Fig 2: Clinical relevance of MCM and Ki67 expression in SI-NENs. (A) MCM expression in normal mucosa tended to be higher than in neoplastic small intestinal tumors, whereas Ki67 was not significantly different between any tissue type (*P < .05 versus normal mucosa). (B) Ki67 and MCM2 expression was significantly increased (*P < .05) in grade 2 compared to grade 1 tumors. MCM3 was also increased, but this did not achieve statistical significance. (C) Correlation between MCM mRNA expression and Ki67 staining identified a relationship for MCM2 (r2 = 0.44, P < .05) but not for MCM3. Expression of either MCM significantly correlated with Ki67 mRNA expression (MCM2: r = 0.6, P = .0027, MCM3: r = 0.53, P = .0092) and with each other (r = 0.69, P = .004). (D) High copy number variance was noted for all three markers in both primary tumors and in metastases; this was not seen in normal mucosa. Mean ± SD.

Fig 3: Effects of PLK1, MCM2, or MCM3 knockdown on mtDNA content.(A) Western blot analysis of proteins PLK1, MCM2, and MCM3. Total lysates in knockdown cells were extracted 24 h after transfection with siRNA against PLK1, or 72 h after transfection with siRNAs against MCM2 and MCM3, and analyzed using antibodies specific for PLK1, MCM2, MCM3, and Actin (loading control). (B) FACS analysis of DNA content in PLK1, MCM2, and MCM3 knockdown cells. DNA content was analyzed using flow cytometry 24 h after transfection with siRNA against PLK1, or 72 h after transfection with siRNAs against MCM2 and MCM3. (C) Percentage of cells at each phase during the cell cycle. (D) Southern blot analysis of mtDNA content in cells transfected with either control siRNA or the indicated siRNAs. Total cellular DNA in knockdown cells was extracted 24 h after transfection with siRNA against PLK1, or 72 h after transfection with siRNAs against MCM2 and MCM3, and digested with BamHI. Signals of mtDNA and nuclear DNA were detected with probes as described in Figure 1D. (E) Quantitative analysis of relative mtDNA levels obtained from Southern blot and real-time PCR analyses. Relative mtDNA levels were estimated by both Southern blot and quantitative real-time PCR analyses as described in Figure 1E. For real-time PCR analysis, data are shown as the mean ± standard error of the mean (SEM) from three independent experiments.

Fig 4: Effect of PUFA treatment on MCM2, p-cyclin D1, cyclin D1 and VE-cadherin in subconfluent EA.hy926 cells grown on Matrigel-coated plates.EA.hy926 endothelial cells were seeded on Matrigel-coated plates at 9000 cells/cm2 and grown for 4 days. Cells were treated with PUFAs (LA, AA, ALA, EPA and DHA) individually at 125 µM for 8 h. Protein levels of MCM2, p-cyclinD1, cyclin D1 and VE-cadherin were determined by Western blotting of cell lysates prepared after treatments. Representative blots are shown in Panel A. Densitometry was used to quantify the intensity of the bands in the upper panel and data were normalized to a band visualized by Ponceau staining. Data are presented as means ± SEM (n = 3) for MCM2 (Panel B), p-cyclin D1/cyclin D1 (Panel C) and VE-cadherin (Panel D). *Significantly different (p <0.05) from vehicle control.

Fig 5: Survival curves for AQUA analysis of MCM2 and MCM3 in SI-NENs.Low expressions of MCM2 (A) and MCM3 (B) were both associated with notably longer OS. Using a combination of proliferative markers, patients with low–proliferation score tumors (0-1; n = 21) had prolonged survival (median OS 12 years) compared to those with high–proliferation scores tumors (2-3; n = 24) (median OS years 6.5) (C, P = .06, HR 0.46). A high MCM3 expression was correlated with worst prognosis, whereas after more than 25-years of follow-up, the median OS of patients with low MCM3 expression was not reached in the subset of tumors that were only Ki67% positive (D, P = .019, HR 0.13).