80 CC tissues and the matched adjacent non-cancerous tissues were collected from patients at the First Affiliated Hospital of Nanchang University. Permission of this study was given by the Ethics Approval Committee of the First Affiliated Hospital of Nanchang University. The written informed consents had been collected from all patients, who underwent no chemo- or radiotherapy prior to the surgical dissection. After dissection, tissues were instantly snap-frozen in the liquid nitrogen and maintained under −80 °C for subsequent use.
Cell lines and cell culture
CC cell lines (C33A, HeLa, CaSki, SiHa) and human embryonic kidney cell line 293 T (HEK293T) were bought from BeNa Culture Collection (Beijing, China). The cervical epithelial immortalized cells (H8) were provided by the Cell Resource Database of Chinese Academy of Sciences (Shanghai, China). To culture the cells, Roswell Park Memorial Institute (RPMI)-1640 (Thermo Fisher Scientific, Waltham, MA, USA) was used, with the supplementary of 10% fatal bovine serum (FBS; Wisent, Quebec, Canada), streptomycin (100 µg/mL), and penicillin (100 µg/mL). The incubation atmosphere was humidified, with 95% air and 5% CO2 at 37 °C.
MiR-3150b-3p inhibitor and miR-3150b-3p mimics (RiboBio, Guangzhou, China) were used for the knockdown or overexpression of miR-3150b-3p, and the negative controls were anti-NC and NC mimics. LINC00885 and BAZ2A knockdown was realized by specific short hairpin RNAs (shRNAs) targeting LINC00885 and BAZ2A, termed sh-LINC00885#1/2/3 and sh-BAZ2A#1/2/3. The scramble shRNAs (shCtrl) were negative controls. The sequences of sh-LINC00885 and sh-BAZ2A, as well as the corresponding sh-Ctrl, were listed in Additional file. The overexpression of LINC00885 was realized through pcDNA3.1/LINC00885 plasmids, with empty vector pcDNA3.1 (GenePharma, Suzhou, China) as negative controls. The cell transfection was accomplished by the utilization of Lipofectamine 2000 (Invitrogen) as required.
To obtain total RNAs from CC tumors or cells, the Trizol reagent (Invitrogen) was utilized. NanoDrop was applied to determine the purity and concentration of total RNAs. The generation of complementary DNA (cDNA) was realized by utilizing the First Strand cDNA Synthesis kit (Thermo Scientific). Gene expressions were quantified via the real-time PCR applying the SYBR EX TAQ (Takara, Dalian, China). GAPDH (for mRNA) and U6 (for miRNA) were the internal controls. The calculation of relative expression was on the basis of 2−ΔΔCt method. Primers were used as follows:
Cell proliferation assay
The measurement of cell proliferation was carried out by using reagent CCK-8 (Roche, Basel, Switzerland). CC cells were plated in the 96-well microtiter plates (Corning, NY, USA) with the density of 1 × 103 per well. After incubation for 0, 24, 48, 72, and 96 h, CCK-8 solution (10 μL) was added into each well for 2-h incubation. The absorbance (wavelength: 450 nm) was evaluated to examine the cell viability.
The cell proliferation was also examined by utilizing the EdU DNA Proliferation in vitro Detection kit (RiboBio, China). CC cells were seeded into the 96-well plate. Subsequent to transfection for 48 h, CC cells were stained by EdU and the cell nuclei was stained by 4',6-diamidino-2-phenylindole (DAPI) (Cell Signaling Technology, Danvers, MA, USA). The proportion of EdU-positive cells was calculated to determine the cell proliferation.
Colony formation assay
For colony formation assay, cells were seeded in 6-well plate with 5 × 102 cells each well. After incubation for 2 weeks, the cells were subjected to fixation with 4% paraformaldehyde and staining with 0.1% crystal violet. The colonies with over 50 cells were observed and evaluated with a light microscope.
TUNEL kit (Vazyme, TUNEL Bright-Red Apoptosis Detection Kit, A113) was used to evaluate apoptosis of CC cells. In short, cell slides were blocked by the hydrogen peroxide solution after PBS washing. Subsequent to the permeabilization in Trixon-100 (Sigma-Aldrich Co., St Louis, MO, USA), CC cell slides were incubated with the TUNEL solution for 1.5 h, followed by DAPI (Cell Signaling Technology) staining. The TUNEL-positive cells were observed utilizing the fluorescence microscopy (DMI4000B, Leica, Mannheim, Germany).
Subcellular fractionation analysis
Subcellular isolation of RNAs in SiHa and HeLa cells was carried out by utilizing the Cytoplasmic and Nuclear RNA Purification Kit (Norgenbiotek Corporation, Thorold, ON, Canada) according to the manufacturer's instructions. The expression of LINC00885 in nucleus and cytoplasm was evaluated by RT-qPCR, with U6 and GAPDH as the nuclear and cytoplasmic controls.
