Patient tissue specimens
For tissue samples, a total of 189 paraffin-embedded primary specimens of CRC patients were collected in our study. All CRC patients underwent surgical resection from 2000 to 2007 at the Sun Yat-Sen University Cancer Center (SYSUCC).No patient received preoperative radiotherapy or chemotherapy. Written informed consent was obtained from the sample donors, and approval was granted by the Institute Research Medical Ethics Committee of Sun Yat-Sen University.
Cell lines and cell culture
For cells, the human CRC cell lines HCT116 and DLD1 were obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China). HCT116 and DLD1 stable cell lines were cultured at 37 °C in DMEM medium (Invitrogen) with 10% fetal bovine serum (Gibco, USA) in a humidified incubator with 5% CO2.
Plasmids and Antibodies
The full-length cDNAs of human ZBTB7A was cloned into the pSin-EF2-puro vector to generate the stable overexpression of ZBTB7A in HCT116 cells. Two RNA interference lentiviral vectors (shRNA-ZBTB7A 1 and 2) for silencing ZBTB7A in DLD1 cells were constructed and synthesized by Shanghai Genechem Technology Co., Ltd. Human anti-ZBTB7A (ABGENT, USA) and the GAPDH (Abcam, UK) antibodies were used. The targeted sequences of ZBTB7A were as follows: (shRNA-1) 5’-GCAGAAGGTGGAGAAGAAGAT-3’ and (shRNA-2) 5’-CCAGTACTTCAAGAAGCTGTT-3’. The sequence of shRNA-NC was 5’-ccgcag gtatgcacgcgt-3’.
RNA extraction and qRT-PCR
The total RNA from each group was extracted with RNA simple Total RNA Kit (TIANGEN, China). Then the cDNA was synthesized by the PrimeScriptTM RT Reagent Kit (Takara, Japan). And the qPCR was performed to detect the expression level of ZBTB7A mRNA with TB GreenTM Premix Ex Taq™ II (Takara, Japan).The primer sequences involved in our study were as follows：
for GAPDH：5'-ACAGTCAGCCGCATCTTCTT-3' (forward),
Western blot assay
The total protein was extracted from CRC cells by RIPA Lysis Buffer (Beyotime, China). A total of 30μg harvested protein was loaded, subjected to 10% SDS-PAGE, and then transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 1h on shaking table. Next, membranes were incubated with primary antibody (ZBTB7A and GAPDH) at 4 ∘C overnight and secondary antibody for 1 h the next day. Finally, the membranes were detected by the ECL chemiluminescence system (Pierce, Rockford, USA).
Plate colony formation assay
Briefly, 600 cells (DLD1-knock-down, HCT116-overexpression and their control cells) were seed into 6-well plates and cultured for 14 days. The cells were incubated at 37 ∘C for about 14 days. Then the colonies were fixed with 100% methyl alcoholand for 10 minutes then stained with crystal violet for 20 minutes, at room temperature. Cell colonies were calculated and photographed.
The cell viability in vitro was assessed using The CCK8 assay was used to assess the CRC cell viability. In brief, cells (1,000/well) were seeded in 96-well plates and incubated for consecutive five days. Ten microliters of CCK8 solution (Cell Counting Kit-8, Beyotime, China) was added to each well and incubated for 1.5 hours. The absorbance value (OD) of each well was measured spectrophotometrically at 450 nM by automatic microplate reader.
Tumor xenograft formation assay in vivo
All the experiments were approved by the Ethical Committee of the Sun Yat-Sen University Cancer Center. The five-week-old male athymic nude mice were purchased from Shanghai Institutes for Biological Sciences (Shanghai, China). The mice were randomly divided into four groups (n=6 for each group). ZBTB7A-knockdown and control DLD1 cells (5×106/0.2 ml PBS), ZBTB7A-overexpression and control HCT116 cells (5×106/0.2 ml PBS) were injected into the flanks of mice, respectively. Tumor size was measured once every two days with digital calipers. The tumor volume was calculated as 1/2(length×width2). After about 20 days, mice were sacrificed. Then the tumor tissues were preserved for further hematoxylin and eosin (H&E) and IHC staining.
The expression level of ZBTB7A in CRC specimens was measured by IHC. Briefly, the sections were sequentially deparaffinized by dimethylbenzene, rehydrated by graded ethanol, repaired for antigen by citrate buffer (pH 6.0), and incubated with primary anti-ZBTB7A antibody diluted 1:500 at 4 ∘C overnight in a humidified container. After three washes with 1× PBS, sections were incubated with the secondary antibody for 1h at room temperature and then immunostained with 3, 3’-diaminobenzidine tetrahydrochloride (DAB) chromagen kit. The IHC results were calculated by multiplying the intensity degrees and positive rates. The scores were categorized into 0–3 (no staining, weak staining, moderate staining and high staining) for staining intensity and 0–4 (no staining, <10%, 10%–50%, 50%–80%, and >80%) for the positive ratio. The final scores (0 to 12) were grouped into no/low expression (≤1) and high expression (>1). The scores were calculated blindly by two pathologists.
Statistically significant differences were investigated by SPSS software (version 19.0). T-test was used to compare the differences among continuous parameters. Chi-square test or Fisher exact test was used to compare clinicopathological variables between different ZBTB7A expression groups. The endpoint of overall survival (OS) was measured from the first date of treatment to the date of death due to any causes. Survival analysis was assessed by the Kaplan-Meier method and the log-rank test. P value less than 0.05 was considered statistically significant. The graphs were generated by GraphPad Prism 5.0. The raw data of this paper have been uploaded onto the Research Data Deposit (RDD) with an RDD number of RDDB2020000904.