MicroRNA‑15a‑5p‑targeting oncogene YAP1 inhibits cell viability and induces cell apoptosis in cervical cancer cells.

MicroRNAs (miRNAs) have been reported to have important regulatory roles in the progression of several types of cancer, including cervical cancer (CC). However, the biological roles and regulatory mechanisms of miRNAs in CC remain to be fully elucidated. The aim of the present study was to examine the functions of miRNAs in CC and the possible mechanisms. Using a microarray, it was identified that miRNA‑15a‑5p (miR‑15a‑5p) was one of the most downregulated miRNAs in CC tissues compared with adjacent noncancerous tissues. The low expression of miR‑15a‑5p was observed in CC tumor tissues with distant metastasis and in CC cell lines. In addition, the effects of miR‑15a‑5p upregulation on cell viability, apoptosis, invasion and migration of CC cells were investigated using CCK‑8, flow cytometry, Transwell and wound healing assays, respectively. It was demonstrated that upregulation of miR‑15a‑5p significantly suppressed the viability, migration and invasion, and promoted the apoptosis of SiHa and C‑33A cells. Furthermore, yes‑associated protein 1 (YAP1), a well‑known oncogene, was confirmed to be directly targeted by miR‑15a‑5p and was found to be negatively regulated by miR‑15a‑5p. Further correlation analysis indicated that miR‑15a‑5p expression was negatively correlated with YAP1 expression in CC tissues. Notably, overexpression of YAP1 abrogated the tumor suppressive effects of miR‑15a‑5p in CC cells. Taken together, these present findings indicated that the miR‑15a‑5p/YAP1 axis may provide a novel strategy for the clinical treatment of CC.

Introduction

Cervical cancer (CC) is a type of malignant tumor, commonly presenting in women (1-3). In 2012, ~500,000 CC cases are diagnosed each year and it accounts for 7.5% of all female cancer-associated mortalities each year worldwide (4). Despite advances in the therapeutic strategies for CC, including targeted therapies and immunotherapy, the prognosis of CC remains poor due to the abnormal growth of epithelial cells (1,5). Thus, it is imperative to clarify the molecular interactions occurring during the initiation and progression of CC.

MicroRNAs (miRNAs) are a family of short, non-coding RNAs, with an average length of 22 nucleotides, which negatively regulate target gene expression through either translation repression or RNA degradation (6,7). Accumulating evidence has indicated that miRNAs may function as oncogenes or tumor suppressors, depending on their target mRNA, in various types of cancer, including CC (8-11). For example, Yang et al (12) reported that miR-214 inhibits the growth of CC cells by the regulation of its target, enhancer of zeste homolog 2 (12). Dong et al (13) demonstrated a suppressive role of miR-217 in the development of CC cells via targeting Rho-associated protein kinase 1 (13). Chen et al (14) reported that miR-499a promotes the proliferation, cell cycle progres-sion, colony formation, migration and invasion of CC cells by targeting SRY-box transcription factor 6. In addition, several miRNAs serve as diagnostic biomarkers in patients with CC, such as miR-152 and miR-365 (15,16). Despite the aforementioned findings, the roles of miRNAs in the development of CC require further investigation.

In the present study, a miRNA microarray was performed to investigate the expression profiles of miRNAs in CC tissues, and the most downregulated miRNA identified, miR-15a-5p, was selected for further analysis. The potential role and underlying mechanism of miR-15a-5p in CC cells were also investigated. The present results suggest that miR-15a-5p may serve as a therapeutic target for CC.

