CC and corresponding adjacent normal tissues were obtained from 117 patients who received routine surgery at The First Affiliated Hospital of Sun Yat-Sen University during January 2008 to December 2011. None of them had received any therapy before surgery. All tissues were stored at −80°C until RNA extraction. Informed consent was obtained from all the patients and the present study was approved by the Ethics Committee of the Sun Yat-sen University in accordance with the Declaration of Helsinki.

Four CC cell lines (C33A, HeLa, CaSki and SiHa) and a normal human cervical epithelial cell line (H8) were obtained from the Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China) and were cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone, Logan, UT, USA) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) with 1% penicillin/streptomycin (Sigma, St. Louis, MO, USA) at 37°C with 5% CO₂.

The RNA in CC tissues and cell lines was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Reverse transcription of miRNA and mRNA were performed with MiScript II RT kit (Qiagen, Hilden, Germany) and QuantScript RT kit (Tiangen, Beijing, China), respectively. SYBR-Green PCR kit (Qiagen) was used for RT-PCR quantification. The gene expression levels were calculated using the ΔΔCt method with U6 or GAPDH as an internal control. The hsa-miR-1297 primer was synthesized by Sangon (Shanghai, China), and the snRNA U6 qPCR Primer (HmiRQP9001), AEG-1 (HQP016089) and GAPDH (HQP006940) were purchased from GeneCopoeia (Guangzhou, China).

miRNA vectors, including miR-1297 expression and control vector, and miR-1297 inhibitor and the negative control were obtained from GeneCopoeia. The AEG-1 overexpression plasmid and specific siRNA against AEG-1 and a scramble siRNA were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The cells were transfected with the aforementioned vectors using Lipofectamine 2000 reagent (Invitrogen Life Technologies) in accordance with the manufacturer's protocol.

Cellular proteins from CC cells were extracted using RIPA buffer (Beyotime, Shanghai, China). Isolated proteins were subjected to electrophoresis on 4–20% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After being blocked with 5 % non-fat milk/Tris-buffered saline Tween-20 (TBST) the membranes were incubated with the primary antibody (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight and incubated with an HRP-conjugated secondary antibody (1:5,000; ZSGB-BIO, Beijing, China) at room temperature for 2 h. The protein bands were detected and visualized with an enhanced chemiluminescence (ECL) reagent (Amersham, Little Chalfont, UK).

Briefly, 4-µm sections were deparaffinized in xylene, rehydrated by graded ethanol, followed by blocking of endogenous peroxidase activity in 4% hydrogen peroxide for 10 min at room temperature. The corresponding antibody (1:300; Cell Signaling Technology, Inc.) was applied as the primary antibody using a streptavidin peroxidase-conjugated (SP-IHC) method. The staining results were semi-quantitatively evaluated by multiplying the staining intensity and the percentage of positive-stained cells. The percentage of positive cells was scored as follows: 0 for <5%; 1 for 6–25%; 2 for 26–50%; 3 for 51–75%; and 4 for >75%. The staining intensity was assessed as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. Each section was assayed for 10 independent high magnifications (×400) fields to obtain the average scores.

Matrigel-uncoated and -coated Transwell inserts (8-µm pore size; Millipore) were used to evaluate cell migration and invasion. Briefly, 2×10⁴ transfected cells were suspended in 150 µl serum-free RPMI-1640 medium into the upper chamber, and 700 µl RPMI-1640 medium containing 20% FBS was placed in the lower chamber. After 24 h of incubation, the cells were fixed in 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet dye for 15 min. The cells on the inner layer were gently removed with a cotton swab and 5 randomly selected views were counted. Subsequently, the average number of cells per view was calculated.

CC cells seeded into 24-well plates were transfected with 200 ng of miR-1297 mimic or inhibitor or the corresponding control vector along with 50 ng of wild-type (wt) or mutant (mt) 3′-UTR of AEG-1 mRNA. Forty-eight hours after transfection, these cells were collected and the luciferase activity was detected with a Dual-Luciferase Reporter Assay System following the manufacturer's instructions (Promega, Madison, WI, USA).

Data are presented as the mean ± SD of at least 3 independent replicates. SPSS software 16.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA) were used for a two-tailed Students t-test, Pearson's correlation analysis, Kaplan-Meier method and the log-rank test to evaluate the statistical significance. Differences were defined as P<0.05.

We initially investigated the expression level of miR-1297 in 117 pairs of CC and their matched non-tumor tissues. We found that the expression of miR-1297 was significantly lower in CC tissues than those in corresponding non-tumor tissues (P<0.05; Fig. 1A). Moreover, we analyzed the expression of miR-1297 in a panel of CC cell lines and a normal human cervical epithelial cell line (H8). The data revealed that the relative miR-1297 expression was obviously decreased in the CC cell lines compared with the H8 cells (P<0.05; Fig. 1B). These results indicated that decreased miR-1297 expression is potentially correlated with the development and progression of CC.