To determine the cellular localization of LINC00885, FISH was performed by utilizing the RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, California, USA). CC cells were subjected to 24-h incubation on the culture slides (Thermo Fisher Scientific, Inc., Waltham, MA, USA), followed by 0.5-h fixation in 4% paraformaldehyde. Then, the cell slides were treated with Hydrogen Peroxide and Protease III, followed by 2-h hybridization with the LINC00885 FISH probe (RiboBio, Guangzhou, China) in the HybEZ Oven (Advanced Cell Diagnostics). Thereafter, cells were washed and subjected to dehydration in the graded ethanol. Finally, slides were stained with DAPI (Cell Signaling Technologies, Danvers, USA). The observation and photographing of the cell slides was accomplished by using a FV1000 confocal laser microscope (Olympus, Tokyo, Japan).
The interaction of miR-3150b-3p with LINC00885 and BAZ2A was evaluated by RIP assay using the EZMagna RIP Kit (Millipore, Billerica, MA, USA). CC cells were lysed by using the complete RNA immunoprecipitation (RIP) lysis buffer. The magnetic beads conjugated with the antibodies against Argonaute 2 (Ago2) or immunoglobulin G (IgG) (Millipore, Billerica, MA, USA) were incubated with the CC cell extracts for 6 h at 4℃. After the removal of beads and the elution of RNA, the expressions of miR-3150b-3p, LINC00885, and ZAB2A mRNA were analyzed by RT-qPCR.
The biotin-labeled LINC00885 and antisense LINC00885 (LINC00885-AS) were subjected to reverse transcription by utilizing the Biotin RNA Labeling Mix (Roche, Basel, Switzerland) and T7 RNA polymerase (Takara Biomedical Technology), followed by the treatment of RNase-free DNase I (Roche, Basel, Switzerland) and the purification by RNeasy Mini Kit (Qiagen, MD, USA). Subsequent to the incubation of cell lysates with the biotin-labeled probes and the Dynabeads M-280 Streptavidin (Thermo Fisher Scientific), the RNAs pulled down were eluted and measured by RT-qPCR.
Luciferase reporter assay
Full sequence of LINC00885 containing the predicted miR-3150b-3p binding sequences or muated miR-3150b-3p binding sequences were respectively cloned into the pmirGLO Expression Vector (Promega, Madison, WI, USA) to generate the LINC00885-WT/MUT reporter plasmids. Wide type BAZ2A 3’UTR with miR-3150b-3p binding site, or with miR-3150b-3p binding site 1/2 mutated was subcloned into pmirGLO vectors to construct BAZ2A-WT, BAZ2A-MUT-1 (with binding site 1 mutated), BAZ2A-MUT-2 (with binding site 2 mutated) and BAZ2A-MUT-1 + 2 (with binding sites 1 and 2 both mutated). These plasmids were co-transfected with miR-3150b-3p mimics or NC mimics respectively into HEK293T cells using the Lipofectamine 2000 (Invitrogen). A dual-Luciferase Reporter Assay System (Promega) was utilized to evaluate the luciferase activities, with Renilla luciferase activity as normalized control.
RIPA protein extraction reagent (Beyotime, Shanghai, China) with the addition of PMSF (Roche, Basel, Switzerland) was used to lyse the cells. Protein extracts were separated by utilizing the 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and were then transferred onto the nitrocellulose membranes (Sigma, St. Louis, MO, USA), which were incubated with specific primary antibodies for 12 h, followed by 2-h incubation with secondary antibodies. Detection of protein bands was done by the enhanced chemiluminescent (ECL) method. Primary antibodies against total caspase 3, cleaved-caspase 3, total caspase 9, cleaved-caspase 9, BAZ2A, PCNA, Ki67, and GAPDH were all from Abcam (Cambridge, MA, USA).
The five-week-old female athymic BALB/c mice were raised in the pathogen-free condition, and the animal experiment was conducted according to the protocols authorized by the animal center of the First Affiliated Hospital of Nanchang University. All mice were randomly grouped in two, with one group subjected to the subcutaneous injection of SiHa cells transfected with pcDNA3.1 and the other group subjected to the subcutaneous injection of SiHa cells transfected with pcDNA3.1/LINC00885. The SiHa cells with indicated transfection were harvested at a density of 2 × 107 cells/mL. Then, 0.1 mL of the suspend SiHa cells were injected in nude mice at both sides of the posterior flank. Then, tumor volumes were measured every 4 days. At the 20th day after injection, all mice were sacrificed and tumors were dissected for the evaluation of final tumor volume and weight and also for RNA and protein detection via RT-qPCR and western blot analysis.
All experiments were performed for at least 3 times. All data was demonstrated as the mean ± standard deviation (SD). The analyses and presentation of data were conducted by the SPSS 19.0 statistical software and GraphPad Prism V5.0 (GraphPad Software, Inc., La Jolla, CA, USA) software. Student’s t-test or ANOVA were used for the difference measurement between 2 groups or more than 2 groups. The expression correlation was evaluated by Spearman’s correlation analysis. Overall survival of CC patients was analyzed by Kaplan–Meier analysis and log-rank test. P < 0.05 was considered to possess the statistical significance.