Materials and methods

Patients and samples

In total, 40 paired cervical samples (tumor tissues and adjacent noncancerous tissues) were obtained from female patients with CC who underwent cervical surgical resection without preoperative systemic therapy at the Department of Obstetrics and Gynecology, Huashan Hospital North of Fudan University (Shanghai, China) between May 2016 and December 2017. The median age of the patients was 51 years (range, 42-68 years). Among all patients, there were 20 patients with metastatic CC and 20 with non-metastatic CC. The matched non-tumor adjacent tissue was obtained 3 cm beyond the boundary of CC tissue. All tissue samples were immediately snap-frozen in liquid nitrogen and stored at −80°C until use. The experimental protocols were approved by the Ethics Committee of Huashan Hospital North of Fudan University. Written informed consent for participation in the study was obtained from all patients.

miRNA expression profiling

Total RNA from CC tissues (three randomly selected paired tumor tissues and adjacent noncancerous tissues) was extracted using miRNeasy mini kit (Qiagen GmbH). The samples were assessed using the miRCURY LNA™ Array v18.0 (Agilent Technologies, Inc.). The procedure and imaging processes were performed as described previously (17).

Cell culture

Human CC cell lines (HeLa, C-33A, CaSki and SiHa), 293T cells and normal cervical epithelial cells Ect1/E6E7 were obtained from the American Type Culture Collection. All cells were cultured in DMEM (Sigma-Aldrich; Merck KGaA) supplemented with 10% (v/v) FBS (Sigma-Aldrich; Merck KGaA) plus 100 U/ml penicillin/streptomycin at 37°C with 5% CO2.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from tissues or cell lines using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). For miRNA RT, cDNA was generated from 10 ng total RNA samples using TaqMan™ MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) at 42°C for 60 min. For mRNA RT, cDNA was synthesized using PrimeScript RT reagent kit (Takara Bio, Inc.) at 42°C for 60 min. qPCR for miRNA and mRNA was performed using the SYBR-Green I Real-Time PCR kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) on an ABI 7300 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction was performed under the following conditions: 95°C for 5 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 50 sec, and a final extension at 72°C for 20 sec. The primers for qPCR analysis were as follows: miR-15a-5p forward 5′-AAT GTT GCC CGT AAT GCC-3′ and reverse, 5′-CCC AAG CGG AGA AAG GAA-3′; U6 forward, 5′-GCT TCG GCA GCA CAT ATA CTA AAA T-3′ and reverse, 5′-CGC TTC ACG AAT TTG CGT GTC AT-3′; yes-associated protein 1 (YAP1) forward, 5′-CGG TCC ACT TCA GTC TCC-3′ and reverse, 5′-GAG TGT GGT GGA CAG GTA CTG-3′; and GAPDH forward, 5′-GTG GTG AAG ACG CCA GTG GA-3′ and reverse, 5′-CGA GCC ACA TCG CTC AGA CA-3′. The expression levels of miR-15a-5p and YAP1 were normalized to the expression of U6 and GAPDH, respectively. The relative expression of each gene was calculated using the 2−∆∆Cq method (18).

Cell transfection

The miR-15a-5p mimic, mimic negative control (NC), miR-15a-5p inhibitor, inhibitor NC, YAP1 overexpression plasmid (pcDNA-YAP1) and pcDNA-vector were all provided by Guangzhou RiboBio Co., Ltd. When C-33A and SiHa cells (5×105 cells/well) in 6-well plates grew to ~80% confluence, miR-15a-5p mimic (20 nM), mimic NC (20 nM), miR-15a-5p inhibitor (20 nM), inhibitor NC (20 nM), pcDNA-YAP1 (2 µg) or pcDNA-vector (2 µg) were transfected into cells at 37°C for 24 h using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The sequences were as follows: miR-15a-5p mimic, 5′-UAG CAG CAC AUA AUG GUU UGU G-3′; mimic NC, 5′-UUC UCC GAA CGU GUC ACG UTT-3′; miR-15a-5p inhibitor, 5′-CAC AAA CCA UUA UGU GCU GCU A-3′; and inhibitor NC, 5′-CAG UAC UUU UGU GUA GUA CAA-3′.