To explore its clinical relevance in CC tissues, we defined the different miR-1297 groups based on the median expression level. As shown in Table I, low miR-1297 expression was significantly associated with lymph node metastasis (LNM; P=0.008) and lymphovascular space invasion (LVSI; P=0.005). Therefore, these results indicated that decreased miR-1297 was involved in the development and progression of CC. Moreover, survival analysis revealed that the downregulation of miR-1297 was obviously correlated with shorter overall survival (P=0.0002; Fig. 2) in CC patients. Furthermore, the expression of miR-1297 was an independent prognostic factor for predicting the overall survival in CC patients (P=0.003, 0.007, respectively; Table II). These results revealed that miR-1297 could serve as a potential prognostic biomarker in CC patients.

To explore the biological significance of miR-1297 in CC, we transduced CC cell lines with miR-1297 expression or anti-miR-1297 vector which contained different endogenous miR-1297 levels. As determined by qRT-PCR, we confirmed that miR-1297 effectively upregulated miR-1297 in C33A cells (P<0.05; Fig. 3A) or downregulated miR-1297 in CaSki cells (P<0.05; Fig. 3C). As examined by Matrigel-coated (for invasion) and -uncoated (for migration) Transwell assays, miR-1297 overexpression significantly inhibited the migration and invasion of C33A cells (P<0.05; Fig. 3B), whereas miR-1297 knockdown markedly increased the number of migrated and invaded CaSki cells (P<0.05; Fig. 3D). In conclusion, these data revealed that miR-1297 regulated CC cell migration and invasion and may exert an antimetastatic effect on CC.

EMT has been proposed to have a critical role in the initiation of metastasis progression of cancer (20,21). To gain a mechanistic illustration of the potential role of miR-1297 in modulating CC metastasis, EMT markers were assessed. We found that miR-1297 overexpression facilitated the epithelial marker E-cadherin and suppressed N-cadherin and vimentin expression (P<0.05; Fig. 4A). In contrast, miR-1297 knockdown decreased E-cadherin expression and increased N-cadherin and vimentin expression (P<0.05; Fig. 4B). In addition, we further explored the correlation between miR-1297 expression and EMT markers in CC tissues. We found that the expression of E-cadherin in the high-expresssing miR-1297 group was higher than that in the low-expressing miR-1297 group. Conversely, the expression level of vimentin in the high-expressing miR-1297 group was markedly lower than that in the low-expressing miR-1297 group (P<0.05; Fig. 4C). Collectively, these results revealed that miR-1297 functioned as a suppressor of EMT in CC cells.

To elucidate the molecular mechanisms responsible for the functional influence of miR-1297 in CC cells, we searched the publicly available database TargetScan to explore the candidate target genes. Among them, AEG-1 was known to play an important role in CC invasion and metastasis. As shown in Fig. 5A, the sequence complementary to the binding sites of miR-1297 was revealed in the 3′-UTR of AEG-1. To confirm this, we performed a luciferase reporter assay to ascertain that miR-1297 could bind to the 3′-UTR of AEG-1. The results revealed that miR-1297 overexpression significantly decreased the luciferase activity of the wild-type (wt) AEG-1 3′-UTR while it had no influence on that of the mutant (mt) AEG-1 3′-UTR (P<0.05; Fig. 5B). On the contrary, miR-1297 knockdown increased the luciferase activity of the wt AEG-1 3′-UTR (P<0.05; Fig. 5B), but it did not affect the luciferase activity of the mt AEG-1 3′-UTR constructs. In addition, miR-1297 overexpression markedly decreased the mRNA and protein levels of AEG-1 in C33A cells (P<0.05, respectively; Fig. 5C and D). By contrast, the expression of AEG-1 mRNA and protein were significantly increased by the downregulation of miR-1297 in CaSki cells (P<0.05, respectively; Fig. 5C and D).

To further evaluate the relationship between miR-1297 and AEG-1 in CC tissues, we assessed the AEG-1 mRNA and protein expression in two groups of miR-1297. As expected, our data revealed that both the AEG-1 mRNA and protein expression level in the high-expressing miR-1297 group were significantly lower than those in the low-expressing miR-1297 group in CC (P<0.05; Fig. 6A and B). Moreover, we demonstrated that the mRNA level of AEG-1 in the CC tissues was inversely correlated with miR-1297 expression (R²=0.6845, P<0.0001, Fig. 6C). Consistently, as assessed by IHC assay, the AEG-1 protein expression in the miR-1297 high-expressing tumors was obviously lower than that in the miR-1297 low-expressing tumors (P<0.05, Fig. 6D), which was similar with previous studies (18). In conclusion, these data revealed that AEG-1 was a direct downstream target of miR-1297 in CC.

To confirm that AEG-1 is a functional target of miR-1297, AEG-1 was overexpressed by a plasmid vector in miR-1297-overexpressing C33A cells (P<0.05; Fig. 7A). Furthermore, AEG-1 overexpression abrogated the inhibitory effect induced by miR-1297 on cell migration and invasion (P<0.05, respectively; Fig. 7B) and promoted EMT progression (P<0.05; Fig. 7E). Similarly, AEG-1 knockdown by a specific siRNA in the miR-1297-suppressive CaSki cells (P<0.05; Fig. 7C) significantly reversed the promotive function induced by miR-1297 knockdown on cell migration and invasion (P<0.05, respectively; Fig. 7D) and EMT progression (P<0.05; Fig. 7F). These data demonstrated that AEG-1 is not only a downstream target of miR-1297, but also a functional mediator of miR-1297 in CC.