The human cervical cancer cell lines C-4I, and SiHa were purchased from the American Type Culture Collection. SKG-II was provided by Keio University (Tokyo, Japan). Cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (Equitech-Bio, Inc.) at 37°C in a humidified incubator with 5% CO₂.

Pre-miR™ miRNA precursor molecules (pre-miR-22-3p; cat. no. AM17101), negative (non-specific) control (pre-miR-negative control #1; cat. no. AM17110) and inhibitor miRNA (anti-miR-22-3p; cat. no. AM17001) were ordered from Ambion (Thermo Fisher Scientific, Inc.). These were designed to mimic endogenous mature miRNAs, but the sequences are not publicly available. The mature miRNA sequence of miR-22-3p is 5′-AAGCUGCCAGUUGAAGAACUGU-3′. Cervical cancer cells were transfected with pre-miR-22-3p, anti-miR-22-3p or pre-miR-negative control (30 nM) for 24 h. Oligonucleotide transfection was performed using siPORT NeoFX Transfection Agent (Ambion; Thermo Fisher Scientific, Inc.).

C-4I and SiHa cells were used for the 3′UTR reporter assay. The full length MYCBP 3′UTR was inserted downstream of a Gaussia luciferase (Gluc) reporter in the pEZX-MT05 vector (GeneCopoeia, Inc.). The secreted alkaline phosphatase (seAP) reporter gene was also present in the vector as an internal control for transfection normalization. As a control (pEZX-MT05-CT), miRNA target clone control vector (CmiT000001-MT05) was purchased from GeneCopoeia, Inc. Cells (1×10⁵/ml) seeded in 24-well plates were co-transfected using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) complexed with the pEZX-MT05 vector and pre-miR-22-3p, anti-miR-22-3p or pre-miR-negative control (cont miR) according to the manufacturer's protocol. Culture medium (DMEM, Gibco; Thermo Fisher Scientific, Inc.) was collected after 48 h and the relative luciferase activity (Gluc:seAP ratio) was analyzed using the Secrete-Pair Dual Luminescence Assay kit (GeneCopoeia, Inc.) according to the manufacturer's protocol.

The putative human target genes of miR-22 were analyzed using the TargetScan (version 6.0; targetscan.org/) and miRDB (version 5.0; mirdb.org/) web-based bioinformatics algorithms. TargetScan predicts biological targets of miRNAs by searching for the presence of conserved 8mer, 7mer and 6mer sites that match the seed region of each miRNA (17). The cumulative weighted context ++ score <-0.1 was applied as the cut-off criteria (17). In miRDB, the prediction of miRNA-mRNA pair is based on both the 3′UTR and 5′UTR regions of conserved and non-conserved genes, the base composition in the regions flanking the seed pairing sites, secondary structure, and the location of the site within the 3′UTR (18).

Total RNA was extracted from C-4I, SKG-II and SiHa cells using a RNeasy kit (Qiagen, Inc). A Super Script II Reverse Transcriptase kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used to synthesize cDNA using random primers according to the manufacturer's protocol. A total of 1 µg of RNA, 125 ng random primers and 1 µl 10 mM dNTPs in 12 µl total volume were denatured at 65°C for 5 min before 2 min on ice. Then, 4 µl 5× first strand buffer and 2 µl of 0.1 M DTT were added followed by 1 µl Superscript II. Reactions were incubated 10 min at 25°C, 42°C for 50 min and 70°C for 15 min. Subsequently, TaqMan qPCR was performed in triplicate using the StepOne Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The mixed primers for TaqMan qPCR used in the present study were purchased from Applied Biosystems in the form of a probe mix (MYCBP: Hs 00429315_g1; c-Myc: Hs00153408_m1; hTERT: Hs00972650_m1) and GAPDH (Hs02786624_g1, Applied Biosystems) was used as a housekeeping control gene. The sequences of these primers are not publicly available. PCR conditions were 95°C for 10 min, followed by 60°C for 1 min for 40 cycles following the manufacturer's protocol.

miRNA extraction was performed using a mirVana miRNA isolation kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. miRNA was then reverse transcribed using the microRNA reverse transcription kit according to the manufacturer's protocol (Applied Biosystems; Thermo Fisher Scientific, Inc.) in combination with the stem-loop primer for miR-22-3p (5′-GGCUGAGCCGCAGUAGUUCUUCAGUGGCAAGCUUUAUGUCCUGACCCAGCUAAAGCUGCCAGUUGAAGAACUGUUGCCCUCUGCC-3′) and the endogenous control RNU48 (5′-GATGACCCCAGGTAACTCTGAGTGTGTCGCTGATGCCATCACCGCAGCGCTCTGACC-3′) (Applied Biosystems; Thermo Fisher Scientific, Inc.) and served as a template for the quantification of the expression of mature miRNA. qPCR of miR-22 was performed according to the manufacturer's instructions (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR conditions were 95°C for 10 min, followed by 60°C for 1 min for 40 cycles. Data analyses were performed using the 2^−ΔΔCq method (19).