In addition, small interfering RNA targeting YAP1 (si-YAP1) and the negative control targeting a non-specific sequence (si-Scramble) were provided by Thermo Fisher Scientific, Inc. SiHa and C-33A cells were transfected with the siRNAs (100 nmol/l) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The sequences of si-YAP1 and si-Scramble were as follows: si-YAP1, 5′-CTC AGG ATG GAG AAA TTT A-3′; and si-Scramble, 5′-TTC TCC GAA CGT GTC ACG T-3′. At 24 h post-transfection, the cells were harvested for further analysis, and the inhibition efficiency was deter-mined by western blotting.

Cell viability

The C-33A and SiHa cells were seeded in 96-well plates at a density of 5×103/well overnight. Following transfection, the cell viability was measured using a CCK-8 assay. Briefly, 10 µl CCK-8 solution was added to each well and cultured for 4 h at 37°C. The absorbance of the samples at 490 nm was detected using a microplate reader (Bio-Rad Laboratories, Inc.).

Caspase-3 activity

Following transfection, C-33A and SiHa cells were harvested and the caspase-3 activity was measured using a Caspase-3 Activity assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol.

Cell apoptosis

The apoptosis of C-33A and SiHa cells was examined using flow cytometry. Following transfection, C-33A and SiHa cells were collected and the apoptotic cells were identified using an Annexin V-FITC Apoptosis Detection kit (Abcam) according to the manufacturer's protocol. After washing with cold PBS, the cells were resuspended in binding buffer followed by staining with Annexin V and propidium iodide for 15 min in the dark at room temperature. The fluorescence was measured using a FACScan flow cytometer (Beckman Coulter, Inc.) and then analyzed by FlowJo v8.7.1 software (FlowJo LLC).

Immunofluorescence assay

Following transfection, C-33A and SiHa cells were fixed in absolute ethyl alcohol for 30 min at room temperature. After washing twice with PBS, the fixed cells were stained with primary antibody targeting cleaved-caspase-3 (1:1,000; cat. no. 9664; Cell Signaling Technology, Inc.) for 1 h at room temperature. Subsequently, an anti-rabbit conjugated antibody with FITC (cat. no. F0382; 1:100; Sigma-Aldrich; Merck KGaA) was added for 2 h in the dark. Fluorescence images were obtained using an inverted fluorescence microscope (magnification, ×200).

Cell invasion assays

Transwell chambers (8-µm pore; BD Biosciences) coated with Matrigel (BD Biosciences) were used for the invasion assay. Briefly, C-33A and SiHa cells (8×104) were seeded in the top chamber with serum-free medium, while the lower chamber contained culture medium with 20% FBS. Following incubation for 24 h, the cells were fixed in 4% paraformaldehyde solution (Beyotime Institute of Biotechnology) for 30 min and stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) for 15 min at room temperature. Images were captured with an inverted microscope (Olympus Corporation; magnification, ×100).

Wound healing assay

For the wound healing assay, C-33A and SiHa cells were seeded onto 12-well plates (2×105 cells/well), and 24 h after transfection, a scratch was made using a 10-µl pipette tip in the confluent cell monolayer. Then, cells were washed twice with PBS and incubated in DMEM without FBS. The wound healing images were captured at 0 and 48 h after scratching using an inverted light microscope (Olympus Corporation; magnification, ×100). The wound healing rate was calculated using ImageJ software (v1.46; National Institutes of Health).

Dual-luciferase reporter assay

miRNA target prediction tools, including MiRanda (http://miranda.org.uk) and TargetScan 7.0 (http://targetscan.org/), were used to search for the putative targets of miR-15a-5p. pGL3-YAP1 wide-type or pGL3-YAP1 mutant type (pGL3-YAP1-mut) (Promega Corporation) were co-transfected with miR-15a-5p mimics into 293T cells in 24-well plates (2×105/well) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 24 h post-transfection, the luciferase activities were analyzed using the Dual-Luciferase Reporter assay system (Promega Corporation) with Renilla luciferase activity as an internal control.

