MicroRNA-454-3p inhibits cervical cancer cell invasion and migration by targeting c-Met.

Increasing evidence has demonstrated that microRNAs (miRNAs) have a crucial role in the initiation and progression of tumors. The present study aimed to investigate the expression and the role of miRNA-454-3p in human cervical cancer. Human cervical cancer cells were transfected with miRNA-454-3p mimics or negative control miRNA. MTT, Transwell and wound healing assays were performed to investigate the effects of miRNA-454-3p overexpression on cell proliferation, invasion and migration, respectively. The results indicated that miRNA-454-3p was down-regulated in human cervical cancer cell lines, while its ectopic overexpression significantly inhibited their proliferation, migration and invasion. Furthermore, a luciferase reporter assay confirmed that c-met was a novel target of miRNA-454-3p in HeLa cells. In conclusion, the results of the present study suggested that miRNA-454-3p exhibits significant tumor-suppressive effects in cervical cancer by targeting c-met, and may be a potential means of treating cervical cancer.

Introduction

Based on the GLOBOCAN estimates for 2012, cervical cancer affects ~527,600 individuals per year worldwide, it is also the leading cause of cancer-associated death among females in less developed countries (1). In spite of developments in radiotherapy, chemotherapy and surgery for the treatment of cervical cancer, the 5-year survival rates for patients with cervical cancer at stages III and IV remain <40% (2). Hence, it is urgent to explore the underlying molecular mechanisms of the initiation and progression of cervical cancer and identify potential therapeutic strategies.

Increasing evidence has demonstrated that microRNAs (miRNAs), which are non-coding RNAs of 18–22 nucleotides in length (3), are involved in the regulation of numerous cellular processes, including cell proliferation, apoptosis, invasion as well as migration (4). miRNAs post-transcriptionally regulate gene expression by base-pairing with the 3′-untranslated regions (3′-UTRs) of their target mRNAs (5). Recent studies have indicated that miRNAs act as tumor suppressors or oncogenes in numerous cancer types, and the aberrant expression of miRNAs is associated with the genesis and progression of tumors (6,7).

Previous studies have indicated that miRNA-454-3p functions as a tumor-suppressive or oncogenic miRNA in various types of cancer, including non-small cell lung cancer (8), glioblastoma (9), hepatocellular carcinoma (10) and colorectal cancer (11). For instance, Zhou et al (12) demonstrated that miRNA-454-3p was upregulated in hepatocellular carcinoma tissues and is correlated with a low 5-year overall survival. On the contrary, overexpression of miRNA-454-3p significantly inhibited the proliferation and cell cycle progression of human glioblastoma cells (9). However, the expression and the biological roles of miRNA-454-3p in cervical cancer have remained elusive.

The present study determined the expression of miRNA-454-3p in three human cervical cancer cell lines and investigated the effects of miRNA-454-3p on the proliferation, migration as well as invasion of human cervical cancer cells. The results demonstrated that miRNA-454-3p was downregulated in human cervical cancer cells, while its ectopic overexpression significantly inhibited the proliferation, migration and invasion of human cervical cancer cells.

Materials and methods

Cell culture

The C33A, SiHa and HeLa human cervical cancer cell lines as well as H8 normal cervical cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Science (Shanghai, China). All cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; GE Healthcare, Little Chalfont, UK) and cultured at 37°C in a humidified incubator containing 5% CO2.

Transfection

miRNA-454-3p mimics and negative control miRNA (NC) were synthesized by GenePharma (Shanghai, China). The 6 h transient transfection was performed in HeLa cells using Lipofectamine 2000® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

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

Total RNA was isolated with an RNeasy Mini kit (Qiagen, Hilden, Germany) and reverse-transcribed using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). qPCR was performed to determine the expression of gene and miRNA. The primers used were as follows: miR-454-3p forward, 5′-ACCCTATCAATATTGTCTCTGC-3′ and reverse, 5′-GCGAGCACAGAATTAATACGAC-3′; and U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. U6 small nuclear RNA was used as endogenous control for miRNA analysis. Additionally, the Taq DNA polymerase (cat. no. 10342053; Thermo Fisher Scientific, Inc.) was used in PCR reaction. The thermocycling conditions were as follows: 97°C for 5 min, 95°C for 30 sec, 65°C for 30 sec and 75°C for 90 sec for 33 cycles. The PCR product was subsequently stored at 4°C for the following experiments. The comparative 2−∆∆Cq method was used for relative quantification and statistical analysis (13).

