Cell lines and cell culture
The HCT116 cell line was procured from Procell (Wu Han, China) and cultured in complete McCoy's 5A medium (PM150710B, Procell). 293 Phoenix Ampho (293PA) and HEK-293 cells were kindly provided by Prof. Tong-Chuan HE (University of Chicago, Chicago, USA) and maintained in complete Dulbecco's Modified Eagle's medium-supplemented with 10% fetal bovine serum (FBS, Hyclone, CA, USA), 100 units of penicillin, and 100 mg of streptomycin-in an incubator at 37 °C with 5% CO2.
Retrovirus production and stable cell lines
lncRNA-KCNQ1OT1 siRNA (siKCNQ1OT1) was designed and cloned into the pSEB61 vector (pSEB61-siKCNQ1OT1) and synthesized by Decoding Therapeutics Corp (Mt Prospect, USA). Retroviral vector pSEB61-siKCNQ1OT1 was packaged into a retrovirus in 293PA cells. Briefly, 293PA cells (70-80% density) were seeded onto 100-mm dishes. pSEB61-siKCNQ1OT1 and pCL-Ampho vectors were co-transfected into 293PA cells to harvest the retrovirus at intervals of 12 h. HCT116 cells, at a density of 50%, were infected with packaged retrovirus pSEB61-siKCNQ1OT1 concentrated using PEG8000 (89510, Sigma). Blasticidin S (0.5 μg/ml) selection yielded stable knockdown expression of KCNQ1OT1 or control cell lines, designated as HCT116-siKCN and HCT116-NC, respectively.
Total RNA extraction and real-time polymerase chain reaction (qPCR)
Total RNA was extracted using TRIzol Reagents (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and used to generate cDNA templates via reverse transcription. qRT-PCR was performed as described in a instructions. The primers used in this study are shown in Table 1. SYBR Green-based qPCR analysis was carried out using the thermo cycler CXF-Connect (Bio-Rad, CA). GAPDH was used as an internal control.
RNA isolation, cDNA library construction, sequencing, and data analysis
Total RNA was extracted from HCT116 and HCT116-SiKCN cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. After RNA quality was assessed using NanoDrop2000, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. The fragments per kilobase of transcript per million fragments mapped (FPKM) value was used to evaluate gene expression. Differentially expressed genes (DEGs) were identified using the tool Cuffdiff. The significant genes were selected based on the criteria of |log2(fold change)| ≥ 1 and false discovery rate < 0.01. The statistical enrichment of DEGs in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was compared to the whole genome background.
Immunofluorescence and Immunohistochemical staining
Immunofluorescence staining was performed as previously described instructions. Briefly, cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 10% Block AidTM (B10710, Invitrogen), followed by incubation with primary antibodies against the following antigens: B-cell lymphoma 2 (BCL2) (1:200, ab182858, Abcam, CA, USA), proliferating cell nuclear antigen (PCNA) (1:200, ab92552, Abcam, CA, USA), and cyclin E2 (CCNE2) (1:200, ab40890, Abcam, CA, USA ). Cells were then washed and incubated with 2 µg/mL Donkey Anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (A-21207-1, Thermo Scientific,CA, USA) for immunofluorescence staining or ?????for immunohistochemical staining . Nuclei were counterstained with diamidino-2-phenylindole (DAPI) at a concentration of 1.43 µM. The stained cells were imaged using a laser scanning confocal microscope. Negative controls were stained without primary antibodies or with control IgG.
Total proteins were extracted and quantified using RIPA lysis buffer and a BCA detection kit (Beyotime, China) according to the manufacturer’s instructions. Next, equal amounts of protein were separated using 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Subsequently, the membranes were blocked and incubated with primary antibodies against BCL2 (1:1000, ab182858, Abcam), caspase3 (CASP3) (1:1000, 9662s, Cell Signaling Technology), PCNA (1:1000, ab92552, Abcam), CCNE2 (1:1000, ab40890, Abcam), and GAPDH (1:1000, 8457s, Cell Signaling Technology) at 4 °C overnight and then with the appropriate peroxidase–conjugated secondary antibody at room temperature . Chemiluminescence (Beyotime) reagent was used to detect the bands, and Image J software was employed for protein band quantification.
Crystal violet staining to determine cell proliferation
HCT116 and HCT116-siKCN cells were plated into 35-mm dishes at a density of 2 × 105 cells/dish and stained with crystal violet/formalin solution at 24, 48, 72, and 96 h for 20-30 min after gently washing with phosphate-buffered saline (PBS). Crystal violet was desorbed from the stained cells using acetic acid, following which the number of cells was quantified at 570-590 nm using a microplate reader.
Cell cycle distribution assay
HCT116 and HCT116-siKCN cells were digested with 0.25% trypsin and washed with 0.1 M PBS. Approximately 1 × 106 cells were fixed using 75% ethyl alcohol at 4 °C overnight. Cells were washed with cold PBS and then stained with propidium iodide (C1052, Beyotime) at 37 °C for 30 min in the dark. Cell analysis was performed using a flow cytometer (FACScan) equipped with CellQuest software (both from BD Biosciences).
In vivo bioluminescence tumor xenograft
All animal studies were conducted in accordance with the guidelines approved by the Ethics Committee of Wannan Medical College(No.2021012). Ad-FLuc was kindly provided by Prof. Tong-Chuan HE (University of Chicago). Both HCT116 and HCT116-siKCN cells were infected with Ad-Fluc to yield HCT116-FLuc and HCT116-siKCN-FLuc, respectively. Cells (1.5 × 107 per injection) were collected and injected subcutaneously into the flanks of 4–6-week-old BALB/c nude mice. After 2 and 14 days of injection, the animals were intraperitoneally injected with luciferase (ab145164, Abcam) and bioluminescence images were obtained using the Berthold LB983 imaging system. Data from bioluminescence images were analyzed using INDIGO software. Mice were sacrificed 20 days after injection, and the tumors were collected, making paraffin section for further analyses. Immunostaining with anti-PCNA (1:400, ab92552, Abcam) and anti-CCNE2 (1:400, ab40890, Abcam) was performed to detect the positive cells, which were quantified in three different high-power fields of each section.
All data are expressed as the mean ± standard deviation. The statistical analysis was performed using SPSS 22.0 software. The statistical significance of the data was evaluated using Student’s t-test or one-way analysis of variance. P < 0.05 was considered statistically significant.