Western blot analysis

Western blotting was performed as previously described (19). Briefly, cells were lysed using radio immunoprecipitation assay buffer (Beyotime Institute of Biotechnology) and the protein concentration was determined using the bicinchoninic acid assay. Total protein (40 µg/lane) was separated by 8% SDS-PAGE and electrophoretically transferred onto a polyvinylidene difluoride membrane (EMD Millipore). Subsequently, membranes were blocked with 5% skim milk for 2 h at 4°C overnight. Each membrane was probed with primary antibodies against YAP1 (1:1,000; cat. no. 14074) and β-actin (1:2,000; cat. no. 4970) at 4°C overnight. All primary antibodies were obtained from Cell Signaling Technology, Inc. Subsequently, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000; cat. no. 205718; Abcam) at room temperature for 1 h. β-actin served as the loading control and for normalization of protein expression. The protein bands were developed using ECL kit (GE Healthcare) and expression levels were quantified using ImageJ (v1.46; National Institutes of Health).

Statistical analysis

All data are presented as mean ± standard deviation. The correlation between miR-15a-5p and YAP1 levels was evaluated using Spearman's correlation analysis. Pairwise comparisons were performed by Student's t-test, and comparisons among groups were analyzed by one-way ANOVA followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

miR-15a-5p is downregulated in CC

To examine the potential involvement of miRNAs in the development of CC, microarray analysis was performed to evaluate the miRNA expression profiles between CC tissues and adjacent noncancerous tissues. Of 54 differently expressed miRNAs identified in the tumor group, 27 miRNAs exhibited decreased expression and 27 miRNAs demonstrated increased expression compared with that in adjacent noncancerous tissues (Fig. 1A). Among the aberrant miRNAs, the present study focused on miR-15a-5p for subsequent experiments due to its suppressive role in a variety of other cancer types, such as endometrial cancer and chronic myeloid leukemia (20,21).

Figure 1

miR-15a-5p is downregulated in CC tissues and cell lines. (A) Heat map presents significant differentially expressed 54 miRNAs in CC tissues and matched adjacent noncancerous tissues (n=3). Green indicates downregulation and red indicates upregulation. (B) miR-15a-5p expression was measured by RT-qPCR in 40 pairs of CC tissues and matched adjacent noncancerous tissues. (C) miR-15a-5p expression was measured in 20 tumor tissues with distant metastasis and 20 tumors tissues without distant metastasis by RT-qPCR. (D) miR-15a-5p expression was detected in four cervical cancer cell lines (HeLa, C-33A, CaSki and SiHa) and the normal cervical epithelial cells Ect1/E6E7. Data are expressed at the mean ± standard deviation (n=3) of one representative experiment. **P<0.01 vs. Ect1/E6E7 cells. miR, microRNA; CC, cervical cancer; RT-qPCR, reverse transcription-quantitative PCR.

Subsequently, RT-qPCR was performed to detect the expression of miR-15a-5p in 40 pairs of tumor tissues and adjacent noncancerous tissues. The results revealed that the level of miR-15a-5p was significantly lower in tumor tissues compared with that in adjacent noncancerous tissues (Fig. 1B). It was also observed that miR-15a-5p was expressed at a significantly lower level in tumor tissues with distant metastasis compared with in tumors tissues without distant metastasis (Fig. 1C), indicating that miR-15a-5p downregulation is associated with CC metastasis. In addition, RT-qPCR was used to examine the miR-15a-5p level in four CC cell lines (HeLa, C-33A, CaSki and SiHa) and the normal cervical epithelial cell line Ect1/E6E7, which was used as a control. As expected, miR-15a-5p was significantly lower in the four CC cell lines compared with Ect1/E6E7 cells (Fig. 1D). SiHa and C-33A cells were selected for further experiments as they demonstrated the lowest expression of miR-15a-5p among all cell lines examined.