Western blot analysis was performed as described previously (20). In brief, total proteins from C-4I, SKG-II and SiHa were prepared using Pierce RIPA Buffer (Thermo Fisher Scientific, Inc.). The protein concentration was quantified by DC Protein Assay (Bio-Rad Laboratories, Inc.). Protein samples (15 µg/lane) were separated using 4–15% gradient gel electrophoresis (Bio-Rad Laboratories, Inc.) and transferred to PVDF membranes. After being blocked with 10% bovine serum albumin (New England BioLabs, Inc.) for 1 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies diluted at 1:200 (anti-MYCBP: Sigma-Aldrich, HPA041188) or 1:1,000 (anti-MYC: Cell Signaling, 13987 and anti-hTERT; LifeSpan BioSciences, Inc., LS-B11086). After 1 h incubation with horseradish peroxide-conjugated anti-rabbit secondary antibody (1:4,000; goat anti-rabbit IgG; sc-2030; Santa Cruz Biotechnology, Inc.) at room temperature, the blots were visualized using enhanced chemiluminescence (ECL Plus; GE Healthcare Life Sciences).

The clonogenic assay was performed using the technique described previously by Franken et al (21). In brief, C-4I and SKG-II cells (1.0×10²/well for 2 Gy-2.4×10³/well for 8 Gy) transfected with miR-22, anti-miR-22 or cont miR were plated onto 6-well plates. Each group of cells was irradiated with various doses of X-ray (0, 2, 4, 6 and 8 Gy) from an X-ray generator (M-150WE; Softex Co., Ltd.) and incubated at 37°C in a humidified incubator with 5% CO₂ for 14 days. Fixation and staining of colonies was performed using a mixture of 0.5% crystal violet in methanol for 30 min at room temperature. Plates were rinsed with water and left to dry at room temperature. Counting of colonies was done on the following day. The cell survival was measured by standard colony formation after radiation treatment. Colonies containing >50 cells counted under a light microscope (CK40-F100, Olympus) at ×40 magnification were defined as derived from clonogenically viable cells. The survival fraction of the cells was calculated by normalizing the plating efficiency of treated cells by that of control cells as described previously (21). Each experiment was performed at least three times in triplicate wells.

Lentivirus (1×10⁷ plaque forming units/ml) expressing LentimiRa-GFP-hsa-miR-22-3p (L-miR22-C-4I; cat. no. mh15295) and Lnti-III-miR-GFP Control (L-cont-C-4I; cat. no. m002) were purchased from Applied Biological Materials, Inc. Lentiviral transduction was conducted at a multiplicity of infection of 200 with a ViraDuctin Lentivirus Transduction kit (Cell Biolabs, Inc.), according to the manufacturer's protocol. In brief, 5.0×10⁴ C-4I cells were seeded in 24-well plates overnight at 37°C in a humidified incubator with 5% CO₂. LentimiRa-GFP-hsa-miR-22-3p or Lenti-III-miR-GFP Control was added to the cells. After 48 h, purification was performed using puromycin until antibiotic-resistant colonies were identified. Post-transfection cells were further selected in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing puromycin for 2 weeks to establish stably transduced cells.

Female 6-week-old athymic nude mice (BALB/c ^nu/^nu) (average body weight 16 g) were purchased from Japan SLC, Int. A total of 10 animals were divided into two groups, each consisting of 5 mice (n=5). Mice were housed under standard environmental conditions at Osaka Medical College Division of Research Animal Laboratory (temperature, 22°C; humidity, 40–60%; light/dark cycle, 12 h light 12 h darkness) with ad libitum access to food and water. All of the animal studies were carried out in compliance with the guidelines of the Osaka Medical College Animal Care and Use Committee, and followed the institutional guidelines for animal welfare and experimental conduct. Mice were monitored daily for signs of discomfort and pain by laboratory personnel as well as by the staff at the Division of Research Animal Laboratory. In addition to the pathological status, the mice were monitored to ensure that a humane endpoint was reached (defined as complete inability to ambulate). All mice gained weight over the entire study period while appearing generally healthy throughout the experiments. Under anesthesia with 2% isoflurane, C-4I cells infected with L-miR22-C-4I or L-cont-C-4I were injected subcutaneously into the flanks of nude mice (4×10⁶ cells in 100 µl PBS per mouse).

