Data on clinical significance of GAS2 in colorectal cancer cells

The growth arrest-specific 2 (GAS2) was cloned and found to be upregulated in the feces of recurrent CRC patients. This overexpressed GAS2 induced different patterns of gene expressions in CRC cells. Briefly, one cell proliferation marker, Ki-67 antigen (Ki-67), was upregulated in the cells with overexpressed GAS2, “Correlation between proliferation markers: PCNA, Ki-67, MCM-2 and antiapoptotic protein Bcl-2 in colorectal cancer” [1]. Whereas, the expression of another cell proliferation marker, proliferating cell nuclear antigen (PCNA), changed insignificantly [1]. In addition, the mRNA level of one cyclin involving in both cell cycle G1/S and G2/M transitions was also not affected by GAS2 overexpression, “Cdc20 and Cks direct the spindle checkpoint-independent destruction of cyclin A” [2]. The experimental design and procedures in this article can be helpful for understanding the molecular significance of GAS2 in SW480 and SW620 CRC cells.

The data would be valuable to correlate the cell proliferation markers and the expression levels of GAS2.
The data might support the irrelevance of GAS2 in G2 phase of cell cycle.

Data
The data shared in this article are the experimental analyses of GAS2 expression in clinical samples, patient's feces, and CRC cell lines. The level of GAS2 would be up-regulated in the cases with recurrent CRC by microarray analysis (Fig. 1) [3]. The relative mRNA levels of interested genes (proliferating cell nuclear antigen: PCNA; Ki-67 antigen: Ki-67; cyclin A2: CCNA2) in different cells are presented in Figs. 2 and 3.

Expression difference in feces of nonrecurrent and recurrent CRC patients
Candidates in feces of recurrent patients were clustered from each comparison in relative to those fecal expressions of non-recurrent patients. The case numbers and stages in these bioinformatic analyses were 15 non-recurrent patients (6 stage I, 5 stage II, and 4 stage III) and 3 recurrent patients (stage III). Candidates in CRC tissues were extracted from GEO: GSE17536, GEO: GSE17537, GEO: GSE17538_GPL570 (Moffitt Cancer Center and Vanderbilt University Medical Center, respectively), GEO: GSE12032 (individual comparison at each stage for non-recurrent vs. recurrent), and GEO: GSE27854 at the public website of Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo) [4][5][6][7]. Genes with potentially significant changes were selected by comparing the data from non-recurrent and recurrent CRC tissues. 22 genes were selected due to the cross-compared results from fecal and CRC tissue microarrays.

The significance of GAS2 in cell proliferation
We established two SW480 cell lines to overexpress GAS2. Firstly, GAS2 cDNA was amplified from a human placenta library (Sigma-Aldrich, St. Louis, MO, USA) ( Table 1) and the polymerase chain reaction (PCR) bands for GAS2 were sequenced to confirm gene identity. Then, SW480 cells expressing GAS2 were generated either by lentiviral transduction using the all-in-one tetracycline-inducible plasmid (pAS4.1w.Ppuro-aOn) (Academia Sinica, Taipei, Taiwan) or by stable transfection using a plasmid encoding green fluorescent protein (pEGFP C3) (Takara Bio, Shiga, Japan).
The relative mRNA levels of PCNA (NM_002592) and Ki-67 (NM_002417) in cells were quantified by quantitative real-time PCR and relative to the mRNA level of GAPDH (NM_002046). As shown in Fig. 2, the level of Ki-67 in SW480-C3.GAS2 was significantly higher than that in control cells without GAS2 overexpression (SW480-C3). Primer sequences used in these gene quantifications were  Table 1 Primers for cloning growth arrest-specific 2 into pAS4.1w.Ppuro-aOn plasmid.

The correlation of GAS2 in the expression of CCNA2
The molecular marker for G2 phase of cell cycle, CCNA2 (NM_001237), was specific quantified by quantitative real-time PCR. The relative mRNA levels of CCNA2 were also calibrated with that of GAPDH as described before. However, no significant difference was found in the cells with overexpressed GAS2 (Fig. 3). Sequences of primer pair for CCNA2 were 5 0 -CCATACCTCAAGTATTTGCCATC-3 0 (forward primer) and 5 0 -TCCAGTCTTTCGTATTAATGATTCAG-3 0 (reverse primer). The number of universal was #67.