Tissue samples
A total of 46 pairs of cervical cancer tissue and nearby non-tumor tissue were collected from patients at the First Affiliated Hospital of Bengbu Medical College, China, during 2016 and 2017. This study was approved by the Ethic Committee of the Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University and patients provided informed consent. After excision, all tissues were immediately frozen in liquid nitrogen until RNA extraction.
Bioinformatic analysis
The expression level of CCND2 AS1 in tissues was analyzed using Gene Expression Profiling Interactive Analysis (GEPIA), a web server for cancer and normal gene expression profiling and interactive analyses [19]. The |Log2FC| cutoff was 1 and the p-value cutoff was 0.01. A box plot was generated to show the differential expression of CCND2 AS1 between 306 cervical cancer tissues and 13 normal cervical tissues.
Quantitative real-time PCR (qRT-PCR)
RNA was extracted from 100 mg of tissue samples or 5 × 106 cells using Trizol reagent (Invitrogen, USA) as previous described [20]. The quality and quantity of the RNA were assessed by agarose gel electrophoresis and NanoDrop spectrophotometry (ND-1000) (Thermo Fisher Scientific, Waltham, MA, USA). RNA was reverse transcribed to cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (TAKARA, Japan) according to the manufacturer’s instruction. LncRNA expression levels were examined using a SYBR Green PCR kit (TAKARA, Japan). Primers were synthesized by Genscript Corporation (Nanjing, China). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was analyzed as an internal control for normalization. The primer sequences were (5′ to 3′): CCND2 AS1 forward CAAGCTGGAACCCTGCAAGA, reverse AAGGGTATACCTTCCTCCCCAA; GAPDH forward AGAAGGCTGGGGCTCATTTG, and reverse GCAGGAGGCATTGCTGATGAT. The amplification conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 30 s and 60 °C for 30 s. Relative lncRNA expression levels were quantified by the 2−ΔΔCt method.
Cell culture and transfection
The human cervical cancer cell lines HeLa and SiHa were obtained from the cell bank of the Chinese Academy of Science (Shanghai, China) and were cultured in DMEM medium (Wisent, China) with 10% fetal bovine serum (FBS, Wisent) and 1% penicillin and streptomycin at 37 °C in a humidified 5% CO2 incubator. CCND2 AS1 and a control scramble sequence were synthesized by Genscript and cloned into pcDNA3.1 vector to produce pCCND2 AS1 and pcDNA3.1 plasmids, respectively. For transfection, HeLa and SiHa were plated at 1.5 × 105 and 2 × 105 cells per well, respectively, in a 6-well plate for 24 h, and then transfected with pCCND2 AS1 or pcDNA3.1 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. G418 (Gibco, Grand Island, USA) was added at 400 μg μl−1 to generate the stable HeLa and SiHa cell lines. HeLa and SiHa cells were also transiently transfected with small interfering RNAs (siRNAs; GenePharma, China) against CCND2 AS1 and negative control (NC) sequences. The sequence were (5′ to 3′): SiRNA1 sense GGGCUGGUCUCUUUGAGUUTT, antisense AACUCAAAGAGACCAGCCCTT; SiRNA2 sense: GCCAAGAAACGGUCCAGAATT, antisense UUCUGGACCGUUUCUUGGCTT; SiRNA3 sense GCAAAUCUGAAGCCACAAATT, antisense AACUCAAAGAGACCAGCCCTT.
Cell proliferation assay
Cell proliferation was detected using a colorimetric Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan) following the manufacturer’s protocol. The kit contains WST-8 [2-(2-methoxy-4-nitrophenyl)-3(4-nitrophenyl) -5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt], which produces a water-soluble formazan dye upon reduction in the presence of an electron mediator, as is reduced by dehydrogenases in cells to give an orange colored product (formazan), which is soluble in the culture medium. The amount of the formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells. Cells were added to 96-well plates at 1 × 103 100 μl−1 per well (n = 3) and cultured for 1, 2, 3, 4, 5 or 6 and 7 days. The medium was then replaced with a mixture of 90 μl of fresh DMEM medium and 10 μl of CCK‑8 reagent and the cells were incubated for an additional 2 h. The absorbance at 450 nm, which is proportional to cell number, was measured using a microplate reader (iMark, USA).