The in vivo growth of C-4I xenografts was monitored by measuring their volumes and calculated using the modified ellipse formula (volume=length × (width)²/2). When the xenograft volumes reached ~100 mm³, the tumor was irradiated with X-rays (6 Gy) following the intraperitoneal administration of a mixture of three anesthetic agents (0.3 mg/kg medetomidine, 4 mg/kg midazolam and 5 mg/kg butorphanol). After irradiation, the tumors were measured with calipers every 7 days. A total of 35 days after irradiation, all mice were euthanized by cervical dislocation under anesthesia with 5% isoflurane for sample collection. Death was verified by the absence of a heart beat and the onset of rigor mortis. The tumors were excised, weighed (the maximum percentage of tumor weight of total body weight was <0.15%) and fixed in 10% neutral buffered formalin for 24 h at room temperature, and 4 µm-thick paraffin sections were prepared for immunohistochemistry and the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay.

The aforementioned section from paraffin-embedded xenograft tissues were subjected to immunostaining. Tissue samples were formalin-fixed and embedded in paraffin. Deparaffinized and rehydrated sections were autoclaved in 0.01 mol/l citrate buffer (pH 6.0) for 15 min at 121°C for antigen retrieval. The endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide solution in methanol for 30 min at room temperature, then sections were incubated at room temperature for 30 min with rabbit anti-Ki67 antibody (1:300; AB9260; Merck KGaA). The sections were then washed once with phosphate-buffered saline (PBS) and incubated with secondary antibody Histofine Simple Stain MAX PO (MULTI) (ready to use; cat. no. 414151F; Nichirei Corporation) for 30 min at room temperature. Finally, the sections were washed once with PBS and visualized by incubating with H₂O₂/diaminobenzidine substrate solution for 5 min. The sections were counterstained with hematoxylin for 20 sec at room temperature prior to dehydration and mounting. The Ki-67 index and percentage of apoptotic cells reflected the percentage of the total number of tumor cells with nuclear staining in viable regions per 5 high-power fields using a fluorescence microscope (BZ-X700, KEYENCE) at ×400 magnification.

Apoptotic cell death was determined by TUNEL assay using a in situ Apoptosis Detection kit (Wako Pure Chemical Industries, Ltd.), according to the manufacturer's protocol. The sections were deparaffinized for 10 min, dehydrated in 100% ethanol for 10 min and proteins of the sections were digested using pre-warmed protease solution for 5 min at 37°C. After washing, the sections were incubated with 50 µl TdT reaction solution (consisting of TdT Enzyme 1 µl + TdT Substrate Solution 49 µl) for 10 min at 37°C. After washing, the endogenous peroxidase activity was blocked using 3% H₂O₂ for 5 min at room temperature. After washing, the sections were reacted with 100 µl of POD-conjugated antibody solution for 10 min at 37°C. After removing the antibody solution and washing, immunoreactivity was visualized using 3,3′-diaminobenzidine. The sections were counterstained with 0.5% methyl green for 5 min at room temperature. The TUNEL index was calculated as the percentage of TUNEL-positive cells in 1,000 carcinoma cells in the areas of highest nuclear labeling under a fluorescence microscope (BZ-X700, KEYENCE) at ×400 magnification.

The statistical analyses were performed using the StatView software program (version 5.0; SAS Institute, Inc.). The data are presented as the mean ± standard deviation of three independent experiments. The statistical analysis was performed using the Student's paired t-test and P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR was performed to evaluate the expression profile of miR-22 in several cervical cancer cell lines. The endogenous miR-22 expression was higher in the SiHa cell line compared with the SKG-II and C-4I lines (Fig. 1A). Accordingly, SKG-II and C-4I were used for overexpression experiments of miR-22, whereas SiHa was used for reduced experiments of miR-22.

The overexpression of miR-22 was induced by the transfection of pre-miR-22, and an increased level of miR-22 in SKG-II and C-4I cells was confirmed (Fig. 1B). By contrast, the transfection of anti-miR-22 reduced the miR-22 expression level in SiHa cells (Fig. 1B).

Subsequently, it was determined whether or not expression of MYCBP could be altered by the overexpression or suppression of miR-22. RT-qPCR revealed that the level of MYCBP was decreased under conditions of miR-22 overexpression, while MYCBP mRNA was increased under conditions of miR-22 suppression (Fig. 1C).

