2.1 Cell lines and cell cultures
RKO, HCT 116, SW480, and SW620 human CRC cell lines (purchased from Wuhan Genechem company) were cultured in a 37°C constant temperature incubator (containing 5% CO2) in a dulbecco's modified eagle medium (DMEM) medium containing 10% fetal bovine serum (FBS).
2.2 Analysis of the differential expression of WDR12 in CRC and non-tumor tissues
The RNA sequencing (RNA-seq) and RNA sequencing version 2 (RNAseqV2) data of CRC and para-cancerous tissues were downloaded from the TCGA database. The limma R software package was applied for the Wilcoxon test to compare the differential expression of WDR12 in cancer tissues and para-cancerous tissues (|logFC|>2 and P<0.001) and paint the volcano map by using R language.
2.3 Quantitative real-time polymerase chain reaction (PCR)
Total RNA was extracted using Trizol (Shanghai Pufei Inc, Shanghai, China). Complementary deoxyribonucleic acid (cDNA) was generated with random hexamers from mRNA using appropriate RNAs dose (1 μg). The mRNA was quantified by SYBR Green PCR Master Mix (TAKARA Inc.) and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels.
2.4 Western blot
HCT-116 cells were harvested and then lysed in ice-cold radio immunoprecipitation assay (RIPA) buffer which including 50mM Tris (pH 7.4), 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS). The cell suspension was then placed on ice for 15 minutes and centrifuged for 5 minutes at 14,000 rpm. The supernatant was harvested, and the protein concentration was analyzed using a bicinchoninic acid (BCA) protein assay kit. Resolved the protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred it onto polyvinylidene fluoride (PVDF) membrane, which was then blocked with 5% skim milk powder for 1 h. The primary antibody was diluted with blocking solution and then incubated with the blocked PVDF membrane at room temperature for 2 h or 4 ℃ overnight, and the membrane was washed 4 times with TBST for 8 min each. Then dilute the corresponding secondary antibody with blocking solution, incubate the PVDF membrane for 1.5 h at room temperature, and wash the membrane 4 times with TBST for 8 min each. The PVDF film was placed on the lay-up plastic wrap, and the liquids A and B were mixed at a ratio of 1:40. The mixed liquid was added dropwise to the PVDF film and protected from light for 5 minutes. Take out the film, drain the excess enhanced chemiluminescence (ECL) substrate reaction solution slightly, put it in a dark box, cover it with plastic wrap (to avoid air bubbles), put X-ray film (to avoid X-ray film movement), close the film box, and expose 1-2 minutes. Take out the X-ray film, put it in the developer, take it out after about 1 minute, rinse it in clean water for a few seconds, and then put it in the fixing solution for at least 2 minutes (the exposure time needs to try a few times, depending on whether the fluorescence can be seen by the naked eye and the intensity of the fluorescence is not properly adjusted for the exposure time). Remove the X-rays, allow them to dry, and analyze.
2.5 Lentivirus construction and infection
Insert short hairpin RNAs (shRNAs) targeting WDR12 into the lentiviral vector GV115. The target sequence of WDR12 was as follows: 5′-GCTGATTTCAGGATCTTTA-3′. Performed lentivirus construction and infection as previously described[14]. Mix the WDR12 shRNA plasmid with pHelper packaging plasmids, and lentivirus-assisted transfection of HCT116 and SW620 cell lines with knockdown WDR12 (shWDR12) was performed using Poly Jet reagent. At the same time, empty lentivirus transfection control (shCtrl) was also prepared. Separately collect supernatant containing lentivirus at 48 hours and 72 hours post-transfection. The lentivirus was filtered and then used for later infection assays.
2.6 Cell proliferation assay
Celigo assay: shCtrl group and shWDR12 group were prepared and the cell concentration was adjusted to 3000 cells/100 ml. After the 96-well plate was inoculated, the reading plate was detected with Celigo (full fifield cell analyzer) once a day, starting from the next day for 5 consecutive days.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay: shCtrl group and shWDR12 group were prepared and the cell concentration was adjusted to 3000 cells/100 ml. Five 96-well plates were laid (tests for 5 days), they were marked, and divided into two groups. Then added 10 μl MTT (Sigma) to each well. After 4–6h incubation, discarded the medium. 100μl dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan particles and then incubated in the dark for 20 min. And the absorbance was determined by an enzyme-labeling measuring instrument at 570 nm. Each group consisted of 5 duplicates and repeated at least three separate experiments.