Upregulation of miR-15a-5p inhibits cell viability and promotes cell apoptosis

In an attempt to understand the biological function of miR-15a-5p, miR-15a-5p expression was upregulated or downregulated in the cultured SiHa and C-33A cells by transfection with miR-15a-5p mimic or inhibitor, respectively. miR-15a-5p expression was significantly increased after miR-15a-5p mimic transfection, whereas it was significantly decreased following miR-15a-5p inhibitor transfection in both SiHa and C-33A cells (Fig. 2A). The present study then investigated the effect of miR-15a-5p expression on cell viability and the results demonstrated that the viability of SiHa and C-33A cells was significantly inhibited by overexpression of miR-15a-5p, whereas it was significantly enhanced by knockdown of miR-15a-5p compared with the negative control group (Fig. 2B and C). To assess the effects of miR-15a-5p upregulation on the apoptosis of SiHa and C-33A cells, caspase-3 expression level and activity were analyzed by immunofluorescence and Caspase-3 Activity assays, respectively. As presented in Fig. 2D and E, the expression of cleaved caspase-3 and caspase-3 activity was increased in SiHa and C-33A cells transfected with miR-15a-5p mimic, compared with the mimic NC groups. Furthermore, the results of flow cytometry demonstrated that the extent of apoptosis was significantly increased after miR-15a-5p mimic transfection compared with the mimic NC groups (Fig. 2F). Taken together, these results indicate that overexpression of miR-15a-5p inhibits cell viability by inducing cell apoptosis.

Figure 2

Overexpression of miR-15a-5p suppresses cell viability and promotes cell apoptosis. SiHa and C-33A cells were transfected with the miR-15a-5p mimic or inhibitor for 48 h, and then cells were used for analysis. (A) Transfection efficiency was assessed by reverse transcription-quantitative PCR. Cell viability was measured by CCK-8 assay at indicated times for (B) SiHa and (C) C-33A cells. (D) The expression of cleaved caspase-3 was determined by immunofluorescence assay. Magnification, ×200. (E) The caspase-3 activity was detected by a commercial caspase-3 activity kit. (F) Cell apoptosis was measured by flow cytometry. Data are expressed at the mean ± standard deviation (n=3) of one representative experiment. *P<0.05, **P<0.01 vs. mimic NC. #P<0.05, ##P<0.01 vs. inhibitor NC. miR, microRNA; NC, negative control; OD, optical density; PI, propidium iodide.

Upregulation of miR-15a-5p inhibits the invasion and migration of CC cells

The present study further investigated whether overexpression of miR-15a-5p could reduce the invasiveness and migratory potential of CC cells. Using a Transwell assay, it was identified that the invasive capacities of SiHa and C-33A cells were significantly inhibited by miR-15a-5p mimic, whereas they were increased by miR-15a-5p inhibitor compared with the NC groups. Furthermore, the wound healing assay results also demonstrated a significant reduction of cell migration in SiHa and C-33A cells following miR-15a-5p overexpression. However, the migration of SiHa and C-33A cells was significantly enhanced by miR-15a-5p inhibition (Fig. 3C and D). Collectively, the present data suggest that overexpression of miR-15a-5p suppresses the invasive and migratory abilities of CC cells.

Figure 3

Overexpression of miR-15a-5p suppresses cell invasion and migration. SiHa and C-33A cells were transfected with the miR-15a-5p mimic or inhibitor for 48 h, and then cells were used for analysis. Invasion of (A) SiHa and (B) C-33A cells was measured by a Transwell assay. Magnification, ×200. The migration of (C) SiHa and (D) C-33A cells was assessed by a wound healing assay. The images were taken at 0 and 48 h after gaps were generated. Wound healing was quantified by the distance of the wounded region with an absence of cells. Data are expressed at the mean ± standard deviation (n=3) of one representative experiment. **P<0.01 vs. mimics NC. ##P<0.01 vs. inhibitor NC. miR, microRNA; NC, negative control.