MTT assay

To investigate the effect of miR-454-3p on the viability of HeLa cells, the MTT assay was performed with a Cell proliferation Kit I (GE Healthcare) according to the manufacturer's protocol. Cell viability was determined at an absorbance at 570 nm by a VersaMax (Molecular Devices, Sunnyvale, CA, USA).

Invasion assay

The invasive capacity of HeLa cells was assessed using 24-well Transwell plates (cat. no. 3071528; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). For the invasion assay, 1×105 HeLa cells were suspended in serum-free DMEM and then seeded into the upper chamber of each insert coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA), while the lower chamber was filled with DMEM supplemented with 20% FBS. After 24 h of incubation, cells on the bottom side of the membrane were fixed with 4% polyoxymethylene for 15 min and then stained with 0.1% crystal violet dye for 10 min at 37°C and finally visualized using a light microscope (Leica Microsystems, Wetzlar, Germany). Three independent experiments were performed.

Wound healing assay

The wound healing assay was performed to assess cell migration. In brief, HeLa cells seeded in six-well plates (8×105/per well). After 24 h, the cell monolayer was scraped with a sterile micropipette tip to create separate wounds, and the wells were washed with PBS to remove cell debris. Representative images at 48 h after wounding were captured under a light microscope (Leica Microsystems). Three independent experiments were performed.

Western blot analysis

Total protein was extracted from HeLa cells using a radioimmunoprecipitation buffer (cat. no. 20101ES60; Shanghai Qcbio Science & Technologies Co., Ltd., Shanghai, China) at 96 h after transfection and the protein concentration was measured using a BCA kit (cat. no. 20201ES76; Shanghai Qcbio Science & Technologies Co., Ltd.). A total of 2 µg protein/per lane was separated by 10% SDS-PAGE followed by transfer onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Inc.). After blocking with 5% non-fat dried milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h, the membranes were incubated with primary antibodies overnight at 4°C and further incubated with secondary antibody. The anti-c-Met (cat. no. AF1432; 1:1,000), -p-Akt (cat. no. AA331; 1:1,000), -MMP-2 (cat. no. AF1420; 1:10,000) and -GAPDH (cat. no. AF1186; 1:10,000) primary antibodies were purchased from Beyotime Institute of Biotechnology, Haimen, China. While the anti-MMP-9 (cat. no. ab76003; 1:10,000) primary antibody and goat anti-rabbit IgG H&L horseradish peroxidase-conjugated secondary antibodies (cat. no. ab205718; 1:2,000) were purchase from Abcam, Cambridge, MA, USA. Bands were visualized using an enhanced chemiluminescence kit (cat. no. 32209; Suzhou Biotsith Bioscience Co., Ltd., Suzhou China). The images were captured by an ChemiDoc™ XRS imaging system (Bio-Rad Laboratories, Inc.) and analyzed by ImageJ software version 1.8 (National Institutes of Health, Bethesda, MD, USA).

Luciferase reporter assay

The miRNA-454-3p, c-Met 3′-UTR-WT and MUT plasmids were constructed by Biomics Biotechnologies Co., Ltd. (Nantong, China). The constructed plasmids (100 ng) were transfected with HeLa cells (106) using Lipo6000™ (0.2 µl; Beyotime Institute of Biotechnology) and incubated at 37°C with 5% CO2 for 6 h in 96-well plates. The 3′UTR was from the mRNA sequence of c-Met and the transfected cells were subsequently cultured for 72 h prior to the luciferase activity measurement. A luciferase assay kit (cat. no. RG005; Beyotime Institute of Biotechnology) was used to measure the reporter activity according to the manufacturer's protocol. The luciferase activity was normalization to Renilla luciferase activity.

