MicroRNA-217 functions as a tumor suppressor in cervical cancer cells through targeting Rho-associated protein kinase 1.

The abnormal expression of microRNAs (miRNAs/miRs) has been widely reported in various tumor types. miR-217 was demonstrated to be aberrantly expressed in a number of tumors, including pancreatic adenocarcinoma and osteosarcoma; however, its specific expression pattern has never been investigated in cervical cancer cells. Compared with normal control, the level of Rho-associated protein kinase 1 (ROCK1) expression was markedly increased in cervical cancer tissues and cells compared with that in non-cancerous tissues and cells. The expression of miR-217 was significantly reduced in cervical cancer tissues and cell lines. Overexpression of miR-217 could suppress colony formation and the cell invasion capacity of SiHa and HeLa cells. Flow cytometry indicated that miR-217 significantly increased cell apoptosis in SiHa and HeLa cells. Dual-luciferase reporter assays demonstrated that ROCK1 was a target gene of miR-217. In addition, overexpression of ROCK1 also led to an increased invasion capacity in SiHa cells, even when miR-217 was inhibited, indicating that the anti-invasive effects of miR-217 were mediated through ROCK1. In summary, the results of the present study indicated that miR-217 functions as a tumor suppressor in cervical cancer cells, primarily by targeting ROCK1.

Introduction

Cervical cancer (CC) accounts for the second most common cancer among women around the world, with an estimated incidence of 5 million newly diagnosed female patients and a mortality incidence of ~2 million annually (1,2). Evidence has indicated that the development of novel diagnostic and therapeutic methods has markedly improved the life quality of a number of patients; however, the molecular etiology of CC remains largely unknown (3–5).

MicroRNAs (miRNAs/miRs) are a class of small single-stranded non-coding RNAs of ~22 nucleotides (6). miRNAs are reported to be widely involved in multiple biological processes, including cellular proliferation, differentiation, migration and invasion, primarily through binding the 3′-untranslated region (3′UTR) of target mRNAs, inhibiting their translation (7–9). The altered expression of miRNAs was found to be associated with the progression of various tumors, including pancreatic, lung and breast cancer (10–12). miR-25-3p was reported to target Sema4C in CC cells thereby regulating the epithelial-mesenchymal transition (1). miR-143 was determined to be negatively associated with CC cell proliferation and apoptosis (13). miR-217 was demonstrated to be aberrantly expressed in a number of tumor types, including pancreatic adenocarcinoma and osteosarcoma (14,15). However, to the best of our knowledge its specific expression pattern has never been investigated in CC tissues.

Rho-associated protein kinase 1 (ROCK1) belongs to the Rho-associated serine/threonine kinase family, which function as oncogenes and participate in malignant processes that include cellular migration, invasion and metastasis (16,17). ROCK1 expression is enhanced in various tumors, including glioma, osteosarcoma, prostate cancer and gastric cancer (18). Previous studies have indicated that ROCK1 was the target gene of miR-135a, miR-145, and miR-148a (19–21). To the best of our knowledge, the present study was the first to demonstrate that ROCK1 was a novel target gene of miR-217 and that miR-217 acted as a tumor suppressor mainly by targeting ROCK1 in cervical cancer cells.

Materials and methods

Ethics statement

Human samples used in the present study were obtained from the patients with written informed consent. The present study was approved by the Ethics Committee of Tengzhou Central People's Hospital (Tengzhou, China) and was conducted according to The Declaration of Helsinki.

Cell lines and samples

The immortalized human cervical epithelial Ect1/E6E7 cell line and the human cervical cancer SiHa, Caski and HeLa cell lines, as well as 293T cells, were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with streptomycin (100 µg/ml) and penicillin (100 U/ml) (Life Technologies; Thermo Fisher Scientific, Inc.).

A total of 40 patients (mean age, 58.4 years; range, 42–69 years) diagnosed with CC who underwent resection in the Department of Gynecology, Tengzhou Central People's Hospital were recruited between February 2015 and December 2016. Among the 40 patients, there were 23 cases with metastasis cervical cancer and 17 with non-metastatic cervical cancer. None of the patients received anticancer treatment, including radiotherapy or immunotherapy. Inclusion criteria: Patients with histopathologically diagnosed cervical carcinoma. Exclusion criteria: Patients with active infections, HPV infections, chronic inflammatory disease and histopathologically undetermined cervical abnormalities. Tumor tissues were obtained from patients with CC (>3 cm away from the tumor). Adjacent normal epithelial tissues were used as controls. The tissues were frozen in liquid nitrogen following surgery and stored at −80°C. Overall survival was defined as the period of time between surgery and mortality.

