Cervical cancer cell lines: Ca Ski, Hela, Siha, and C33a were purchased from Procell Life Science & Technology Co., Ltd. and the normal cell line H8 was purchased from Shanghai Yu Bo Biotech Co., Ltd. Ca Ski and H8 were cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS (fetal bovine serum) (Gibco; Thermo Fisher Scientific Inc.). Hela, Siha and C33a were cultured in Minimum Essential Medium (Gibco; Thermo Fisher Scientific Inc.) supplemented with 10% heat-inactivated FBS. Penicillin (100 U/ml)/streptomycin (100 µg/ml) (GE Healthcare Life Sciences) was used in the culture medium and cells were grown in a humidified atmosphere containing 5% CO₂ at 37°C. Cells were passed to the next generation every three days. In the present study, tissues were collected from Department of Gynecological Oncology in Sun Yat-sen Memorial Hospital (Guangzhou, China) between June 2016 and May 2017. The inclusion criteria of the patients were as follows: i) Females diagnosed with CC using the International Federation of Gynecology and Obstetrics (FIGO, 2018) system (19); and ii) underwent biopsy or trachelectomy surgery. The exclusion criteria of the patients were as follows: i) Diagnosed with other tumors; ii) underwent radiotherapy or chemotherapy or any other treatment prior to surgery; and iii) CC recurrence. Peritumoral tissue samples were taken at least 1 cm distal to tumor margins. Tissue histology was independently evaluated by two pathologists. A total of 29 CC tissues (mean age, 37.6 years; age range, 21–77 years) and peritumoral tissues were collected and immediately placed and stored in liquid nitrogen. All of the 29 CC tissues and peritumoral tissues were used for reverse-transcription quantitative (RT-q) PCR analysis, 3 CC tissues and corresponding peritumoral tissues were used for microarray analysis and 26 CC tissues were used for ISHH. All patients underwent biopsy or trachelectomy surgery at the Sun Yat-sen Memorial Hospital (Guangzhou, China). All specimens were obtained with the approval of the Sun Yat-sen Memorial Hospital Review Board (approval no. 2015132) (Guangzhou, China) and a written informed content was obtained from each patient.

Human 12×135k Long Non-coding RNA Array was manufactured by NimbleGen Systems Inc. Each array represented all long transcripts, both protein coding mRNAs and lncRNAs in the human genome. In total, >23,000 lncRNAs were collected from the authoritative data sources including NCBI RefSeq (GRCh37 (hg19); http://www.ncbi.nlm.nih.gov/refseq/), UCSC (GRCh37 (hg19); http://genome.ucsc.edu/cgi-bin/hgTables), RNAdb 2.0 (http://research.imb.uq.edu.au/rnadb/), lncRNAs from literatures (20,21) and UCRs (NCBI Build 35 (hg17); http://users.soe.ucsc.edu/~jill/ultra.html).

In total 3 CC tissues and 3 corresponding peritumoral tissues were used to synthesize double-stranded complementary DNA (cDNA). Double-strand cDNA (ds-cDNA) was synthesized from 5 µg of total RNA using a SuperScript ds-cDNA synthesis kit (Invitrogen; Thermo Fisher Scientific Inc.) in the presence of 100 pmol oligo dT primers according to the manufacturer's instructions. Ds-cDNA was cleaned and labeled in accordance with the NimbleGen Gene Expression Analysis protocol (NimbleGen Systems, Inc.). Briefly, ds-cDNA was incubated with 4 µg RNase A at 37°C for 10 min and cleaned using phenol:chloroform:isoamyl alcohol (25:24:1), followed by ice-cold absolute ethanol precipitation. The purified cDNA was quantified using NanoDrop ND-1000 (Thermo Fisher Scientific Inc.). For Cy3 labeling of cDNA, the NimbleGen One-Color DNA labeling kit (NimbleGen Systems, Inc.) was used according to the manufacturer's instructions detailed in the Gene Expression Analysis protocol (NimbleGen Systems, Inc.). In brief, 1 µg ds-cDNA was incubated for 10 min at 98°C with 1 optical density (OD) of Cy3-9mer primer. Next, 100 pmol of deoxynucleoside triphosphates and 100 U of the Klenow fragment (New England Biolabs Inc.) were added and the mixture incubated at 37°C for 2 h. The reaction was stopped by 0.5 mol/l EDTA (0.1 times the volume of the previous mixture), and the labeled ds-cDNA was purified by isopropanol/ethanol precipitation. Microarrays were hybridized at 42°C for 16–20 h with 4 µg of Cy3 labelled ds-cDNA in hybridization buffer/hybridization component A (NimbleGen Systems, Inc.) in a hybridization chamber (hybridization system; NimbleGen Systems, Inc.).