Cell cycle
Cells were cultured at 1 × 106 well−1 in 6-well plates and synchronized by starvation by culturing in medium with 1% FBS for 2 days. The cells were then fixed in cold 70% alcohol at −20 °C, washed twice with phosphate-buffered saline (PBS) containing 1% FBS, and then treated with PBS containing 0.02% TritonX-100, 0.1 mg ml−1 RNase (Sigma-Aldrich, St. Louis, Missouri, USA), and 10 mg ml−1 propidium iodide (Sigma-Aldrich, St. Louis, Missouri, USA) for 30 min at 37 °C. Cell cycle distribution was examined by flow cytometry using a FACScan flow cytometer (Becton–Dickinson, San Jose, CA, USA). The relative number of cells in each phase of the cell cycle was analyzed using the Modfit program (Verity Software House, Topsham, ME, USA) [21].
Tumor xenograft mouse model
Female BALB/c nude mice at 4-week-old were purchased from Laboratory Animal Center of Yangzhou University. For each cell type, 107 cells in 100 μl PBS were injected into the right dorsal flank of mice. Tumor size was monitored weekly using a Vernier caliper to measure the length (L) and width (W) of the tumors. Volume was calculated as: V = 0.5 × L × W2. The mice were euthanized 4 weeks after injection and the tumors were removed and weighed. The experiments were conducted in accordance with the guidelines of the Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University.
Western blot analysis
At 72 h after transfection, aliquots of 5 × 106 cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology, China), and the lysate was determined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). A total of 30 μg protein per lane was resolved by 10% SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, USA). The membrane was blocked with 5% non-fat milk and then incubated overnight with primary antibodies against β-actin (Sigma-Aldrich, St. Louis, Missouri, USA), CDK-4 (Cell Signaling Technology, USA), CCND1 (Cell Signaling Technology, USA), or CCND2 (Cell Signaling Technology, USA). The membrane was washed in TBST and incubated with the secondary goat anti-mouse IgG antibody (Beyotime Institute of Biotechnology, Shanghai, China). An enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) was used to detect the protein bands using a FluorChem E System (ProteinSimple, CA, USA).
Methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS)
Genomic DNA was extracted from cells using an Axygen Miniprep kit and then treated with bisulfite using a Methylamp DNA Modification Kit (Epigentek, USA) according to the manufacturer’s instruction. Samples of 50 ng modified DNA were amplified to determine the proportion of methylated (M) and unmethylated (U) DNA in the promoter region of CCND2 AS1. The primer sequences (5′ to 3′) for MSP were: CCND2 AS1 (M) forward TATAGTTTTTTCGCGGTTAGC, reverse TAAAATCCCGACTCCGAA; CCND2 AS1 (U) forward ATGTATAGTTTTTTTGTGGTTAGT, and reverse AACTAAAATCCCAACTCCAAA. For BGS, the primers were CCND2 AS1 forward GTATTTAGGAGTTGTAGATGGG, and reverse CCCCAAACATTTTT TCCAATTAT. The PCR conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 30 s and 60 °C for 30 s. PCR products were analyzed by agarose gel electrophoresis. For BGS, PCR products were purified and subcloned into a pEASY-T3 vector (TransGen Biotech, Beijing, China). Ten colonies were randomly chosen and sequenced to assess methylation at each CpG site.
Statistical analysis
Data were expressed as the mean ± SD of at least three independent experiments. Analyses were performed using SPSS Statistics 17 (SPSS, Chicago, IL, USA). Differences between group means were assessed using Student’s t-test, and the associations between CCND2 AS1 expression and clinicopathological features were analyzed using the Chi squared (χ2) test. All p values were two-sided and p < 0.05 was considered to be statistically significant.