Transient transfection

SiHa and HeLa cells were seeded at 1×106 cells/well in the 6-well plates. Meanwhile, a small interfering RNA (siRNA; 5′-CTTGTGGAAAGGACGAAACACCGG-3′) targeting ROCK1 or a negative control siRNA (NC; 5′-TTCTCCGAACGTGTCACGT-3′) were mixed with HiperFect transfection reagent Qiagen, Inc. (Valencia, CA, USA) at a final concentration of 20 nM and incubated at room temperature for 10 min. Next, the complex was added in to the culture medium for 48 h. Following transfection for 48 h, the cells were collected for subsequent experiments.

Colony formation assay

SiHa and HeLa cells were suspended in 0.3% agar (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in DMEM at 37°C for 24 h at a density of 1×106 cells/dish in a 10 cm dish, which was preloaded with a thin layer of 1.0% agar. Subsequently, cells were transfected with pcDNA3.1-ROCK1 by Lipofectamine® 3000 (0.5 µg/µl; Thermo Fisher Scientific, Inc.) in the presence or absence of miR-217 mimic for another 48 h. Cells were kept in culture medium during the assay and monitored for colony formation. Following culture for 7 days, colony formation was observed. The clones were stained with trypan blue (Sigma-Aldrich; Merck KGaA) at room temperature for 1 min to evaluate colony formation. An Olympus light microscope was used to capture images at magnification, ×200.

Western blotting

Total proteins were isolated from tissues using an active protein extraction kit (KGP1050; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). A BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to determine the protein concentration. A total of 20 µg protein per lane was separated using 10% SDS-PAGE, transferred onto polyvinylidene difluoride membranes and then blocked with 5% fat-free milk at room temperature for 2 h. Membranes were then incubated with primary antibodies detecting ROCK1 (sc-374388; 1:1,000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and GAPDH (sc-293335; 1:1,000 dilution; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Following two washes with Tris-buffered saline with 0.5% Tween-20 (TBS-T), the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; ZB-2306; OriGene Technologies, Inc., Beijing, China) for 2 h at room temperature and then washed twice with TBS-T. Proteins were detected using enhanced chemiluminescence RapidStep™ ECL, according to the manufacturer's protocol (cat. no. 345818; Merck KGaA). ImageJ (version 1.8.0; National Institutes of Health, Bethesda, MD, USA) was applied to quantify the relative protein levels. GAPDH was used as an internal control. The integral optical density ratio of ROCK1/GAPDH indicated the relative expression of ROCK1 protein.

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

Total RNA (10 µg) from the CC tissues or Ect1/E6E7, SiHa, Caski and HeLa was isolated using RNAVzol (Vigorous Biotechnology Beijing Co., Ltd., Beijing, China) in accordance with the manufacturer's protocol. The concentration and purity of the RNA samples were determined by the OD260/OD280 ratio using a microplate reader (Model 3550; Thermo Fisher Scientific, Inc.).

For synthesis of cDNA of the specific miR, 1 µg total RNA was reverse transcribed using TaqMan™ MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) with specific primers for miR-140-5p and U6 (Sangon Biotech Co., Ltd., Shanghai, China). To quantify the miR-140-5p, a qPCR assay was performed using iQ™ SYBR® Green Supermix in an iCycler iQ™ qPCR detection system (both Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR amplifications were performed in a 10 µl reaction system containing 5 µl SYBR Green Supermix, 0.4 µl forward primer, 0.4 µl reverse primer, 2.2 µl double-distilled water and 2 µl template cDNA. The thermal cycling conditions were as follows: 95°C for 10 min; followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min; annealing at 55°C for 30 sec; and elongation at 72°C for 3 min. The relative level of miR-140-5p was determined using the 2−ΔΔCq analysis method (22). U6 was selected as the internal control. The primers used in the present study were as follows: miR-140-5p-RT (stem loop primer), 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTACCA-3′; U6-RT (step loop primer), 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATG-3′; miR-140-5p forward, 5′-GCGCGCAGUGGUUUUACCCUA-3′; U6 forward, 5′-GCGCGTCGTGAAGCGTTC-3′; and universal reverse primer, 5′-GTGCAGGGTCCGAGGT-3′.

