2.1 Human tissue samples
This study was conducted in accordance with the principles embodied in the Declaration of Helsinki. The experimental protocol involving human tissues for this study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University. Additionally, for this study, written informed consent was obtained from each human tissue provider. Forty pairs of CRC tumor tissue and adjacent non-tumor tissue samples were collected from patients who had undergone resection of the primary CRC at the First Affiliated Hospital of Chongqing Medical University (Chongqing, China). Cases were screened according to the following criteria: the resected specimen was confirmed to be CRC by pathological examination, patients did not receive chemotherapy or radiotherapy preoperatively, and patients with hereditary CRC such as Lynch syndrome were excluded. After harvesting, tissue specimens were immediately frozen in liquid nitrogen and stored at -80℃ until use.
2.2 Cell lines and culture conditions
Five CRC cell lines, including SW480, SW620, Caco2, HT29, and LoVo, the human normal colon epithelial cell line NCM460, and the human embryonic kidney cell line HEK293T were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, NY, USA) with 10% fetal bovine serum (FBS, Gibco, NY, USA) and 1% penicillin streptomycin solution (Beyotime, Shanghai, China. The cultural environment was a 37℃ incubator with 5% CO2. No mycoplasma contamination was found in any of the cell lines, as indicated by mycoplasma detection kits (Lonza, Switzerland).
2.3 RNA extraction and Real-Time Quantitative Reverse Transcription PCR (qRT-PCR)
Total RNA from cells and tissues was extracted with Trizol reagent (Invitrogen, CA, USA) and stored at -80℃. RNA concentration and the optical density (OD) value were measured with the Nano-500 microspectrophotometer (Allsheng, Hangzhou, China). Next, 1 µg RNA was used to synthesize cDNA using the Prime-Script™ RT kit(Takara,Tokyo,Japan)as directed by the manufacturer's instructions. In addition, 2x SYBR Green qPCR Master Mix (Bimake, TX, USA) was employed for qPCR experiments performed in the ABI StepOne Real-time Detection System (LTC, Carlsbad, USA). There were three replicate holes for each tested sample, and relative RNA levels were compared using the comparative 2 − ΔΔCT method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified as the internal reference. The primer sequences for qRT-PCR are shown in Table S1 and were synthesized by the TsingKe Company (Chongqing, China)
2.4 Subcellular fractionation
The cytoplasmic and nuclear fractions of the HT29 and LoVo cells were isolated using the Minute™ Cytoplasmic and Nuclear Extraction Kit (Invent, Plymouth, USA) in accordance with the instruction manual. Cytoplasmic and nuclear RNA were then extracted by Trizol reagent. qRT-PCR was performed to measure the relative expression levels of cytoplasmic and nuclear specific RNAs in CRC cells. GAPDH and U6 small nuclear RNA were considered as cytoplasmic and nuclear controls, respectively.
2.5 Plasmid construction and transfection
Short hairpin RNA (shRNA) directed against human IGFL2-AS1 and HIF-1α and negative control short hairpin negative control RNA (sh-nc) were designed (the sequences are shown in Table S2) and then inserted into the lentiviral vector pGreenPuro (SBI, CA, USA) at the EcoR I/BamH I site. For the overexpression vector (oeIGFL2-AS1 and oeCA9), the full-length cDNA of IGFL2-AS1 and the cDNA sequence containing the CDS region of CA9 were synthesized, PCR-amplified by the TsingKe Company (Chongqing, China), and ligated into the pCDH-CMVMCS-EF1-CopGFP-T2A-puro vector (SBI, CA, USA). It was verified that the nucleotide sequences were correct in all constructed vectors by sequencing.
The packaging plasmid psPAX2, envelope plasmid pMD2.G (Addgene, MA, USA), and HEK293T cells were used for lentivirus packaging. The lentiviral vector, auxiliary packaging plasmid, and Lipofectamine 2000 (Invitrogen, CA, USA) were co-transfected according to the manufacturer's instructions, and the lentiviral supernatant of the HEK293T cells was harvested 48 h later. HT29 and LoVo cells were incubated with the viral fluid containing 10 µg/mL polybrene (Solarbio, Beijing, China) for 72 h and subsequently screened with 2 mg/mL puromycin (Beyotime, Shanghai, China) for 14 days to construct stable transfection target plasmid CRC cell lines.
2.6 Cell counting kit-8 (CCK-8) assay
Cell viability was examined by the cell counting kit (CCK)-8 assay (Beyotime, Shanghai, China). HT29 and LoVo cells from the control and treatment groups in the logarithmic growth phase were inoculated into 96-well plates at a density of 2×103 cells/well, with three replicate wells for each group. After the cells became adherent (seeding for 6 h), each well was supplemented with 10 µL of CCK-8 reagent and incubated at 37℃ for another 2 h. The OD value of all cells at 450 nm was measured using a spectrophotometric plate reader (BioTek, USA). Next, CCK-8 reagent was added to the corresponding wells at 24, 48, 72, and 96 h after cell-plating, and cell viabilities were further measured according to the OD value.
2.7 5-Ethynyl-2’-deoxyuridine (EdU) incorporation assay
Cell proliferation was tested by EdU assay using an EdU Assay Kit (C0075S, Beyotime, Shanghai, China). Following the manufacturer's protocols, 2 × 105 CRC cells that were stably transfected with lentiviral vectors were seeded in 12-well plates, which were plated with cell climbing slices and cultured overnight. Then, 10 µM EdU working solution was added to the medium and incubated at 37℃ for 2 h to complete the EdU labeling. Next, cells in each group were fixed in 4% paraformaldehyde for 15 min. After the fixative was washed off with potassium buffered saline (PBS), cells were permeabilized with 0.3% Triton X-100 for 15 min. After washing, cells were incubated with the Click Reaction Cocktail configured according to the instructions away from light for 30 min. Thereafter, the nuclei were counterstained with Hoechst 33342 for 10 min while avoiding light. Finally, fluorescence images were taken by confocal laser scanning microscopy (Leica, Germany).
