Immortalized non-tumorigenic endocervical End1/E6E7 (CRL-2615) cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in keratinocyte-serum free medium (Gibco-BRL 17005-042; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 0.1 ng/ml human recombinant EGF, 0.05 mg/ml bovine pituitary extract and additional calcium chloride 44.1 mg/l (final concentration 0.4 mM). HeLa and C-33A cervical cancer cells were obtained from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). HeLa cells have been reported to contain human papilloma virus 18 (HPV-18) sequences, while C-33A cells and CRL-2615 were negative. Both HeLa and C-33A cells were cultured in DMEM (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Sijiqing Biotechnology Co., Ltd., Hangzhou, China), 100 U/ml penicillin and 100 mg/ml streptomycin, in a humidified atmosphere containing 5% CO₂, at 37°C.

Cervical cancer samples were obtained between January 2013 and January 2016 at the First Affiliated Hospital of Nanjing Medical University. Twenty cervical cancer tissues and fifteen specimens of normal cervical tissue were excised surgically from patients who had previously provided informed written consent. The study was approved by the Research Ethics Committee of Nanjing Medical University, and informed consent was obtained from all patients. Tumor samples and normal tissues were immediately frozen and stored at −80°C until used. The tumor stage and grade was in consistence with the International Federation of Gynecologists and Obstetricians (FIGO).

To investigate the effect of SNP on the expression of miR-146a, pre-miR-146a consisting of a 370-bp DNA fragment was amplified from a genomic DNA sample with a miR-146a rs2910164 G allele or rs2910164 C allele, and cloned into pcDNA3.1(+) expression plasmid vector (Invitrogen Life Technologies, Carlsbad, CA, USA). Amplified DNA fragments were then sequenced to confirm that there were no mutations. For transfections, CRL-2615, HeLa and C-33A cells were transfected with expression plasmid vector containing either the G or C allele of miR-146a rs2910164 (pre-miR-146a-G or pre-miR-146a-C), using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer's instructions. The pcDNA3.1(+) vector without an insert was used as a negative control. Transfection efficiency was verified by real-time PCR.

Total RNA was extracted from the cultured cells using TRIzol reagent (Takara, Otsu, Japan), according to the manufacturer's instructions. Bulge-loop miRNA quantitative real-time (qRT)-PCR primer sets specific for miR-146a were designed by Guangzhou RiboBio (Guangzhou, China) and expression levels were normalized against U6. The following primers were used: TRAF6 forward, 5′-TTTGCTCTTATGGATTGTCCCC-3′ and reverse, 5′-CATTGATGCAGCACAGTTGTC-3′; IRAK1 forward, 5′-TGAGGAACACGGTGTATGCTG-3′ and reverse, 5′-GTTTGGGTGACGAAACCTGGA-3′; GAPDH forward, 5′- GAAGGTCG GAGTCAACGGATTT-3′ and reverse, 5′-CTGGAAGATGGTGATGGGATTTC-3′. cDNA was synthesized from 1 µg total RNA using random primers and Superscript II Reverse Transcriptase (Takara) within 20 µl reaction volumes. Quantitative real-time PCR was performed in an Applied Biosystems StepOne Real-time PCR system (Thermo Fischer Scientific), using SYBR-Green PCR kit (Takara) by normalizing to GAPDH. The 2^−∆∆Ct method was used to analyze the relative miRNA or mRNA expression. All reactions were prepared in three independent experiments.

Cells were harvested in RIPA lysis buffer containing protease and phosphatase inhibitors, and the protein concentrations were determined by BCA protein assay (Beyotime Institute of Biotechnology, Shanghai, China). Protein samples were separated by 10% SDS-PAGE, and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA), blocked with TBST containing 5% skimmed milk for 1 h at 37°C. Cells reacted with the following primary antibodies: rabbit anti-TRAF6 antibody (1:500; cat. no. BS3684; Bioworld Technology, Inc., St. Louis Park, MN, USA), rabbit anti-IRAK1 antibody (1:1,000; cat. no. BS1541; Bioworld Technology), mouse anti-cleaved caspase-3 antibody (1:1,000; cat. no. 9661; Cell Signaling Technology, Danvers, MA, USA), cyclin D1 (1:1,000; cat. no. 2922; Cell Signaling Technology) and mouse anti-β-actin antibody (1:5,000; cat. no. ab8226; Abcam, Cambridge, MA, USA). After being washed in TBST at room temperature, the membranes were incubated for 1 h at 37°C with a secondary HRP-conjugated antibody (1:5,000; Beijing ZhongShan Golden Bridge Biotechnology Co., Ltd., Beijing, China) and were visualized using an ECL detection kit (Amercontrol Biosciences, London, UK). Each experiment was performed in triplicate.