Apoptosis analysis

Following transfection of SiHa and HeLa cells with miR-217 mimic or NC for 48 h, the cells were washed with ice-cold PBS three times and collected. An Annexin V-FITC/PI Apoptosis kit (Invitrogen; Thermo Fisher Scientific, Inc.) was applied to determine cell apoptosis. Briefly, cells were washed with 1X PBS three times and suspended at 2–3×106 cells/ml in 1X Annexin V binding buffer [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2]. Annexin V-FITC and Propidium Iodide Buffer (Invitrogen; Thermo Fisher Scientific, Inc.) were added to the cells, which were then incubated for 15 min at room temperature in darkness. Untransfected cells were used as the internal control. Following incubation, the cells were filtered by a filter screen and the cells were analyzed using a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) within 1 h of staining and data were analyzed using ModFit software (version 4.1; Verity Software House, Inc., Topsham, ME, USA). 10,000 cells were evaluated in each sample.

Invasion and motility assays

SiHa and HeLa cells were seeded in the top chamber (8-µm pore filter; Corning Incorporated, Corning, NY, USA) of each insert at 1.0×105 cells/well. The filter surfaces with 8.0-µm pores were precoated with Matrigel (BD Biosciences) for the motility assay. For the invasion assays, 2.0×105 cells were cultured in a chamber (8-µm pore filter; Corning Incorporated) pre-coated with 0.2% Matrigel at 37°C. Cells in the upper chamber were cultured in DMEM without FBS. As a chemoattractant, 10% FBS was added to DMEM in the lower chamber. After 24 h, the cells that remained in the upper compartment were removed by cotton swabs, and those that migrated or invaded through the membrane were stained with a dye solution containing 20% methanol and 0.1% crystal violet for 1 h at 37°C. Images of the cells were then captured using a light microscope (magnification, ×40); 10 individual fields were counted per insert. The results are presented as an average of three separate experiments.

Bioinformatic prediction

To investigate the possible target gene of miR-217, the online prediction system, TargetScan (http://www.targetscan.org), was applied.

Dual-luciferase reporter assay

The 3′UTR of ROCK1 containing the predicted target site 1 (pmirGLO-ROCK1-3UTR-1) or site 2 (pmirGLO-ROCK1-3UTR-2) for miR-217, was cloned into the pmirGLO luciferase reporter vector (Promega Corporation, Madison, WI, USA), which had been cleaved at the SacI and XhoI sites. Prior to conducting the luciferase reporter assay, 5×104 293T cells/well were seeded in 24-well plates in 500 µl DMEM with 10% FBS and cultured at 37°C for 18 h. The cells were transfected with the modified firefly luciferase vector (500 ng/µl) mixed with Vigofect transfection reagent (Vigorous Biotechnology Beijing Co., Ltd.), according to the manufacturer's protocol. Following 48 h of continuous exposure, the luciferase activities from firefly and Renilla were measured with the Dual-Luciferase Reporter assay system (Promega Corporation). Renilla activity was used as the normalized parameter.

Statistical analysis

Data are presented as mean ± standard deviation from 3 independent experiments. Two-tailed unpaired Student's t-tests were used for comparisons of two groups. Analysis of variance was used to perform multiple comparisons followed by Turkey's post hoc test. SPSS 13.0 (SPSS, Inc., Chicago, IL, USA) was used to perform all analyses. P<0.05 was considered to indicate a statistically significant difference.

Results

miR-217 and ROCK1 expression in CC specimens

The present study first investigated the expression levels of ROCK1 protein in CC and non-cancerous tissues. Compared with normal control, the level of ROCK1 was markedly increased in CC tissues (1.852±0.3875) compared with that of non-cancerous tissues (1.000±0.258) (Fig. 1A). Differences in the levels of ROCK1 expression between metastatic tissues and non-metastatic CC tissues were also obtained. Compared with non-metastatic CC tissues (1.000±0.126), higher expression of ROCK1 was observed in non-metastatic CC tissues (2.456±0.435) (Fig. 1B). The expression level of miR-217 was also investigated in non-metastatic CC tissues, metastatic CC tissues and non-cancerous tissues. As shown in Fig. 1C, the expression of miR-217 was significantly decreased in metastatic (0.438±0.0656), compared with non-metastatic CC tissues (0.876±0.379) and non-cancerous tissues (1.000±0.189).