Washing was performed three times using the SeqCap EZ Hybridization and Wash kit (cat. no. 05634261001; NimbleGen Systems, Inc.). After being washed in an ozone-free environment, the slides were scanned using the Axon GenePix 4000B microarray scanner (Molecular Devices, LLC.). Raw data were extracted as pair files using NimbleScan software version 2.5 (Roche NimbleGen, Inc.) and normalized through quantile normalization and the Robust Multichip Average algorithm included in the NimbleScan software. All gene level files were imported into GeneSpring GX software version. 11.5.1 (Agilent Technologies Inc.) or further analysis. Differentially expressed lncRNAs with statistical significance between two groups were identified through volcano plot filtering. Differentially expressed lncRNAs between two samples were identified through fold change filtering. P-value was calculated using the paired t-test. The threshold set for up- and downregulated genes was a fold change ≥2.0 and a P-value ≤0.05. Hierarchical clustering was performed using the GeneSpring GX software version 11.5.1 (Agilent Technologies Inc.).

A total of 4 AK001903-specific small interfering (si)RNAs (si-AK001903-713, siRNA-AK001903-1306, siRNA-AK001903-1051 and siRNA-AK001903-1605) were used to knock down lncRNA-AK001903, and a non-targeting siRNA (si-NC) oligonucleotide was used as a negative control (all Shanghai Gene Pharma Co. Ltd.). Sequences were listed in Table I. Ca Ski were plated at 60% confluence (5×10⁵ cells/well) on a 6-well plate. The following day, the culture medium was replaced with 500 µl of Opti-MEM (Gibco; Thermo Fisher Scientific Inc.). A total of 250 µl of Opti-MEM and 5 µl of Lipofectamine^® RNAiMAX (Invitrogen; Thermo Fisher Scientific Inc.) were mixed in one tube, 20 pM siRNA (Shanghai GenePharma Co. Ltd.) and 250 µl of Opti-MEM were mixed in the other tube. Then they were combined and incubated for 5 min at room temperature. After 6 h incubation in 37°C, the transfection medium was replaced with 2 ml of standard growth medium. Cells were haversted 48 h post transfection and subsequent experiments performed.

Total RNA was extracted from tissues or cell lines (Ca Ski, Hela, Siha, C33a and H8) using TRIzol reagent (Invitrogen; Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. Total RNA (500 ng) was reverse transcribed into cDNA using a PrimeScript^® RT reagent kit with gDNA eraser (Takara Bio Inc.). The protocol for RT was as follows: 37°C for 15 min, 85°C for 5 sec and termination at 4°C. Quantitative PCR was performed using an ABI 7500 Real-Time PCR system and a SYBR^® Premix Ex TaqTM II kit (Takara Bio Inc.). PCR thermocycling conditions were as follows: predenaturation at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec and annealing at 60°C for 30 sec. β-actin was used as an endogenous reference. Fold-changes of the relative expression of target genes were calculated using 2^−ΔΔCq method (22). All experiments were performed in duplicate and repeated twice. Primers for quantitative PCR were presented in Table II.