2.7 Cell apoptosis assay
Cell apoptosis detected by flow-cytometry assay: At room temperature in the dark, cells were stained with 100 μl cell suspension containing 10 μl Annexin V-APC (Cat. 88-8007, eBioscience, USA) for 10–15 min at day 5 after lentivirus transfection, the Guava easyCyte HT flow cytometry system (Millipore) was used for flow cytometry analysis.
Cell apoptosis detected by Caspase-3/7 assay: The Caspase-Glo® Glo Assay kit (Promega) was used for the measurement of Caspase-3/7 activities according to the manufacturer's protocol. Cells were plated in triplicate in 96-well cell culture plates. Cells were incubated with 100 µl caspase-Glo reagent at room temperature for 30 minutes at day 3 after small interfering RNA (siRNA) transfection. Assays were measured by detection with a fluorescence microplate reader (TECAN infinite M2009MR).
2.8 Tumor formation experiment in nude mice
The experimental animal model of tumor formation in nude mice was 4-week-old male BALB/ C nude mice (purchased from Shanghai Genomics Company), and there were 20 nude mice in total, with 10 nude mice in each group. After 1 week of acclimatization, each group of 10 nude mice is housed in the separated individual standard cleaned cages under automatically controlled air condition system with temperature (22 ± 2 °C), humidity (about 60%), and lighting (12:12-h light–dark cycle). Diet and sterilized water are provided ad libitum throughout the experiments. Tumor cells were prepared according to the experimental groups, and nude mice were injected subcutaneously with some cells at the same time after randomization of the size of the mice. Animals were bred to tumors visible to the naked eye, and animal weights and tumor sizes (long diameter and short diameter) were measured, and live imaging was performed. According to the Luciferase luminescence detection needs, D Luciferin (15mg/mL) is injected intraperitoneally at a dose of 10 μl/g lasting for about 15 to 20 minutes. The nude mice were injected intraperitoneally with a 0.7% sodium pentobarbital solution at a dose of 10 μl/g. After about 5 minutes, the nude mice were completely in a coma. The index finger gently pressed the animal's chest, and the heartbeat was tested to ensure that the animal was alive. Use the software that comes with the imager to perform imaging: set various parameters of the living body imaging, adjust the focal length, and perform live body imaging detection on the animal according to the set detection program to observe the fluorescence expression in the animal or subcutaneously. Use software for quantitative analysis and data collection. After the data were recorded, the nude mice were sacrificed by cervical dislocation, the tumor tissues were dissected and weighed, and the nude mice and tumors were photographed. The data were statistically analyzed to compare the differences in tumor volume and weight between the two groups. The animal study protocol was approved by the Institutional Review Board of Shanghai Genechem Co.,Ltd (protocol code GSZE0167660 and date of July 8, 2018).
2.9 Ingenuity pathway analysis
The protein networks and biological pathways upstream regulators affected by WDR12 knockdown in HCT 116 cells were got insight through Ingenuity pathway analysis (IPA; Ingenuity H Systems, Redwood City, CA, USA; http://www.ingenuity.com). The graph represented networks and pathways. The proteins was represented as nodes, and the lines represented the biological interaction between two nodes. We selected networks and upstream regulatory scoring<=-2. This study Used a linear model based on empirical Bayes distribution17 to calculate the significant difference level P-value. The screening criteria for Differentially Expressed Genes (DEGs) is P-value < 0.001.
2.10 Statistical analysis
IBM SPSS Statistic 23.0 software was used for statistical analysis of the experimental data, and Graph Pad Prism8.0 software was used for making images. Data are shown as the mean ± standard deviation. The differences between the two groups were evaluated using t-test or analyzed by one‑way analysis of variance (ANOVA) when there were more than two groups. P < 0.05 was considered to indicate a statistically significant difference.