Immunochemistry. Colorectal multi-tumor tissue microarrays (TMAs) were purchased from Genechem Co., Ltd. The TMAs were blocked with 5% goat serum dissolved in PBS for 60 min and subsequently incubated with Rabbit polyclonal anti-WDR43 antibody (cat no. ab174906; dilution, 1:50; Abcam) overnight at 4˚C. Following staining, the samples were assessed by a senior pathologist, and a blinded manner were taken to record the immunohistochemical results. The final score of each microarray was based on the proportion of positively colored cells (0, 0–5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%) and staining intensity (0, no staining; 1, thin staining; 2, middling staining; and 3, strong staining). Low expression was defined as a final score of < 6, and high as a final score of ≥ 6. The present study was authorized by the Ethics Committee of the Nanjing Drum Tower Hospital.
Cell culture. The DLD-1 CRC cell line was purchased from Procell Life Science & Technology Co., Ltd., and the HCT116 CRC cell line from Nanjing KeyGen Biotech Co., Ltd.. Both cell lines were STR-authenticated. Tumor cells were incubated at 37˚C, 5% CO2, DMEM or McCoy’s 5A medium (Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco, USA) for DLD-1 and HCT116 cells, respectively. The medium was renewed every 3 days and the cells were passaged when the cell density reached 80–90%.
Lentiviral transfection. The shRNA plasmid vector was constructed for lentiviral (GeneChem, Inc.) infection to stably knockdown WDR43. The whole transfection procedure was then performed following the manufacturer’s instructions. Puromycin was used to eliminate the untransfected cells 3 days after transfection. The transfection of the surviving cells was confirmed by RT-qPCR.
RNA extraction and RT-qPCR. Total RNA of cells was extracted using TRIzol® reagent (Merck KGaA) and detected by BioDrop µLITE+ (Biochrom, Ltd.). RT was then performed using HiScript III RT SuperMix for qPCR (Vazyme Biotech Co., Ltd.) to synthesize cDNA. Next, RT-qPCR was executed on a ViiA™ 7 Real-Time PCR System (Thermo Fisher Scientific, Inc.) using the comparative threshold cycle (2-ΔΔCt) method. Target gene expression at the mRNA level was relatively quantified using GAPDH as the internal reference gene. Following is primer sequences : WDR43, 5’-CCAGGGCTTAGAAAGTAACGA-3’ forward and 5’-TCAGGCAACGTGGACAGGTAT-3’ reverse (final product size, 228 bp); GAPDH, 5’-TGACTTCAACAGCGACACCCA-3’ forward and, 5’-CACCCTGTTGCTGTAGCCAAA-3’ reverse (final product size, 121 bp). Acquired data were finally analyzed by GraphPad Prism 7.0 (GraphPad Software, Inc.).
Protein extraction and immunoblotting. Proteins were extracted from cells with 100 µl RIPA buffer containing 1:100 Complete™ Protease Inhibitor Cocktail (Roche Diagnostics). Following lysing and centrifugation, the protein-containing supernatant was mixed with 5X SDS-PAGE loading buffer (1:4 ratio; Biosharp Life Sciences) and boiled for 5 min to denaturate the protein. BCA assay (Nanjing KeyGen Biotech Co., Ltd.) was used for protein quantification. Equal amounts of proteins (15 µg) from each sample were loaded in 10% gel (PG112, epizyme) and transferred on PVDF membrane (10600021, GE Healthcare). The membranes were then cut into stripes and blocked with 10% skim milk (1172GR500, Biofroxx). Then the stripes were incubated with primary antibodies against WDR43 (cat. no. ab174906; dilution, 1:300; Abcam) and the internal reference protein GAPDH (cat. no. sc-32233; dilution, 1:2000), followed by incubation with secondary antibodies for two hours at room temperature (7074S, dilution, 1:4000, Cell-Signaling Technology, Inc.). 20X LumiGLO® Reagent and 20X peroxide (7003S; Cell-Signaling Technology, Inc.) were used in development following the instruction, and the bands were imaged using a Tanon 5200 Chemiluminescent Imaging System (Tanon Science & Technology Co., Ltd).
