RPL5 expression in normal tissues and pan-cancer

The levels of RPL5 mRNA (transcript RNA-seq datasets) across the pan-cancer were analyzed using data obtained TCGA from UCSC Xena (https://xena.ucsc.edu). Considering the small number of normal samples in TCGA, we combined the normal tissue data from GTEx database with the TCGA tumor tissue data to investigate the differential expression of RPL5 in 33 cancerous tissues and adjacent tissues.

RPL5 expression and its relationship with clinical stage of COAD

Based on the above pan-cancer analysis, we further focused on the impact of RPL5 expression on the clinical stage of COAD patients in the database cohort. Data on COAD cases and normal controls were downloaded from the UC Santa Cruz website (https://xena.ucsc.edu). Therefore, tumor samples were matched to clinical stage to obtain data on RPL5 expression and clinical stage, and their relationship was analyzed.

Clinical samples

Formalin-fixed paraffin-embedded tissue blocks of COAD tumors and adjacent normal mucosal tissues were obtained from 11 patients between March 2021 and November 2021 at the Yan’an People’s Hospital (Yan’an, China). None of the patients received prior chemotherapy, radiotherapy or systemic therapies, or had additional malignant tumors. The study protocol was approved by Medical School of Yan’an University’ Ethics Committee (NO.2022057), with written informed consent of all patients.

Cell culture and siRNA transfection

Human colon cancer cell lines (HCT116 and RKO) were gifted by the Key Laboratory of Environment and Genes Related to Diseases of Xi’an Jiaotong University. RKO cells were maintained in DMEM medium (Biological Industries, Beit Haemek, Israel) supplemented with 10% fetal bovine serum (Biological Industries, Beit Haemek, Israel). HCT116 cells were maintained in RPMI-1640 medium (Biological Industries, Beit Haemek, Israel) supplemented with 10% fetal bovine serum (Biological Industries, Beit Haemek, Israel). All cells were cultured in a humidified 5% CO2 incubator at 37 °C. Human RPL5 siRNAs (siRPL5–1 sense, 5′-GGGAGCUGUGGAUGGAGGCTT-3′; siRPL5–1 antisense, 5′-GCCUCCAUCCACAGCUCCCTT-3′; siRPL5–2 sense, 5′-CUGCCAAAAUAUGGUGUGATT-3′; siRPL5–2 antisense, 5′-UCACACCAUAUUUUGGCAGTT-3′;) and negative control siRNA (siNC sense, 5′- UUCUCCGAACGUGUCACGU-3′; and siNC antisense, 5′-ACGUGACACGUUCGGAGAA-3′) were chemically synthesized by GenePharma (Shanghai, China). Transfection of siRNAs was performed using jetPRIME reagent (Polyplus-transfection SA), according to the manufacturer’s protocol.

Western blotting

Cells were lysed in RIPA buffer (Pioneer, Shanghai, China) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (TargetMol, USA). Protein concentration determined using a BCA protein assay kit (Proteintech, Wuhan, China). The protein samples were transferred to PVDF membrane by SDS-PAEG, membranes were incubated with specific primary antibody at room temperature for 30 min and then overnight at 4 °C. Then they were incubated with corresponding anti-rabbit/anti-mouse secondary antibody (TransGen Biotech, Beijing) at room temperature for 1 h. The membranes were incubated with ECL (Boster Biological Technology co.ltd, California, USA) in the dark for chemiluminescence detection. Luminescent signals were detected and recorded by Syngene GBox (Syngene, Cambridge, UK). The primary and secondary antibodies used are listed as follows: RPL5 (Proteintech, 29,092–1-AP), MMP2 (Proteintech, 10,373–2-AP), MMP9 (Proteintech, 10,375–2-AP), CDK4 (Proteintech, 11,026–1-AP), CyclinD1 (Proteintech, 26,939–1-AP), MEK1/2 (Proteintech, 11,049–1-AP), p-ERK (Proteintech, 28,733–1-AP), ERK (Proteintech, 67,170–1-Ig), c-Myc (Proteintech, 10,828–1-AP), p-MEK1/2 (Cell Signaling Technology, 9154), FOXO3 (Boster, BM4734) and β-Tubulin (Proteintech, 66,240–1-Ig).

RNA isolation and RT-qPCR

Total RNA from the human CRC cells was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. mRNA was reverse transcribed into cDNA using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing) according to the manufactures instruction. The primers were synthesized by Tsingke Biotech, and the sequences were as follows: RPL5 forward primer-GCTTATGCCCGTATAGAGGGG，RPL5 reverse primer-GCCAAACCTATTGAGAAGCCTG; GAPDH forward primer-GGAGCGAGATCCCTCCAAAAT, GAPDH reverse primer -GGCTGTTGTCATACTTCTCATGG. qPCR was performed using PerfectStartGreen qPCR SuperMix (TransGen Biotech, Beijing) on a Cobas z480 instrument, according to the manufacturer’s instructions. The relative expression of RPL5 to β-actin (internal control) was calculated using the 2-ΔΔCt method [10].

CCK-8 assay

In brief, colon carcinoma cells HCT116 and RKO (3 × 103/cells per well) were seeded in 96-well culture plates. About 10 μl of CCK8 (TargetMol, USA) solution was added to the 96-well at 24, 48 and 72 h after transfection and cells were incubated in 37 °C for 2 h. The absorbance was measured on a microplate reader (MD, USA) at 450 nm.

Clone formation assay

Transfected HCT116 and RKO cells were plated in 6-well plates at a density of 800 cells in triplicate and incubated at 37 °C with 5% CO2 for 7–10 days until they reached 80% confluence. Cells were then fixed with 4% paraformaldehyde and stained with 0.5% crystal violet (Sigma Aldrich; Merck KGaA) for 30 min at room temperature. Images were captured on a G: BOX XR5 Gel Imaging System (Syngene, UK). After dissolving in DMSO, colonies were quantified and the absorbance was measured on a microplate reader (MD, USA) at 570 nm.

Wound healing assay

Wound healing assay was performed to measure cell migration capacity. Briefly, once cells had grown to 80–90% confluence in 6-well plates, a single scratch wound was generated with a 10 μl disposable pipette tip. Images of the cells were captured at 40× magnification (Nikon, Tokyo, Japan) at 0, 24, 48 and 72 h and used to determine cell migration.

Cell cycle assay

HCT116 and RKO cells were seeded in 6-well plates (30 × 104/cells per well). 24 h after transfection, cells were collected by trypsinization, washed with PBS and fixed ice-cold 70% ethanol at 4 °C overnight. Then cells were washed with PBS twice. And the cells were incubated with 150 μl RNase for 15 min at room temperature and stained with 150 μl propidium iodide for 30 min at 4 °C in the dark. Cell cycle distributions were measured using a flow cytometer (Syngene, USA).

TBHQ treatment

Tert-Butylhydroquinone (TBHQ; HY-100,489) purchased from MedChemExpress (Shanghai, China) and was dissolved in DMSO. For western blotting, TBHQ was added 6 h after transfection and incubated for 48 h. For CCK8 assay and cell scratch assay, TBHQ was added 6 h after transfection and the cell viability and cell migration ability were detected by incubating for 24 h and 48 h respectively.

Statistical analysis

All experiments were performed with three independent replicates. The data were expressed as the mean ± standard deviation and analyzed with SPSS 22.0 software (SPSS, Inc.). Student’s t-test was used for comparison of two groups and the one-way analysis of variance (ANOVA) followed was performed for multi-group comparison P < 0.05 was considered to be statistically significant.