YAP1 is a direct target of miR-15a-5p

Using the TargetScan 7.0 and miRanda algorithms, YAP1 was found to have a putative target site of miR-15a-5p in its 3′-UTR (Fig. 4A). To validate the possibility that YAP1 is a direct target gene of miR-15a-5p, a luciferase reporter assay was then performed. The data revealed that miR-15a-5p mimic significantly inhibited the luciferase activity in the constructs containing the wild-type binding site of YAP1-3′UTR, while it had no evident effects on the activity of YAP1-3′UTR-Mut. By contrast, miR-15a-5p inhibitor significantly increased luciferase activity, without any evident effects on YAP1-3′UTR-Mut activity (Fig. 4B). Subsequently, to further detect the potential regulation of YAP1 by miR-15a-5p, the expression of YAP1 protein was measured in CC cells by western blotting. As presented in Fig. 4C, the expression of YAP1 was significantly decreased upon ectopic expression of miR-15a-5p, suggesting that high expression of YAP1 was partly due to the downregulation of miR-15a-5p in CC cells. In addition, it was identified that the mRNA level of YAP1 was significantly increased in cervical cancer compared with the control, and inversely correlated with miR-15a-5p expression levels in cancer tissues (Fig. 4D and E). These results indicated that YAP1 is a down-stream gene of miR-15a-5p in CC.

Figure 4

YAP1 is a direct target of miR-15a-5p. (A) Schematic of the YAP1 3′UTR containing the miR-15a-5p binding sites. (B) Luciferase assay of 293T cells co-transfected with firefly luciferase constructs containing the YAP1 wt or mut 3′-UTRs and miR-15a-5p mimics, mimics NC, miR-15a-5p inhibitor or inhibitor NC, as indicated (n=3). **P<0.01. (C) SiHa and C-33A cells were transfected with the miR-15a-5p mimic and mimic NC for 48 h, and the expression levels of YAP1 protein were determined by western blotting. **P<0.01 vs. mimic NC. (D) YAP1 expression was measured by reverse transcription-quantitative PCR in CC tissues and matched adjacent noncancerous tissues (n=40). **P<0.01. (E) Spearman's analysis was used to analyze the correlation between the expression of YAP1 and the expression of miR-15a-5p expression in cervical cancer tissues. (r = -0.8195; P<0.01). Data are expressed at the mean ± standard deviation (n=3) of one representative experiment. YAP1, yes-associated protein 1; miR, microRNA; 3′UTR, 3′-untranslated region; wt, wild-type; mut, mutant; NC, negative control.

YAP1 inhibition suppresses cell viability, promotes cell apoptosis and inhibits invasion and migration

Previous evidence has shown that YAP1 exerts an oncogenic function in several types of human cancer, such as breast and lung cancer (22,23). As the findings of the present study revealed that YAP1 is upregulated in CC, it was hypothesized that YAP1 may act as an oncogenic gene in CC. To confirm this hypothesis, SiHa and C-33A cells were transfected with si-YAP1 or si-Scramble. Western blot assay revealed that YAP1 was notably downregulated following transfection with si-YAP1 (Fig. 5A). Functionally, YAP1-knockdown significantly suppressed the cell viability and induced cell apoptosis compared with the si-Scramble group (Fig. 5B and C). Furthermore, knockdown of YAP1 significantly suppressed the invasive and migratory abilities of SiHa and C-33A cells (Fig. 5D and E), suggesting that YAP1 may play an oncogene role in the development of CC.

Figure 5

YAP1 inhibition suppresses cell viability, promotes cell apoptosis and inhibits invasion and migration. SiHa and C-33A cells were transfected with si-YAP1 or si-Scramble, and then cells were harvested for further study. (A) The expression of YAP1 was measured by western blotting. (B) Cell viability was measured by CCK-8 assay. (C) The cell apoptosis was assessed by flow cytometry. (D) Cell invasion was measured by Transwell assay. (E) Cell migration assessed by a wound healing assay. Data are expressed at the mean ± standard deviation (n=3) of one representative experiment. **P<0.01 vs. si-Scramble. YAP1, yes-associated protein 1; miR, microRNA; si, small interfering RNA.