TargetScan and miRDB were utilized to predict that the MYCBP gene has a putative miR-22 target site at its 3′UTR region. To confirm that miR-22 directly targeted the MYCBP 3′UTR in cervical cancer cells, a luciferase reporter assay was performed. C-4I cells were co-transfected with either the precursor miRNA or control and with either a plasmid containing a luciferase reporter driven by the wild-type human MYCBP 3′UTR (pEZX-MT05-MYCBP; Fig. 2A) or a control plasmid (pEZX-MT05-CT). Treatment of C-4I with complexes of pre-miR-22 and pEZX-MT05-MYCBP significantly reduced the luciferase activity compared with that in the combination of cont miR and pEZX-MT05-MYCBP (Fig. 2B). Knockdown of miR-22 via the transfection of anti-miR22 significantly increased the luciferase activity of MYCBP 3′UTR compared with transfection of cont miR (Fig. 2C). The current results indicate that miR-22 suppresses the expression of MYCBP mRNA via direct targeting of the MYCBP 3′UTR in the cervical cancer cell lines.

MYCBP has been revealed to promote the activation of the c-Myc target gene via E-box (22). Therefore, the current study investigated whether or not the suppression of MYCBP by miR-22 results in subsequent suppression of the E-box-dependent c-Myc target gene expression. The hTERT gene is an E-box-dependent target gene (23) and does not contain a predicted target site for miR-22 in its 3′UTR, according to TargetScan and miRDB. Thus, the effect of the overexpression or suppression of miR-22 on hTERT expression levels was examined. It was revealed that the overexpression of miR-22 significantly reduced the expression level of hTERT, whereas suppression of miR-22 increased hTERT expression (Fig. 3A and B).

Subsequently, whether or not the miR-22-induced hTERT suppression was dependent on the interaction of miR-22 with c-Myc 3′UTR was investigated, and in silico analyses using TargetScan and miRDB predicted that the c-Myc gene had no target site for miR-22. Moreover, RT-qPCR and western blot analyses revealed that neither the overexpression nor the suppression of miR-22 affected c-Myc levels at the mRNA or protein level (Fig. 3B and C). The present results do not support the possibility of a direct interaction between miR-22 and c-Myc mRNA, thus indicating that the inhibition of MYCBP by miR-22 resulted in the subsequent reduction of the c-Myc target gene hTERT.

Telomerase activity reportedly influences the radiosensitivity of the cervical cancer cell line SiHa (24). To investigate the effect of miR-22 on radiosensitivity, C-4I and SKG-II cells were transfected with miR-22 or control miRNA and irradiated with various radiation doses (2, 4, 6 and 8 Gy). Compared with the control miRNA, the survival fraction of the miR-22-transfected group was significantly lower (Fig. 4A and B). Conversely, SiHa cells with a suppressed miR-22 expression exhibited a higher survival fraction following irradiation compared with cells transfected with the control miRNA (Fig. 4C).

Given that the overexpression of miR-22 improved the radiosensitivity of cervical cancer cells by the subsequent reduction of hTERT, the therapeutic potential of miR-22 was assessed in a cervical cancer xenograft model. C-4I cells were stably transduced with lentiviruses containing precursor miR-22 (L-miR22) or control lentiviral vector (L-cont). Stable transduction efficiency was confirmed by the expression of GFP (Fig. 5A). Significant upregulation of miR-22 in L-miR22-transduced C-4I cells (L-miR22-C-4I) was confirmed using RT-qPCR (Fig. 5B). Transduced C-4I cells (L-miR22-C-4I or L-cont-C-4I) were injected subcutaneously, and then the tumor was irradiated with an X-ray dose of 6 Gy once the xenograft volume reached 100 mm³. The tumor growth was significantly inhibited in the L-miR22-C-4I mice compared with that in the L-cont-C-4I mice (between 7 days and 21 days after irradiation; P<0.05, after 28 days or later; P<0.01), indicating that miR-22 had a radiosensitizing effect in vivo (Fig. 5C). A total of 35 days after irradiation, tumor nodules were excised and weighed (Fig. 5D). It was revealed that the L-miR22-C-4I tumors were significantly smaller compared with the L-cont-C-4I tumors (P<0.05; Fig. 5D). In addition, paraffin sections were prepared from the excised tumor and immunostaining of Ki-67 (Fig. 5E) was performed, in addition to a TUNEL assay (Fig. 5F), to investigate the effect on proliferation and apoptosis. The Ki-67 index was significantly lower in the L-miR22-C-4I tumor compared with that in the L-cont-C-4I tumor group (P<0.05; Fig. 5E). Furthermore, the TUNEL index was higher in the L-miR22-C-4I tumor group compared with that in the L-cont-C-4I tumor group, suggesting that miR-22 may influence apoptosis (P<0.05; Fig. 5F).

The present study suggests a novel mechanism for direct miR-22 mediated suppression of MYCBP expression resulting in the subsequent reduction of c-Myc-mediated transactivation (Fig. 6).