2.8 Colony formation assay
A total of 2 × 103 cells in the control and treatment groups were seeded in 6-well plates and cultured in complete medium at 37°C in a humidified atmosphere with 5% CO2. The cells were harvested after 14 days and fixed with 4% paraformaldehyde at room temperature for 20 min. After the fixative was washed off with PBS, 0.1% crystal violet was used to stain all cells for 15 min. The cells were washed three times again with PBS buffer, and the number of cell colonies (> 50 cells/colony) was counted using ImageJ software (NIH, MD, USA).
2.9 RNA sequencing analysis
Total RNA was extracted from the control group and IGFL2-AS1 knockdown LoVo cells using Trizol reagent. RNA samples were subjected to RNA Sequencing Analysis by Personalbio Technology Co., Ltd (Shanghai, China).
2.10 Protein extraction and Western blotting
Total proteins from cells and mice tissues were extracted by ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) containing 1% phenylmethylsulfonyl fluoride (PMSF, Beyotime, Shanghai, China). The concentration of proteins was determined using the Bicinchoninic Acid Assay (BCA) Kit (Beyotime, Shanghai, China) according to the manufacturer's protocols. Then, the cell lysates were supplied with 5× loading buffer and boiled for 8 min to fully denature the proteins.
Subsequently, 30 µg of protein sample was separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes in a semi-wet system. Membranes were blocked with 5% skimmed milk in 1× Tris Buffered Saline with Tween (TBST) for 120 min at room temperature. After blocking, membranes were incubated with specific primary antibodies against CA9 (1:1000, ab243660, Abcam, Cambridge, UK), HIF-1α (1:2000, 66730-1 -Ig, Proteintech, Wuhan, China), and GAPDH (1:8000, 10494-1-AP, Proteintech, Wuhan, China) at 4℃ overnight (14–16 h). Next, the membranes were washed with 1× TBST three times, followed by incubation with horseradish peroxidase-labeled goat anti-rabbit (1:2000, SA00001-2, Proteintech, Wuhan, China) or goat anti-mouse secondary antibody (1:2000, Beyotime, Shanghai, China) for 90 min at room temperature, after which any residual antibody was washed away by 1× TBST. Finally, specific protein bands were detected with the enhanced chemiluminescence ECL Kit (Advansta, CA, USA) and visualized with ChampChemi imaging system (SCS, Beijing, China). All antibodies were diluted in 1x TBST containing 5% bovine serum albumin (BSA).The gray values of all protein bands were analyzed using ImageJ software.
2.11 Immunohistochemical (IHC) staining
Human CRC tissues and nude mouse tumor tissues were fixed with 4% paraformaldehyde for 24 h, embedded in paraffin, and cut into 4 µm sections. The paraffin sections were baked at 60℃ for 2 h and dewaxed in fresh xylene for 30 min. Then, the slides were hydrated with gradient alcohol, followed by boiling in a pressure cooker containing sodium citrate for 2 min to retrieve the antigens. Next, sections cooled to room temperature were incubated with an endogenous peroxidase blocking agent for 15 min and blocked with goat serum blocking solution for 30 min. The slices were incubated with specific primary antibodies against CA9 (1:2000, ab243660, Abcam, Cambridge, UK), HIF-1α (1:300, 66730-1-Ig, Proteintech, Wuhan, China) and Ki67 (1:500, ab92742, Abcam, Cambridge, UK) overnight (14–16 h) at 4℃. After washing with PBS buffer, the slices were incubated with biotin-labeled secondary antibodies for 30 min and subsequently incubated with horseradish enzyme-labeled chain avidin solution for 30 min. Finally, positive staining was visualized with brown 3,3’-diaminobenzidine tetrahydrochloride (DAB, ZSGB, Beijing, China), and counterstaining was performed with hematoxylin. The sections were mounted with 60% neutral resin. The stained sections were scanned with the Pannoramic DESK scanner (3DHISTECH, Budapest, Hungary). The images in 200× and 400× were observed and acquired with the CaseViewer 2.4 software module (3DHISTECH, Budapest, Hungary).
2.12 Xenograft mouse model
The animal experiment protocol of this study was authorized by the Animal Experiment Ethics Committee of Chongqing Medical University. Male BALB/c-nu nude mice 4–5 weeks of age were purchased from HFK Bioscience Co., Ltd. (Beijing, China). All animals were divided randomly into four groups (n = 3 mice per group) and reared under SPF-grade sterile conditions in the Laboratory Animal Center of Chongqing Medical University. Next, 1×107 LoVo cells stably transfected with sh-nc, shIGFL2-AS1#1, pCDH, and oe IGFL2-AS1 were resuspended in 100 µL of PBS solution and subcutaneously injected into the right armpit of the nude mice in each group. Tumor volumes were measured every 4 days from day 7, which was calculated as follows: volume (mm3) = 0.5 × (largest diameter) × (smallest diameter)2. On day 27, all mice were sacrificed by cervical dislocation; the tumor tissues were collected, weighed, and photographed. Proteins from tumor tissues were extracted as previously described, and IHC staining was conducted on mice tumor sections.
2.13 Statistical analysis
Statistical analysis was performed with GraphPad Prism 8 software (GraphPad software, CA, USA). All statistical differences were analyzed using the unpaired two-tailed Student's t-test, and results were presented as means ± standard deviation (SD). The differences were considered significant for p-values < 0.05. Significance is indicated as follows: *p < 0.05, **p < 0.01 and ***p < 0.001.