Figure 1.

Association between miR-217 and ROCK1 in CC specimens. (A) The level of ROCK1 was markedly increased in CC tissues compared with that of non-cancerous tissues. (B) Western blot analysis of ROCK1 protein expression in metastatic tissues and non-metastatic CC tissues. (C) Reverse transcription-quantitative polymerase chain reaction analysis of miR-217 in CC tissues and non-cancerous tissues. *P<0.05, **P<0.01. miR-217, microRNA-217; ROCK1, Rho-associated protein kinase 1; CC, cervical cancer; NC, negative control.

Levels of ROCK1 and miR-217 in CC cell lines

The levels of ROCK1 and miR-217 expression were determined in CC SiHa, Caski and HeLa cell lines. Compared with the immortalized human cervical epithelial cell line Ect1/E6E7, the levels of ROCK1 protein expression were markedly increased in CC cell lines compared with Ect1/E6E7 cells (Fig. 2A). RT-qPCR analysis demonstrated that the levels of miR-217 were markedly reduced in SiHa, Caski and HeLa cells compared with those in Ect1/E6E7 cells (SiHa, 0.479±0.076; Caski, 0.514±0.086; HeLa, 0.703±0.046; Ect1/E6E7, 1.000±0.206) (Fig. 2B).

Figure 2.

Levels of ROCK1 and miR-217 in CC cell lines. (A) Western blot analysis of ROCK1 in CC cell lines, SiHa, Caski and HeLa, compared with the immortalized human cervical epithelial cell line, Ect1/E6E7. (B) Reverse transcription-quantitative polymerase chain reaction analysis of miR-217 in CC cell lines and Ect1/E6E7 cells. *P<0.05, **P<0.01. miR-217, microRNA-217; ROCK1, Rho-associated protein kinase 1; CC, cervical cancer.

miR-217 suppresses invasion and induces apoptosis of CC cells

To investigate the role of miR-217 in the regulation of CC cell malignancies, miR-217 mimic was transfected into SiHa and HeLa cells. As depicted in Fig. 3A, overexpression of miR-217 markedly suppressed the colony-formation capacity of SiHa and HeLa cells. Furthermore, the cell invasion capacity was also decreased in SiHa and HeLa cells transfected with the miR-217 mimic (Fig. 3B). The flow cytometry assay indicated that transfection with miR-217 significantly increased the proportion of cells undergoing apoptosis in SiHa and HeLa cells (Fig. 3C).

Figure 3.

miR-217 suppresses invasion and induces apoptosis of CC cells. (A) Colony formation assay in SiHa and HeLa cells transfected with miR-217 mimic or NC. (B) Cell invasion assay. (C) Flow cytometry assay. *P<0.05, **P<0.01 vs. NC. miR-217, microRNA-217; CC, cervical cancer; NC, negative control; 217 m, miR-217 mimic.

miR-217 functions as a tumor suppressor mainly by targeting ROCK1

Whether miR-217 regulated the malignancies of CC through ROCK1 was investigated. On the basis of TargetScan, two possible bindings sites were identified in the 3′UTR of ROCK1 (Fig. 4A). Next, the 3′UTR containing the two binding sites was cloned into pmirGLO plasmid, pmirGLO-ROCK1-3′UTR-1 and pmirGLO-ROCK1-3′UTR-2 (Fig. 4B). The results of the dual-luciferase reporter assay demonstrated that miR-217 markedly suppressed the relative luciferase activity of pmirGLO-ROCK1-3′UTR-1 and pmirGLO-ROCK1-3′UTR-2 compared with blank vector (Fig. 4B). Western blot analysis also revealed that overexpression of miR-217 significantly reduced the expression level of ROCK1 protein, whereas inhibition of miR-217 expression enhanced the expression of ROCK1 (Fig. 4C and D). These data indicated that ROCK1 was the target gene of miR-217.

Figure 4.