Overexpression of YAP1 moderates the negative functions of miR-15a-5p on cell viability, migration and invasion

To ascertain whether YAP1 is involved in the inhibitory effects of miR-15a-5p on CC cells, the present study co-transfected pcDNA-YAP1 and/or miR-15a-5p mimic, as well as their controls into SiHa and C-33A cells. The overexpression efficiency was verified by western blotting. As shown in Fig. 6A, YAP1 was notably increased in SiHa and C-33A cells after pcDNA-YAP1 transfection. Subsequently, the cell viability, apoptosis, invasion and migration were evaluated. Overexpression of YAP1 significantly abolished the inhibitory effects of miR-15a-5p upregulation on the viability of SiHa and C-33A cells (Fig. 6B). The increased apoptosis induced by miR-15a-5p overexpression was also reversed by overexpression of YAP1 (Fig. 6C). Furthermore, overexpression of YAP1 significantly reversed the inhibitory effects of miR-15a-5p on cell invasion and migration (Fig. 6D and E). In addition, it was identified that overexpression of YAP1 alone significantly promoted CC cell viability, inhibited cell apoptosis, and enhanced the invasion and migration compared with blank control group, suggesting the oncogenic role of YAP1 in CC cells. These results indicate that miR-15a-5p exerts its tumor suppressive role in CC at least partially through YAP1.

Figure 6

miR-15a-5p inhibits cell viability and induces cell apoptosis by targeting YAP1. (A) SiHa and C-33A cells were transfected with the pcDNA-YAP1 plasmid for 48 h and then the protein expression of YAP1 was measured by western blotting. Subsequently, SiHa and C-33A cells were co-transfected with the pcDNA-YAP1 plasmid and miR-15a-5p mimic for 48 h, and then cells were used for analysis. (B) Viability of SiHa and C-33A cells was measured by CCK-8 assay at indicated times. (C) The cell apoptosis was assessed by flow cytometry. (D) Cell invasion was measured by Transwell assay. (E) Cell migration was measured by a wound healing assay. Data are expressed at the mean ± standard deviation (n=3) of one representative experiment. *P<0.05, **P<0.01 vs. blank group. ##P<0.01. miR, microRNA; YAP1, yes-associated protein 1.

Discussion

In the present study, miR-15a-5p was shown to be decreased in CC tissues and cell lines, and associated with CC metastasis. Furthermore, overexpression of miR-15a-5p inhibited the CC cell viability, invasion and migration, and promoted cell apoptosis, while inhibition of miR-15a-5p demonstrated the opposite effects. Additionally, YAP1 was confirmed as a functional target of miR-15a-5p, ectopic expression of which significantly reversed suppression of miR-15a-5p. The present data indicated that miR-15a-5p may function as a tumor suppressor in CC progression by inhibiting YAP1 expression.

