Exploring PYCR2 prognostic ability and expression pattern in CRC
ENCORI Pan-Cancer Analysis Platform (http://starbase.sysu.edu.cn/panCancer.php) is designed for decoding pan-cancer networks of lncRNAs, miRNAs, pseudogenes, snoRNAs, RNA-binding proteins, and protein-coding genes by assessing their expression profiles across 32 cancer types integrated from The Cancer Genome Atlas (TCGA) project. In this study, we used this platform to study PYCR2 expression pattern and prognostic ability in CRC.
Target gene prediction and data screening
To improve the efficiency of target gene prediction, we used four databases: STRING[11, 12] (https://string-db.org/), BioGRID (https://thebiogrid.org/), IntAct (https://www.ebi.ac.uk/intact/), and GeneMANIA (https://genemania.org/). Further, Venn diagram was created through a web tool (http://bioinformatics.psb.ugent.be/webtools/Venn/) and target genes chosen which were overlapped in at least 2 out of 4 databases to enhance the accuracy of the prediction.
Determining the prognostic significance of PYCR2 overlapping target genes
ENCORI and GEPIA (http://gepia.cancer-pku.cn/) were applied to determine whether the expression levels of PYCR2 overlapping target genes varied between CRC and non-cancerous tissues and whether they affected the prognosis of CRC.
Assessing the co-expression of PYCR2 and related genes and determining the mutational status of target genes
cBioPortal[17, 18] (http://www.cbioportal.org/), a web-based tool to explore, visualize, and analyze multidimensional cancer genomics data, was used to analyze the co-expression of PYCR2 and related genes. P < 0.05 was the cut-off criterion. Further, the mutational status of the target genes of PYCR2 in CRC was investigated using cBioPortal.
Comparing prognosis between patients with and without gene mutations
The International Cancer Genome Consortium (ICGC) (https://dcc.icgc.org/) serves to catalog large-scale cancer genome studies in tumors from 50 distinct cancer types/subtypes, containing clinical data as well as data pertaining to abnormal gene expression, somatic mutations, and epigenetic modifications. We used this portal to compare prognosis between patients with and without gene mutations.
HCT116, HT29, and LoVo CRC cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). LoVo and HT29 cells were maintained in 1640 (HyClone), 10% FBS (Gibco), and 100 units/mL penicillin, HCT116 cells were maintained in McCoy’s 5A (HyClone), 10% FBS (Gibco), and 100 units/mL penicillin.
HT29, LoVo, and HCT116 cells were seeded into a 6well plate and cultivated at 37°C and 5% CO2. Their growth was closely monitored until they reached a fusion rate of 80%, and they were then transfected with siRNA (final concentration = 100 nM; Santa Cruz, Texas, USA) in the presence of liposome 2000 (Invitrogen, California, USA). Briefly, Liposome diluted to 5 µl/well, and then siRNA was diluted at a calculated concentration. We incubated them for 5 minutes separately, and incubated the mixture proportioned for 15 minutes. The mixture was added to the cells and the 6-well plate placed into the incubator. After 8 h, the growth medium was changed to fresh media, and the cells were further incubated at 37°C for 24 h (5% CO2). The cells were subsequently collected for further analyses.
Reverse transcription-quantitative PCR
TRIzol® was used to extract total RNA from HCT116, HT29, and LoVo cells, as per manufacturer instructions. Reverse transcription was performed using the PrimeScript RT reagent kit (Takara Bio, Inc.). rRNA amplification was performed on a C1000 Thermal Cycler Detection System (Bio-Rad Laboratories, Inc.) in triplicate, with the reaction mixture (total volume = 50 µL) comprising 1 ng cDNA, 125 nM forward and reverse primers, and 25 µL 2X SYBR® Premix Ex Taq™ (Takara Bio, Inc.). A NanoDrop 2000 Spectrophotometer (Thermo) was used for RNA quantification, with the A260/280 ratio indicating RNA purity. GAPDH was used as the internal control. The cycling conditions were as follows: initial denaturation at 95°C for 1 min, followed by 42 cycles of 95°C for 15 sec, 56°C for 25 sec, and 72°C for 30 sec. The following primers were used: GAPDH-F: 5′-AGCCACATCGCTCAGAACAC-3′, GAPDH-R: 5′-GAGGCATTGCTGATGATCTTG-3′; PYCR2-F: 5′- CTGTCCCAGGTGTCTAAGGG-3′, PYCR2-R: 5′-CTTGCTCCTCAGCACAGAAC-3′. The relative mRNA expression levels were quantified using the 2−ΔΔCq method.
We inoculated approximately 3 × 105 cells/well into a 6-well plate, with the exact number varying on a cell-to-cell basis. The cells were intervened with siRNA separately. The next day, the plates were scratched vertically using a pipette tip. Scratches were ensured to be straight using a ruler. The cells were washed with PBS. The scratched cells were then removed and a serum-free medium was added into the well. The plates were subsequently incubated at 37°C and 5% CO2. Samples were collected at 0, 24, and 48 h, and images were then captured.
Matrigel(8-12 mg/ml) was placed, which was stored at -20 ˚C, onto the ice to melt (keeping the temperature at 2 ˚Cཞ8 ˚C). Matrigel (100 µl) was aspirated with a pipette tip precooled on ice and expelled into precooled 3000 µl serum-free medium (diluted at 1:30-1:40, total volume diluted according to the number of wells), and mixed thoroughly. The diluted 100 µl Matrigel above was added into the upper chamber of the Transwell plate to cover the whole polycarbonyl ester membrane. The Matrigel was polymerized into gelatin at 37°C for 60 minutes. Any remaining liquid medium on the top was discarded carefully.
The pretreated groups of cells were prepared into single-cell suspension (5 ×10^5 cells/ml) with serum-free medium in the regular way. This suspension (200 µl) was added to the upper chamber of the Transwell culture plate, and 700 µl 10% FBS was added to the lower chamber. The plate was then incubated at 37℃ and 5% CO2 for 48 h. Subsequently, the cells were fixed in 4% paraformaldehyde for 30 min and then stained with 0.1% crystal violet for 3 min.
To measure the number of cells that were able to penetrate the film in the upper chamber, the unpenetrated cells on the upper surface of the film and the Matrigel in the chamber were gently wiped off with a wet cotton swab. Finally, images were captured, and the cells were counted.