MTT and Celigo assays. Cells that had received different treatments were harvested using trypsin with 0.25% EDTA (Thermo Fisher Scientific, Inc.) and counted using Countstar® BioTech Automated Cell Counter (Countstar). The cells were then seeded into 96-well plates (1.5x103 cells/well). For Celigo assays, the number of cells was counted each day using the Celigo Image Cytometer (Nexcelom Bioscience LLC). For MTT assays, the cells were then incubated respectively for 24, 48, 72, 96 and 120 h. MTT reagent (20 µl; 5 mg/ml; Genview) was added 4 h before the incubation was terminated. Once precipitates became visible, the supernatants were carefully discarded and 100 µl dimethyl sulfoxide was added for 2–5 min to dissolve the precipitates. The absorbance value of each well was detected by a microplate reader at 490 nm. Each experiment was repeated at least three times.
Colony formation assay. Transfected cells were cultivated into each well of a 6-well culture plate (1.5x103 cells/well) and cultured for 8 days, with renewal of the medium every 3 days during the incubation. The cells were then washed with 1 ml PBS at a time, fixed by 1 ml 4% paraformaldehyde for 25 min at room temperature, and rewashed once with 1 ml PBS. Then, cells were colored with 1 ml crystal violet solution (Beyotime, C0121) (Sangon Biotech Co., Ltd.) for 15 min at room temperature and then washed with ddH2O several times. The plate was air-dried and photographed with a digital camera to count the number of colonies containing > 50 cells. Following formula was used to calculat the colony formation efficiency: Colony formation efficiency = (number of colonies/number of inoculated cells) x100%.
Flow cytometry. Flow cytometry was used to analyze the apoptotic rate of tumor cells that had received different treatments. Annexin V Apoptosis Detection Kit II (556570; BD Biosciences) was used for incubation with the collected cells to detect the apoptotic rate. The results were detected and obtained by BD AccuriTM C6 Plus cell analyzers (BD Biosciences). FlowJo 10.4 (FlowJo, LLC) was used to analyze the final results.
Cell migration and invasion. To evaluate the effect of WDR43 on migration and invasion of cells, Transwell assays were performed. Briefly, Transwell® chambers with an 8.0-µm Pore Polycarbonate Membrane (cat. no. 3422; Corning) were placed in a 24-well plate, with serum-free culture medium in the upper chamber and medium having 10% fetal bovine serum in the lower chamber; tumor cells (1x105 cells) were placed into the upper chamber. Following 24 h of cultivation, 4% paraformaldehyde was used to fix the cells for 15 min and then cells were colored with crystal violet solution (Beyotime, C0121) for 20 min. Following washing with PBS and wiping, the final results were photographed under Inverted Microscope Solution DMi8 S Platform (Leica Microsystems, Inc.) and analyzed by Leica Application Suite X (Leica Microsystems, Inc.). For cell invasion assays, the chambers were coated with Corning® Matrigel® Basement Membrane Matrix (cat. no. 356324; Corning) and incubated for 30 min; the rest of the procedure was coincident as that for the migration assay.
In vivo experiments. Male BALB/c nude mice aged 4–6 weeks were provided by the Nanjing Medical University. Tumor cells were digested with trypsin and adjusted to 1x107 cells/ml, and then subcutaneously injected into the back of mice. Each group contained a minimum of 5 mice. Mice were sacrificed 4 weeks after treatment, and tumors were subsequently resected to assess weight and volume.
Statistical analysis. All experiments were repeated at least three times. GraphPad Prism 7.0 (GraphPad Software) was used to analyze the data and determine the significance of the differences. For categorical data, student t-test was performed for comparisons between two groups, and one-way ANOVA for comparisons among multiple groups. p < 0.05 was considered statistically significant.