A number of studies have shown that miRNAs participate in the development of CC (24,25). For example, Xia et al (26) reported that miR-374b overexpression suppresses cell proliferative and invasive abilities via affecting forkhead box M1 expression. Yao et al (27) also demonstrated that miR-641 upregulation restricts CC cell growth in vitro and in vivo. Xu et al (28) reported that miR-218-5p suppresses the progression of CC via the LYN/NF-κB signaling pathway. Yuan et al (29), demonstrated that overexpression of miR-138 suppresses CC cell growth in vivo. These findings suggest that targeting miRNAs may be an effective therapeutic strategy for CC. In the present study, based on microarray expression data, it was identified that miR-15a-5p is one of the most markedly downregulated miRNAs in CC tissues. Notably, previous studies have reported that miR-15a-5p functions as a tumor suppressor in several human cancer types (20,30). Although miR-15a-5p has been found to be downregulated in CC (31), to the best of our knowledge, the tumorigenic role and mechanism remain unknown. Therefore, the present study focused on miR-15a-5p in CC for molecular analyses. In the microarray expression data, the expression levels of numerous miRNAs exhibited significant changes, such as miR-137, which demonstrated the most significant upregulation in CC tissues. Miao et al (32) reported that miR-137 upregulation inhibits CC cell invasion, migration and epithelial-mesenchymal transition by suppressing the TGF-β/SMAD pathway. Notably, miR-15a-3p has also reported to exhibit differential expression and induce apoptosis in human CC cells (33,34). Although the present study did not detect the expression change of miR-15a-3p in the microarray expression data, the expression of miR-15a-3p in four CC cell lines was examined and the results demonstrated that miR-15a-3p was also down-regulated in CC cells compared with Ect1/E6E7 cells (data not shown). However, the role and regulatory mechanisms of miR-15a-3p on invasion and migration remain unclear. The function of more miRNAs in CC will be investigated in the future.

Previous studies have reported that miR-15a-5p has the potential to suppress cell growth and inhibit the progression of human cancers by regulating its downstream target genes (35,36). For example, Luo et al (37) demonstrated that overexpression of miR-15a-5p causes cellular growth inhibition and suppression of migration by targeting cyclin E1 in breast cancer. Wu et al (38) and Guo et al (39) found that miR-15a overexpression suppressed the cell proliferation and invasion by suppression of Bmi-1 translation in gastric cancer (GC) as well as pancreatic cancer (PC). Of note, several studies have reported aberrant expression of miR-15a-5p in CC tissues or cells (31,33); however, the role and mechanism of miR-15a-5p in CC remain largely unknown. The present results demonstrated that overexpression of miR-15a-5p inhibited cell viability, cell migration and invasion, and induced cell apoptosis in SiHa and C-33A cells, while inhibition of miR-15a-5p demonstrated the opposite effects, indicating that miR-15a-5p may serve as tumor suppressive role in CC.

YAP1, a transcriptional coactivator and oncogene, has been found to play an important role in different types of carcinoma (40-43). For example, Liu et al (44) reported that YAP1 overexpression promotes the invasion, migration and growth of colon cancer cells. Yu et al (45), demonstrated that knockdown of YAP1 causes a significant inhibition of the growth and migration of renal cell carcinoma cells in vitro and in vivo. Notably, YAP1 has been verified to target miR-15a-5p to suppress cell growth and metastasis in gastric adenocarcinoma (46) and colon cancer (47). However, whether YAP1 is a target of miR-15a-5p in CC remains unclear. In the present study, YAP1 was confirmed to be a target of miR-15a-5p and its protein expression levels were negatively regulated by miR-15a-5p. Further investigation indicated that YAP1 was significantly increased in CC tissues, and inversely correlated with miR-15a-5p in CC tissues. Furthermore, YAP1 was confirmed to act as an oncogene gene in CC cells, and its overexpression partly abrogated the inhibitory effect induced by enhanced expression of miR-15a-5p in CC cells. Taken together, the present study demonstrates that miR-15a-5p exerts its tumor suppressive role in CC cells by targeting YAP1.

Due to the limitation in experimental conditions and funds, further research in the future is required to investigate whether miR-15a-5p serves its role via other downstream targets. In addition, the present study investigated the cellular function of miR-15a-5p and its underlying mechanism in CC, however in vivo studies and clinical trial data are required to validate the preliminary in vitro results obtained. Therefore, the function of miR-15a-5p in CC needs to be further investigated in vivo.

In conclusion, the present results demonstrated that miR-15a-5p suppresses the viability, migration and invasion of CC cells by directly targeting YAP1. Based on these findings, it is proposed that the miR-15a-5p/YAP1 axis may serve as a novel biomarker for new targets in CC therapy.