Fig 2: Level of mRNA and protein expression of TTLL12 (A), CDKN3 (B), CDC16 (C), PTPRA (D), MZT2B (E), and UBE2T (F) in diffuse (n = 103) and intestinal (n = 110) GC tumor samples. Mann – Whitney test was used to compare the relative gene expression levels. In all graphs, the expression in gastric tumors was normalized by matched non-neoplastic gastric tissue. RQ: relative quantification; T: tumor sample; N: normal mucosa sample; The whiskers indicate the minimum and maximum values. red dotted line represents the 1.5 fold-change.

Fig 3: Quantification of MZT2B (A), CDC16 (B) and UBE2T (C) mRNA and protein levels by tumor stage in GC of the diffuse intestinal. *p <0.008, **p <0.001, ***p <0.0002, ****p <0.0001 significant difference between groups (Nonparametric Mann – Whitney test). In all graphs, the expression in gastric tumors was normalized by matched non-neoplastic gastric tissue. RQ: relative quantification; T: tumor sample; N: normal mucosa sample. (D) ROC curve analysis. The cutoff point was chosen as the highest point of the Area-Under-the-Curve (AUC) for MZT2B, AUC: 0.803; (95% CI 0.69-0.82) with 84.2% sensitivity and 50.0% specificity, for CDC16, AUC: 0.798; (95% CI 0.69-0.82) with 86.0% sensitivity and 49.08% specificity and for UBE2T, AUC: 0.549; (95% CI 0.51-0.68) with 52.03% sensitivity and 47.05% specificity. (E) Heat maps showing the average levels of gene expression of ~30 samples of GC tissues of the intestinal-type. The red gradient shows the highest levels of expression while the blue gradient shows the lowest levels. Kaplan-Meier analysis of the overall survival (in months) of patients with gastric cancer of the intestinal-type as a function of the expression of (F) MZT2B, (G) CDC16 and (H) UBE2T. We analyzed high expression (gene expression =1.45; red line), as opposed to low expression (gene expression <1.45; blue line) associated with a higher probability of survival. We found more significant values only for high expression of CDC16 (p <0.0085) when associated with the lower shortest patient survival.

Fig 4: Analysis of protein expression and immunohistochemistry in gastric cancer subtypes. (A) Representative image of Western-blot. Line 1 represents normal gastric tissue and the intensity of the genes are similar to early TNM stages and without metastasis, but much lower than advanced TNM stages (T3/T4). Initial stages with metastasis also have an intensity much higher than that of normal gastric tissue. (B) Positive TTLL12 cytoplasmatic immunostaining in diffuse-type gastric cancer (case 66 T2N3M1); (C) Positive CDC16 cytoplasmic and nuclear immunostaining in diffuse-type gastric cancer (case 140 T3N3M0); (D) Positive CDKN3 cytoplasmatic immunostaining in diffuse-type gastric cancer (case 203 T4N2M1); (E) Positive PTPRA cytoplasmatic immunostaining in intestinal-type gastric cancer (case 5 T1N0M0); (F) Positive MZT2B cytoplasmatic immunostaining in intestinal-type gastric cancer (case 61 T2N3M0); (G) Positive UBE2T cytoplasmatic and nuclear immunostaining in intestinal-type gastric cancer (case 149 T3N3M1) (magnification x40). The differences in band intensity and intensity of immunoreactivity are due to the different stages of TNM in tumor samples of diffuse and intestinal histological types that represent figures (3A, 3B–3G). (H and I) The function of the DEGs in the gastric lines showed a strong correlation between the increased level of protein expression with the human stomach cells in the data from The Human Protein Atlas (HPA) that were accessed and normalized in -log10 p-value (adj.). The levels of protein expression of the DEGs are identified by the red rectangle in 15 subtypes of human stomach cells.

Fig 5: Box plot shows the relationship between the normalized expression of TTLL12, MZT2B, CDC16 and UBE2T with metastatic progression. (A and B) mRNA and protein levels of TTLL12, CDC16 and UBE2T in 103 tissue samples with diffuse-type GC from patients M0 and M1. (C and D) mRNA and protein of MZT2B, CDC16 and UBE2T in 110 tissue samples with intestinal-type GC from patients M0 and M1. The boxes are drawn from the 75th to the 25th percentile. The vertical lines above and below the box define the maximum and minimum values and the dots indicate outliers, the horizontal line inside the box representing the median. (E and F) mRNA and protein of TTLL12, MZT2B, CDC16 and UBE2T in patients with diffuse (n = 103) and intestinal (n = 110) GC that were associated with M0 and M1. The lines with the gray gradient represent the connections of points M0 and M1 that vary according to the normalized expression value. The expression levels of these genes were validated by qRT-PCR and western blot in 213 patients. (M) presence of metastasis. Mann – Whitney test was used to compare the relative gene expression levels.