Tissues were fixed with 4% paraformaldehyde (with 0.1% Diethyl pyrocarbonate) at room temperature for 24 h. Then they were paraffin-embedded and sliced into 20-µm thick slices and spread over PLL-coated glass slides. The paraffin sections were deparaffinized and rehydrated and treated with 0.2 mol/l HCl at 37°C for 20 min, 3 µg/ml proteinase K (PCR grade; Roche Diagnostics) at 37°C for 7 min followed by post-fixation with 4% paraformaldehyde (with 0.1% diethyl pyrocarbonate) for 5 min at room temperature. After acetylation in a solution consisting of 1.5% triethanolamine, 0.25% acetic anhydride, and 0.25% HCl at 40°C for 4 h, the slides were washed by 5X SSC once for 15 min. Pre-hybridization was performed using the pre-hybridization solution (cat. no. AR0152; Boster Biological Technology Co. Ltd.) at 37°C for 4 h. Subsequently, a digoxigenin-labeled oligonucleotide probe (5′-CCACACCCACCTTCCCACATGCATGATA-3′; Shanghai GeneBio Co., Ltd.) was diluted to 200 nM using hybridization buffer (cat. no. AR0062; Boster Biological Technology Co. Ltd.) and hybridized with tissues for 16 h at 55°C. Slides were washed twice at 55°C for 30 min with a solution consisting of 50% formamide, 2X SSC and 0.01% Tween 20, and then 10 µg/ml RNase A was added at 37°C for 1 h in buffer (0.5 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA, and 0.01% Tween 20). Subsequently, the samples were washed in 2X SSC wash buffer with 0.01% Tween 20 at 55°C for 30 min, followed by washing in 0.2X SSC wash buffer with 0.01% Tween 20 at 55°C for 30 min. After additional washing in Tris-buffered saline (TBS, pH 7.6) and blocking in a buffer consisting of 10% Blocking Reagent (cat. no. 11096176001; Roche Diagnostics), 0.1 M maleate, 0.15 M NaCl, and 0.01% Tween 20 in TBS, DIG- labeled probes were detected by biotinylated digoxin antibody (cat. no. AR0147; Boster Biological Technology Co. Ltd.) at 37°C for 1 h, streptavidin-biotin complex-peroxisome (SABC-POD; cat. no. AR0148; Boster Biological Technology Co. Ltd.) at 37°C for 20 min and biotin-horseradish peroxidase (HRP) (cat. no. AR0149; Boster Biological Technology Co. Ltd.) at 37°C for 20 min. The samples were stained using DAB (cat. no. ZLI-9019; Origene Technologies Inc.) and images were captured using light microscopy (Nikon Corporation).

Cell proliferation was assessed using the CCK-8 assay. Ca Ski were plated at 1×10³ cells/well on 96-well plates with three wells for each group. Cell viability was measured over 5 days using a Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies Inc.). A total of 10 µl of CCK-8 (5 mg/ml) was added to each well. After incubating for 4 h at 37°C, the absorbance was determined at 450 nm.

Cell migation and invasion were examined using Polycarbonate Membrane transwell inserts (Costar; Corning Inc.). The upper compartment was pre-coated with RPMI-1640 medium without serum for migation assay and Matrigel for 2 h in 37°C (Corning Inc.) for invasion assay. After 48 h of transfection, 5×10⁴ Ca Ski cells were placed into the upper compartment. RPMI-1640 medium with 20% FBS was used in the lower compartment. Cells were incubated at 37°C for 24 h for the migation assay and 48 h for the invasion assay. Then the compartment were fixed at room temperature with 4% paraformaldehyde for 20 min and stained with crystal violet (1 mg/ml) for 20 min at room temperature, and cells not crossing the membrane were cleaned. Images were captured using light microscopy (Nikon Corporationm).

Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software, Inc.) and SPSS 20.0 software (IBM Corp.). All experiments were performed in triplicate. Comparisons between two groups were performed by paired sample t-tests. Three or more experimental groups were compared by one-way ANOVA followed by the post hoc Bonferroni test. The median value [tissues' CT (cycle threshold) value-corresponding β-actin CT value=7.2] of lncRNA-AK001903 expression level was used to divide patients into high and low lncRNA-AK001903 expression level groups. χ² test was used to compare the association between different clinical features and gene expression level. All data are presented as mean ± standard deviation (SD). P<0.05 was considered to indicate a statistically significant difference.

