradish peroxidase-conjugated goat anti-rabbit and horse anti-mouse secondary antibodies for 1 h at room temperature (cat nos. 7074 and 7076; 1:2,000; Cell Signaling Technologies, Inc.). The proteins were visualized using the Super Signal West Femto kit (Thermo Fisher Scientific, Inc.) and the signal was detected using the Bio-Rad Gel Imaging system (Bio-Rad Laboratories, Inc, Hercules, CA, USA). Image J software (version 1.41; National Institutes of Health, Bethesda, MD, USA) was used to quantify the protein levels.

Wound-healing assay

For the wound-healing assay, OC cells transfected with different vectors were seeded into 6-well plates and cultured to confluence. Wounds were then made using a 100-µl pipette tip and the cells were incubated for 12 h. The width of the wound was visualized using phase-contrast microscopy (magnification, ×100) at 0 and 12 h after the wound was made.

Transwell assays

The migratory and invasive ability of OC cells were evaluated using Transwell assays. SKOV-3 and OVCAR-3 cells (1×10⁴) re-suspended in DMEM were seeded into the upper chamber of the Transwell inserts of 8-µm with (EMD Millipore, Billerica, MA, USA). The lower chamber contained 800 µl DMEM supplemented with 20% FBS. The upper chamber was coated with Matrigel when the invasion assays were performed, but not when the migration assay was performed. At 24 h after the cells were seeded, OC cells on the lower surface were fixed with 4% paraformaldehyde (10 min at room temperature) and stained with 0.1% crystal violet (20 min at room temperature), and the number of migrated and invaded cells were then counted in 5 fields under a light microscope (magnification, ×200).

Dual-luciferase reporter assay

OC cells were seeded in 6-well plates 1 day prior to transfection. These cells were transfected with a PTEN-3′-UTR vector (Promega Corporation, Madison, WI, USA) along with miR-19b mimic, miR-19b inhibitor or mutant miR-19b vector, and pRL-SV40 Renilla plasmid (Promega Corporation) using Lipofectamine 2000. At 48 h after transfection, the Dual-Luciferase Reporter Assay system (Promega Corporation) was used to measure the relative firefly and Renilla (normalization) luciferase activities of OC cells.

Statistical analysis

Data are presented as mean ± standard error of the mean. Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, Inc., USA). P<0.05 was considered to indicate a statistically significant difference. Comparisons between two groups were performed using unpaired Student's t-test; comparisons between multiple groups were performed using one-way analysis of variance with post hoc Tukey's test.

Results

miR-19b is upregulated in OC and is associated with adverse clinicopathological features of OC patients

The expression level of miR-216a in tissue samples from 50 patients with OC and matched non-tumor tissues was examined by RT-qPCR. The results of RT-qPCR analysis demonstrated that miR-19b was significantly overexpressed in OC tissues samples compared with the matched non-tumor tissues (P<0.05; Fig. 1A). The expression level of miR-19b in five OC cell lines and FTE187 cells was also examined. Compared with that in FTE187 cells, the expression of miR-19b in each of the five OC cell lines was significantly increased (P<0.05; Fig. 1B). Among OC cells lines, the expression level of miR-19b was highest in OVCAR-3 cells and was lowest in CAOV3 cells.

Figure 1.

Expression levels of miR-19b in OC tissues and cells. (A) The expression of miR-19b was compared between OC tissues and adjacent non-tumor tissues. (B) miR-19b expression level was compared between five different OC cell lines (CAOV3, SKOV-3, ES-2, HO-8910, and OVCAR-3) and FTE187 cells. *P<0.05. miR-19b, microRNA-19b; OC, ovarian cancer.