Statistical analysis

Values are expressed as the mean ± standard deviation. All statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Comparisons between groups were performed by using one-way analysis of variance followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

miRNA-454-3p is downregulated in human cervical cancer cells

The present study determined the expression of miR-454-3p in three human cervical cancer cell lines and normal cervical H8 cells (Fig. 1). Compared with the H8 normal cervical cell line, the expression of miRNA-454-3p was significantly downregulated in the three human cervical cancer cell lines. In addition, miRNA-454-3p levels in the HeLa cell line were obviously decreased compared with those in the C33A and SiHa cell lines. Therefore, the HeLa cell line was selected for further investigation of the role of miRNA-454-3p in cervical cancer.

Figure 1.

miRNA-454-3p is downregulated in human cervical cancer cell lines. miRNA-454-3p expression levels were detected by reverse-transcription quantitative polymerase chain reaction analysis with U6 used as loading control. Values are expressed as the mean ± standard deviation. **P<0.01; ***P<0.001, compared with H8 cells. miRNA, microRNA.

miRNA-454-3p decreases the proliferation of HeLa cells

As presented in Fig. 2A, after 6 h transfection with miRNA-454-3p mimics, a significant increase in the expression levels of this miRNA was observed (P<0.001), while there was no clear difference between the NC and BL groups. To evaluate the effects of miRNA-454-3p on the proliferation of human cervical cancer cells, HeLa cells were transfected with miRNA-454-3p mimics and the amount of viable cells was determined by an MTT assay. The results demonstrated that transfection with miRNA-454-3p mimics decreased the cell viability in comparison with that in the NC and BL groups (P<0.001; Fig. 2B).

Figure 2.

Overexpression of miRNA-454-3p inhibits the proliferation of HeLa cells. (A) After transfection, the expression of miRNA-454-3p was determined by reverse-transcription quantitative polymerase chain reaction analysis. (B) Cell proliferation in different groups was detected by an MTT assay. ***P<0.001, compared with the NC group. Groups: BL, blank/untransfected group; NC, cells transfected with negative control miRNA; mimic, cells transfected with miRNA-454-3p mimics. miRNA, microRNA.

miRNA-454-3p inhibits invasion and migration of HeLa cells

Next, to investigate the effect of miRNA-454-3p on the invasion and migration of human cervical cancer cells, miRNA-454-3p mimics-transfected HeLa cells were subjected to Transwell and wound healing assays. As illustrated in Figs. 3 and 4, overexpression of miRNA-454-3p induced by transfection of miRNA-454-3p mimics significantly reduced the invasion (Fig. 3) and migration (Fig. 4) of human HeLa cells, compared with that in the BL or NC groups (P<0.05).

Figure 3.

Overexpression of miRNA-454-3p suppresses the invasion of HeLa cells. (A-C) Representative invasion images (magnification, ×200) for (A) BL, (B) NC and (C) Mimics groups. (D) Invasion rate of HeLa cells in each group. ***P<0.001, compared with NC group. Groups: BL, blank/untransfected group; NC, cells transfected with negative control miRNA; mimic, cells transfected with miRNA-454-3p mimics. miRNA, microRNA.

Figure 4.