Hierarchical clustering demonstrated systematic variations in the expression of lncRNAs between CC tissues and corresponding peritumoral tissues (Fig. 1A). Volcano Plots were used to visualize the relationship between fold-change (magnitude of change) and statistical significance (which took both magnitude of change and variability into consideration) (Fig. 1B). According to the microarray expression profiling data, 453 distinctively dysregulated lncRNAs were detected in CC tissues compared to the controls (fold change, ≥2.0 and P<0.05) (Fig. 1A and B). The top 10 of 324 significantly upregulated and 129 significantly downregulated lncRNA transcripts are summarized in Table III. To validate the microarray analysis findings, 5 upregulated and 6 downregulated lncRNAs expression were detected and compared between 9 pairs of CC tissues and their corresponding peritumoral tissues using RT-qPCR. These data confirmed that ASLNC16219 (seqname, AK001903), ASLNC24589 (seqname, U88892), BG419628 (seqname, chr20:1594825-1615675), AI184890 (seqname, HMlincRNA1030), AK097842 (seqname, HMlincRNA810) were upregulated in CC tissues compared to peritumoral tissues, whereas the expression of ASLNC17636 (seqname, AK055280), ASLNC016271 (seqname, AK021467), ASLNC01516 (seqname, NR_024345), ASLNC03532 (seqname, uc001esn), ASLNC14492 (seqname, AY927461) and BF675100 (seqname, chr12:92933675-92975575) was decreased (all P<0.05; Fig. 1C). Thus, the results strongly revealed that expression changes of lncRNAs were involved in the development of cervical cancer.

Among the 20 most significantly differentially expressed lncRNAs (DE lncRNAs) in the CC tissues vs. CP (peritumoral tissues), the most notably upregulated one was lncRNA-AK001903 (fold change, 15.547; Table III). To verify the roles of lncRNA-AK001903 in CC cells, expression of lncRNA-AK001903 in CC cell lines and the normal cell line H8 were determined by RT-qPCR. The results demonstrated that Ca Ski exhibited significantly higher expression level of lncRNA-AK001903 compared with C33a, Hela and Siha cells. Besides, all these cervical cancer cell lines expressed higher level of lncRNA-AK001903 compared with H8 cells (Fig. 2A). In addition, lncRNA-AK001903 expression was assessed in 29 CC tissues and their corresponding peritumoral tissues to confirm the expression change of lncRNA-AK001903 in CC tissues. The result revealed significantly higher levels of lncRNA-AK001903 gene expression compared with corresponding peritumoral tissues (Fig. 2B). Clinicopathologic features of the 29 patients with CC were shown in Table IV. However, according to the results of RT-qPCR, the high and low expression of AK001903 in tumor tissues appeared to be independent of the patient's age, tumor size, FIGO stage, lymph node metastasis, differentiation, and whether the tumor was confined to the cervix (Table IV). These results suggested the probable oncogenic role of lncRNA-AK001903 in cervical cancer tumorigenesis. In addition, an ISHH probe of lncRNA-AK001903 was designed and synthesized to examine the expression differences among CC tissues. The results demonstrated that lncRNA-AK001903 was mostly located in the nucleus of cells and that high lncRNA-AK001903 expression was associated with advanced FIGO stage (Fig. 2C).

As lncRNA-AK001903 was highly expressed in CC cells and tissues compared with H8 cells and peritumoral tissues and related to the FIGO stage of CC, further experimentation was performed to investigate whether lncRNA AK001903 may be a potential oncogene during the progression of CC. Four siRNAs targeting lncRNA-AK001903 at different sites (Table I) were designed and transfected into Ca Ski cells due to the highest expression of lncRNA-AK001903 in Ca Ski. The results demonstrated that of the 4 siRNAs used siRNA-lncRNA-AK001903-1605 produced the most effective interference of lncRNA-AK001903 expression and was then used for further experimentation (Fig. 3A). Next, the effects of knockdown of lncRNA-AK001903 on the proliferation, invasion and migration of Ca Ski cells was investigated. The CCK-8 assay demonstrated that lncRNA-AK001903 knockdown significantly inhibited the proliferation of lncRNA-AK001903 compared to the si-NC group (Fig. 3B). Transwell assay results demonstrated that the ability of migration and invasion were suppressed by knockdown of lncRNA-AK001903 (Fig. 3C).