Next, whether the miR-19b expression level was associated with the clinicopathological features of OC patients was investigated. As shown in Table I, patients with high expression level of miR-19b had a significantly increased percentage to be at an advanced FIGO stage (P=0.018). Furthermore, increased miR-19b expression levels were significantly associated with the lymphatic metastasis of OC patients (P=0.047). These data indicated that an elevated miR-216a level is a promising biomarker for evaluating disease progression for OC patients.

miR-19b promoted the migration and invasion of OC cells

Following the observation of the significantly elevated level of miR-19b in OC samples and cell lines, the biological function of miR-19b was examined in OC cells. A miR-19b mimic and miR-19b inhibitor were transfected to increase and inhibit the miR-19b expression level, respectively, in SKOV-3 and OVCAR-3 cells. The migratory and invasive behavior of OC cells were also examined following alteration the expression of miR-19b in OC cells. First, transfection with the miR-19b inhibitor significantly reduced the expression level of miR-19b in OVCAR-3 cells (P<0.05; Fig. 2A). The results of the wound-healing assay revealed that the migration of OVCAR-3 cells was significantly inhibited following inhibition of miR-19b expression (P<0.05; Fig. 2B). Furthermore, the results of the Transwell assay demonstrated that the migration and invasion of OVCAR-3 cells were significantly inhibited following downregulation of miR-19b (P<0.05; Fig. 2C).

Figure 2.

Inhibition of miR-19b decreases the migration and invasion of ovarian cancer cells. (A) miR-19b inhibitor significantly reduced the expression level of miR-19b in OVCAR-3 cells. (B) Downregulation of miR-19b significantly decreased the migration of OVCAR-3 cells. (C) Inhibition of miR-19b significantly decreased the migration and invasion of OVCAR-3 cells. *P<0.05. miR-19b, microRNA-19b; NC, negative control.

Transfection of the miR-19b expression vector into CAOV-3 cells significantly increased the expression level of miR-19b (P<0.05; Fig. 3A). Subsequently, the migration of CAOV-3 cells was significantly increased (P<0.05; Fig. 3B and C), as was the invasion of CAOV-3 cells (P<0.05; Fig. 3C). These data indicated that miR-19b could increase the migration and invasion of OC cells.

Figure 3.

Overexpression of miR-19b increases the migration and invasion of ovarian cancer cells. (A) miR-19b mimic significantly increased the expression level of miR-19b in CAOV-3 cells. (B) Overexpression of miR-19b significantly increased the migration of CAOV-3 cells. (C) Overexpression of miR-19b significantly increased the migration and invasion of CAOV-3 cells. *P<0.05. miR-19b, microRNA-19b.

miR-19b directly modulates the expression of PTEN and the PTEN/AKT pathway in OC cells

Next the underlying mechanisms responsible for the functional influence of miR-19b on OC cells were examined. Numerous studies confirmed that PTEN and downstream AKT signaling served a notable role in regulating the metastasis of OC cells (23–25). Therefore, whether miR-19b could modulate the expression of PTEN and affect the PTEN/AKT pathway was examined. The results of western blot analysis revealed that overexpression of miR-19b significantly reduced the expression of PTEN in CAOV-3 cells (P<0.05; Fig. 4A). The phosphorylation of AKT, which was under the control of PTEN (26), was significantly increased (P<0.05; Fig. 4A). Inhibition of miR-19b in OVCAR-3 cells significantly increased the expression of PTEN (P<0.05; Fig. 4B) and decreased the phosphorylation of AKT (P<0.05; Fig. 4B). These results indicated that miR-19b could directly regulate the activity of the PTEN/AKT signaling pathway.

Figure 4.

miR-19b inhibits the expression of PTEN in ovarian cancer cells. (A) Results of western blot analysis for PTEN and p-AKT in CAOV3 cells transfected with miR-19b mimics. (B) Western blot results of PTEN and p-AKT in OVCAR-3 cells transfected with miR-19b inhibitor. *P<0.05. miR-19b, microRNA-19b; PTEN, phosphatase and tensin homolog; p-AKT, phosphorylated RAC serine/threonine-protein kinase; NC, negative control.

miR-19b modulates the expression of PTEN by interacting with its 3′-UTR

Following confirmation of the fact that miR-19b could inhibit the expression of PTEN, whether miR-19b could modulate the expression of PTEN by interacting with the 3′-UTR of PTEN was investigated. As shown in Fig. 5A, the 3′-UTR of PTEN contained the complementary sequence for the seed region of miR-19b, indicating that miR-19b could potentially interact with the 3′-UTR of PTEN. A luciferase assay was performed to confirm the interaction between miR-19b and the 3′-UTR of PTEN. Overexpression of miR-19b significantly decreased the luciferase activity of PTEN 3′-UTR (P<0.05; Fig. 5B), whereas inhibition of miR-19b significantly reduced the lucifease activity of PTEN 3′-UTR (P<0.05; Fig. 5B). Next, the mutant miR-19b was transfected into OC cells, which did not affect the luciferase activity of the PTEN 3′-UTR reporter (Fig. 5B). Taken together, these data indicated miR-19b could modulate the expression of PTEN by directly interacting with the 3′-UTR of PTEN.