Overexpression of miRNA-454-3p suppresses cell migration of HeLa cells. (A and B) Representative images of wound healing assay for different groups. (C) Wound healing rate in each group. Magnification, ×200. ***P<0.001, compared with NC group. Groups: BL, blank/untransfected group; NC, cells transfected with negative control miRNA; mimic, cells transfected with miRNA-454-3p mimics. miRNA, microRNA.

miRNA-454-3p directly targets c-Met 3′-UTR

Analysis with the predictive database TargetScan (http://www.targetscan.org/) suggested that c-Met is a putative target of miRNA-454-3p. A luciferase reporter assay was performed to confirm whether miRNA-454-3p directly targets c-Met (Fig. 5A), and the results are presented in Fig. 5B. Compared with the NC group, co-transfection with miRNA-454-3p mimics significantly decreased the luciferase activity of wild-type hc-Met-3′-UTR luciferase vector in HeLa cells (P<0.01). No obvious influence was observed on mutant hc-Met-3′-UTR luciferase activity after miRNA-454-3p transfection (Fig. 5B). Furthermore, compared with those in the blank and NC groups, the protein levels of c-Met were significantly decreased after transfection of miRNA-454-3p (Fig. 5C and D). Taken together, these results suggest that c-Met is a direct target of miR-454-3p in cervical cancer. In addition, the expression of downstream proteins of c-Met, namely Akt, matrix metalloproteinase (MMP-2) and MMP-9, was detected by western blot analysis. As presented in Fig. 5C and E-G, after transfection of miRNA-454-3p, the protein levels of phosphorylated (p)-Akt, MMP-2 and MMP-9 were significantly downregulated compared with those in the blank and NC groups.

Figure 5.

miRNA-454-3p directivity targets c-Met in HeLa cells. (A) The WT or MUT binding sequences of miRNA-454-3p on c-Met 3′-UTR. (B) Luciferase reporter assay results displaying the effect of miRNA-454-3p on c-Met 3′-UTR. (C) Representative western blot image and (D-G) quantified protein levels of (D) c-Met, (E) p-Akt, (F) MMP-2 and (G) MMP-9 in HeLa cells depending on miRNA-454-3p overexpression. ***P<0.001, compared with NC group. Groups: BL, blank/untransfected group; NC, cells transfected with negative control miRNA; mimic, cells transfected with miRNA-454-3p mimics. miRNA, microRNA. WT, wild-type; MUT, mutated; UTR, untranslated region; p-Akt, phosphorylated Akt; MMP, matrix metalloproteinase; hsa, Homo sapiens.

Discussion

Numerous studies have demonstrated that certain miRNAs have important roles in various cancer types. In the present study, the expression and the biological role of miRNA-454-3p in human cervical cancer cells was investigated. The results demonstrated that miRNA-454-3p was downregulated in human cervical cancer cell lines compared with that in a normal cervical cell line. Overexpression of miRNA-454-3p by transfection of miRNA-454-3p mimics significantly inhibited cell proliferation, and suppressed the migration and invasion of human cervical cancer cells, which is consistent with the results of previous studies on glioblastoma (9) and osteosarcoma (14). In addition, the luciferase reporter assay further indicated that c-Met is a direct target of miRNA-454-3p.

A large body of evidence has proven that c-Met is upregulated in several cancer types, including gastric (15), non-small-cell lung (16) and cervical cancer (17). In addition, previous studies demonstrated that overexpression of c-Met has as a significant prognostic value in early-stage invasive (18) and local-regional advanced cervical cancer patients (19). c-Met, acts as an oncogene, and has been widely documented to promote cell proliferation, migration and invasiveness (20). In the present study, miRNA-454-3p was proven to directly target c-Met, and the overexpression of miRNA-454-3p greatly suppressed c-Met expression in human cervical cancer cells. In addition, western blot analysis indicated that after transfection of miRNA-454-3p, the protein levels of downstream effectors of c-Met, including p-Akt, MMP-2 and MMP-9, were significantly upregulated compared with those in the control and blank groups. Collectively, the present study illustrated that miRNA-454-3p suppressed cell proliferation, cell migration and invasion, at least in part due to targeting c-Met, which led to the downregulation of p-Akt, MMP-2 and MMP-9.

In conclusion, the present study revealed that miRNA-454-3p was downregulated in human cervical cancer cell lines, while ectopic overexpression of miRNA-454-3p suppressed cell proliferation, migration and invasion, at least partially by targeting c-Met. These results indicate that miRNA-454-3p may be a potential target for the treatment of cervical cancer.