ROCK1 is the target gene of miR-217. (A) Schematic analysis of miR-217 on the 3′UTR of ROCK1. (B) Results of the dual-luciferase assay. (C) Western blot analysis also revealed that overexpression of miR-217 significantly reduced the protein expression level of ROCK1. (D) The inhibition of miR-217 enhanced the expression of ROCK1. *P<0.05, **P<0.01 vs. blank plasmid or NC. ROCK1, Rho-associated protein kinase 1; miR-217, microRNA-217; 3′UTR, 3′-untranslated region; NC, negative control; 217 m, miR-217 mimic; 217i, miR-217 inhibitor.

ROCK1 partially abolishes the miR-217-induced CC cell invasion inhibition

To investigate the role of ROCK1 in miR-217-mediated suppression of cellular invasion, ROCK1 was overexpressed in SiHa cells. As expected, overexpression of ROCK1 markedly enhanced the protein level of ROCK1 (Fig. 5A). The overexpression of ROCK1 also led to increased invasion capacity in SiHa cells even when miR-217 was inhibited (Fig. 5B). These results demonstrated that the anti-invasive effects of miR-217 were mediated through ROCK1.

Figure 5.

Anti-invasive effects of miR-217 are mediated through ROCK1. (A) The ectopic expression of ROCK1 markedly enhanced the protein level of ROCK1. (B) Overexpression of ROCK1 also led to increased invasive capacity in SiHa cells even when miR-217 was inhibited. *P<0.05, **P<0.01. miR-217, microRNA-217; ROCK1, Rho-associated protein kinase 1; NC, negative control; 217 m, miR-217 mimic.

Discussion

Evidence has demonstrated that miRNAs serve key roles in the malignant progression of various tumor types (23,24). Therefore, it is of great importance for us to analyze the possible target genes of miRNAs. The present study investigated the expression of miR-217 in cervical cancer tissues and demonstrated that it was markedly reduced in metastatic CC cancer cells. To the best of our knowledge, the present study identified that ROCK1 was a novel target gene of miR-217 for the first time. Through targeting ROCK1, miR-217 functions as a tumor suppressor in CC cells.

ROCK-1, a serine/threonine kinase, is a member of the Rho family of GTPase proteins that prompts the reorganization of the actin cytoskeleton and is widely involved in phosphatase and tensin homolog/phosphoinositide 3-kinase signaling, including cell migration, cell death and survival (25–27). Studies have demonstrated that the expression of ROCK1 was significantly enhanced in bladder, lung and prostate cancer (28–30). The increased expression of ROCK1 enhanced cancer cell migration (31), and its targeting has been proposed to have possible therapeutic value in lung cancer. Recently, ROCK1 was demonstrated to be the target gene of multiple miRNAs, including miR-126, miR-335, miR-584, and miR-186 (32–35). These miRNAs were reported to enhance cancer cell proliferation and/or invasion, primarily by suppressing the expression of ROCK1, as reported in colon cancer, osteosarcoma and human renal cell carcinoma (32,33,36). To identify the possible mechanism by which miR-217 elicited its effects in CC, the expression of ROCK1 was analyzed in CC tissues and cells. In line with the data of the previous study, ROCK1 was validated as an oncogene in CC cells (37). Further analysis revealed the presence of two conserved binding sites in the 3′UTR of ROCK1, and a dual-luciferase reporter assay indicated that ROCK1 was a target gene of miR-217. These results revealed that ROCK1 was a target gene of miR-217.

The present study investigated the role of miR-217 in CC cells. The data indicated that overexpression of miR-217 markedly suppressed CC cell proliferation and migration, and also induced CC cell apoptosis. By contrast, overexpression of ROCK1 could partially abolish the miR-217-induced inhibition of cancer cell invasion. The results of the present study indicated that miR-217 functions as a tumor suppressor, primarily by targeting ROCK1.

In conclusion, to the best of our knowledge, the present study is the first to indicate that miR-217 functions as a tumor suppressor, by directly targets oncogene ROCK1. The expression of miR-217 was negatively associated with ROCK-1 expression in primary CC tissues. The experimental data and conclusions in the present study provide valuable information regarding the biological functions of miR-217 and the possible mechanisms of the migration and invasion of cervical cancer. Thus, it is necessary to investigate the molecular network involved in ROCK1 regulation further on a larger quantity of clinical samples.