Figure 5.

miR-19b interacts with the 3′-UTR of PTEN. (A) miR-19b and its complementary sequence in the 3′-UTR of PTEN. (B) The effects of miR-19b mimic, miR-19b inhibitor and mutant miR-19b vector on the luciferase activity of 3′-UTR of PTEN. *P<0.05 vs. miR-control, ^†P<0.05 vs. miR-19b, ^#P<0.05 vs. NC, ^@P<0.05 vs. anti-miR-19b. miR-19b, microRNA-19b. miR-19b, microRNA-19b; PTEN, phosphatase and tensin homolog; 3′-UTR, 3′-untraslated region.

Discussion

Local and systemic metastasis is the main reason for the unsatisfactory survival of OC patients (27). The underlying mechanisms for the metastasis of OC cells are complicated and remain largely unknown (27). miRNAs has been found to serve critical roles in the development and progression of OC (28), and can serve as promising biomarkers and therapeutic targets for OC patients (10).

Of the numerous cancer-associated microRNAs, miR-19b was found to trigger the epithelial-mesenchymal transition in lung cancer cells and could inhibit the growth of lung cancer cells (15). A study into melanoma revealed that miR-19b could regulate human telomerase reverse transcriptase expression and cell proliferation via inhibition of paired-like homeodomain 1 (16). miR-19b also promoted tumor growth and metastasis by targeting tumor protein p53 (29). The present study demonstrated that miR-19b was significantly overexpressed in OC tissues and cell lines. Notably, the increased expression of miR-19b was associated with unfavorable clinical features, including lymphatic metastasis and advanced FIGO stage. Therefore, the results of the present study indicate that miR-19b serves an oncogenic role in OC.

Increased migratory and invasive ability is a hallmark of human cancer (30). miRNAs have been found to be important regulators of metastasis in human cancer (31). The present study used gain- and loss-of-function methods and confirmed that overexpression of miR-19b could increase the migratory and invasive ability of OC cells, whereas the inhibition of miR-19b could decrease it. These data indicated that the miR-19b could promote the progression of OC by potentiating the metastatic ability of OC cells.

PTEN, a tumor suppressor, serves a notable role in the pathogenic processes of OC (32). The present study confirmed that miR-19b could inhibit the expression of PTEN by directly interacting with the 3′-UTR of PTEN. First, overexpression of miR-19b significantly decreased the expression of PTEN, whereas miR-19b inhibition significantly increased it. Second, the complementary sequences of miR-19b were observed in the 3′-UTR of PTEN. Third, altering the expression level of miR-19b could significantly influence the luciferase activity of 3′-UTR of PTEN, whereas the mutant miR-19b vector had no obvious effect on the luciferase activity of 3′-UTR of PTEN. These data indicate that PTEN is a direct downstream target of miR-19b in OC, and that miR-19b could modulate the PTEN/AKT pathway by interacting with the 3′-UTR of PTEN.

In conclusion, the results of the present study demonstrated that miR-19b expression was significantly elevated in OC tissues and cells. The high expression level of miR-19b was associated with poor clinicopathological features of OC patients. Functionally, miR-19b can potentiate the migratory and invasive behaviors of OC cells. Furthermore, the current study confirms that miR-19b could directly modulate the PTEN expression by interacting with the 3′-UTR of PTEN.

Funding

No funding was received.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

DTL conceived and designed the experiments. DTL, HRY, YYL and YYS performed the experiments. DTL and MYS analyzed the data. DTL wrote the paper. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All procedures performed in studies involving human participants were in accordance with the ethical standards of the Institutional Research Ethics Committee of Cangzhou Central Hospital and with the 1964 Helsinki declaration and its later amendments. All written informed consent to participate in the study was obtained from patients with ovarian cancer for samples to be collected from them.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.