Potential targets of miR-146a were predicted and analyzed using TargetScan (http://www.targetscan.org), miRanda (http://www.microrna.org) and PicTar softwares (https://pictar.mdc-berlin.de). TargetScan Release 7.0 was the primary source for target identification and it offered 275 conserved targets, from which we selected 10 top targets with the highest aggregate P-value. miRanda and PicTar softwares were used to predict potential targets based on mirSVR scores <1.50 and PicTar scores >5.0. A wild-type 3′-UTR of the IRAK1 or TRAF6 gene was cloned into the pMIR-Report luciferase vector (Promega, Madison, WI, USA). Constructs carrying the mutated fragment of the 3′-UTR of IRAK1 and TRAF6 mRNA without the putative miR-146a binding sequences were used as the mutated controls. Subsequently, 293 cells were transfected with the reporter constructs together with a miR-146a-5p mimic or negative control (NC), using Lipofectamine 2000 (Invitrogen Life Technologies). Cells were harvested after 48 h and the activity was assessed using a Modulus™ luminometer (Turner Biosystems; Thermo Fisher Scientific). Renilla luciferase activity was used as a control for transfection efficiency. All assays were conducted in triplicate and the experiments were prepared in three independent experiments. IRAK1 and TRAF6 genes were also verified in other malignancies as previously reported (20).

HeLa and C-33A cells were cultured at a density of 10,000 cells/well in 96-well plates. Cell culture continued for 24, 48 and 72 h after transfection, and cell viability were performed using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. The absorbance value of each well was assessed at 450 nm in a microplate reader. The data were obtained from five independent cultures and experiments were repeated in triplicate.

Forty-eight hours after transfection, cells were harvested and washed twice with cold PBS, and the supernatant was removed after centrifugation. Subsequently, the cells were collected and fixed in 75% ice-cold ethanol and incubated at −20 °C overnight. After being washed, they were stained with 50 mg/ml of propidium iodide (PI) for DNA content analysis by flow cytometry on a FACSCalibur system (BD Biosciences, Franklin Lakes, NJ, USA). Results were expressed as a percentage of cells in each cell-cycle phase.

Cells were lifted off the plates, washed twice with PBS and determined with an Annexin V-FITC/propidium iodide (PI) kit (BD Biosciences) at room temperature for 15 min in the dark, and then analyzed by BD Biosciences FACS Calibur Flow Cytometry (BD Biosciences) according to the manufacturer's protocol. All experiments were performed independently, in triplicate.

SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. The results are represented as the means ± SD from three separate experiments or more. Statistical analysis was performed by ANOVA for experiments involving more than two groups. The two-tailed Student's t-test was performed for comparisons with two groups and P<0.05 was considered to indicate a statistically significant difference (*P<0.05; **P<0.01).

Two types of human cervical cancer cells were first analyzed to quantify the expression levels of miR-146a. Our results revealed that the expression of miR-146a was increased in these two cell lines compared with the immortalized non-tumorigenic CRL-2615 cell line (Fig. 1A). Subsequently, we assessed the expression levels of miR-146a in 20 cervical cancer tissues and 15 normal cervical tissue samples. The results demonstrated that the expression of miR-146a was significantly upregulated in cancer tissues than that in normal tissues (P<0.01, Fig. 1B). Collectively, these results indicated that the high expression of miR-146a may be involved as an oncogene in cervical cancer.

Transfection of recombinant expression plasmids into HeLa and C-33A cervical cancer cell lines and the immortalized non-tumorigenic CRL-2615 cell line revealed that the expression of miR-146a was higher with the C allele compared to the G allele in HeLa and C-33A cells, while the expression of miR-146a was lower with the C allele compared to the G allele in CRL-2615 cells (Fig. 1C). Thus the expression of miR-146a was upregulated in cervical cancer, and the G>C substitution in pre-miR-146a increased mature miR-146a expression in cervical cancer cells, but exerted the opposite effect in CRL-2615 cells.

To explore the effect of miR-146a in cervical cancer carcinogenesis, HeLa and C-33A cells were transiently transfected with miR-146a mimic or negative control, and assessed for the expression of miR-146a (Fig. 2A). miR-146a significantly promoted cell growth in HeLa and C-33A cells (Fig. 2B and C), indicating that miR-146a exerted a growth-promoting function in cervical cancer cells.

Given that miR-146a promotes the viability of cervical cancer cells, we examined whether miR-146a influenced the cell cycle progression of cervical cancer cells. The number of cells in the G1 phase was significantly decreased after transfection with miR-146a mimic in HeLa (P=0.048) and C-33A (P=0.022) cells, while cells in the S phase were increased in HeLa cells (P=0.018) and in C-33A cells (P=0.046) compared to the controls (Fig. 2D and E). Therefore, we hypothesized that miR-146a enhanced growth by promoting cell cycle progression at the G1-to-S-phase transition. The expression of the oncogene cyclin D1 in HeLa and C-33A cells was also upregulated following the miRNA-146a mimic transfection (Fig. 2F), indicating that miRNA-146a may be an oncogenic regulator in cervical cancer cells.

The number of early apoptotic cells was assessed by flow cytometry and was not significantly decreased in miR-146a mimic transfected cervical cancer cells (Fig. 3A and B, P>0.05). Western immunoblotting analysis confirmed that cleaved caspase-3 protein was not decreased in either cell line after miR-146a overexpression (Fig. 3C, P>0.05). Thus, miR-146a in cervical cancer cells does not modify apoptosis.

The use of target prediction tools such as TargetScan, miRanda and PicTar predicted that miR-146a was a putative regulator of IRAK1 and TRAF6 (Fig. 4A). To confirm that IRAK1 and TRAF6 were targets of miR-146a, we generated a pMIR-Report luciferase vector containing wild-type 3′-UTR of the IRAK1 and TRAF6 genes. We co-transfected these constructs into 293 cells together with a miR-146a mimic or negative control, and then conducted luciferase activity assays. As displayed in Fig. 4B and C, compared with the control, the overexpression of miR-146a led to a significant reduction in the luciferase activity of the wild-type IRAK1 and TRAF6 3′UTR reporter gene in 293 cells (IRAK1 wild-type, TRAF6 wild-type, P<0.05). To explore the mechanism of viability promotion induced by miR-146a, we investigated whether miR-146a regulated the expression of IRAK1 and TRAF6 in cervical cancer cells. Following miR-146a mimic transfection, the expression of IRAK1 and TRAF6 mRNA (P<0.05; Fig. 4D and E) and the protein expression (P<0.05; Fig. 4F and G) were decreased with ectopic expression of miR-146a.

In HeLa and C-33A cells, the expression of IRAK1 and TRAF6 mRNA was assessed and found decreased compared with CRL-2615 cells (Fig. 5A and B). There was no difference between the HeLa and C-33A cells. RT-PCR was used to investigate the mRNA expression levels of IRAK1 and TRAF6 in cervical cancer tissues (n=20) and normal cervical tissues (n=15). Both IRAK1 and TRAF6 mRNA expression levels were significantly downregulated in cervical cancer tissues (Fig. 5C and D). To investigate whether IRAK1 and TRAF6 were involved in miR-146a-mediated viability of cervical cancer cells, we infected cervical cancer cells with IRAK1 and TRAF6 siRNAs to downregulate IRAK1 and TRAF6 (Fig. 5E and F). The CCK-8 assay demonstrated that si-IRAK1 and si-TRAF6 significantly promoted cell viability in HeLa and C-33A cells (P<0.05; Fig. 5G and H). These findings supported our contention that miR-146a in cervical cancer cells promoted tumor cell viability by inhibiting IRAK1 and TRAF6.