The effects of MEN1 overexpression in islet-epithelial cell clusters was explored using cultured islets isolated from human fetal pancreata 15-19 wks which were transfected with either a control pEGFP vector (Ctrl-EGFP) or a pEGFP-N2 vector with a human full-length MEN1 cDNA insertion (N2-M1). After a 48 hr transfection, qRT-PCR analysis showed a significant two fold increase of MEN1 mRNA levels with N2-M1 transfection (p<0.05 vs. Ctrl-EGFP) (Figure 3.19A). Morphological analysis of islet- epithelial cell cluster sections stained with an anti-menin antibody 48 hrs post- transfection also indicated a significant increase of nMenin+ cells (from approximately 75% to 90%) with N2-M1 treatment (p<0.05 vs. Ctrl-EGFP) (Figure 3.19B). Figure 3.19C, D show representative images illustrating menin overexpression within islet- epithelial cell clusters.
The functional role of menin in regulating proliferation during human fetal pancreatic development was investigated with increasing menin expression (N2-M1 transfection). Quantification of the proliferative cell population displayed a significant increase in KI67+ cells with menin overexpression (p<0.05 vs. Ctrl-EGFP) (Figure 3.20). These results correspond with and confirm those found in Figure 3.13 with a knockdown of menin expression. Our next step was to determine if this increase in proliferation affected a specific endocrine population. Examination of insulin (Figure 3.21A) and
Figure 3.18. Effect of MEN1 siRNA on Cell Death in Islet-Epithelial Cell Clusters.
Islet-epithelial cell clusters, isolated from human fetal pancreata 15 to 19 wks, were transfected with either control siRNA or MEN1 siRNA for a 48 hr treatment period. (A)
Quantitative analysis of the total amount of TUNEL+ cells relative to the total amount of cells present (DAPI stained nuclei, expressed as a percentage) for both control and MEN1
siRNA treatment groups. Data were determined by morphometric analysis. (B) Western blot analysis of cleaved Caspase-3 (cCaspase-3) protein expression (17, 19 kDa) relative to total Caspase-3 (tCaspase-3) protein expression (35 kDa) for both treatment groups, normalized to control. Representative images of TUNEL labelling for (C) control or (D)
MEN1 siRNA treatment groups. Cells were harvested, paraffin-embedded, sections were TUNEL labelled (green) as in Section 2.2, and the nuclei were labelled with DAPI (blue). For each set, the top panel of images shows staining for TUNEL (green), DAPI (blue), and an overlay of images. Scale bars correspond to 25 micrometers. The bottom panels show magnified images corresponding to the boxed section in the above image. Arrows indicate a TUNEL positive cell. Data are expressed as means ± SEM. n=3. **p<0.01 vs. control siRNA treatment.
Figure 3.18. A. B.
C.
Figure 3.19. Effect of MEN1 Overexpression on MEN1 mRNA and nMenin in Islet-Epithelial Cell Clusters.
Islet-epithelial cell clusters, isolated from human fetal pancreata 15 to 19 wks, were transfected with either a control (Ctrl-EGFP) or a MEN1 (N2-M1) vector and cultured for 48 hrs. (A) qRT-PCR analysis of total MEN1 mRNA levels for control and N2-M1 treatments. Data were normalized to 18S rRNA and are expressed as means ± SEM where n=6 per experimental group. (B) Quantitative analysis of the total number of nMenin+ cells relative to the total amount of cells present (nMenin+/DAPI, expressed as a percentage) of both control and N2-M1 groups. Data are expressed as means ± SEM where n=3-4 for each experimental group. *p<0.05 vs. Ctrl-EGFP treatment. (C, D)
Representative images of islet-epithelial cell clusters for (C) control or (D) N2-M1 treatment groups. Cells were harvested, paraffin-embedded, and sections were immunolabelled with an anti-menin antibody visualized with a Cy2 (green) conjugated secondary antibody. Nuclei were labelled with DAPI (blue). For each set, the top panel of images shows staining for menin (green), DAPI (blue), and an overlay of images. Scale bars correspond to 25 micrometers. The bottom panels show magnified images corresponding to the boxed section in the above image. Arrows indicate a nuclear menin positive cell.
Figure 3.19. A. B.
C.
Figure 3.20. Effect of MEN1 Overexpression on Islet-Epithelial Cell Cluster Proliferation.
Islet-epithelial cell clusters, isolated from human fetal pancreata 15 to 19 wks, were transfected with either a control (Ctrl-EGFP) or a MEN1 (N2-M1) vector and cultured for 48 hrs. Cells were harvested, paraffin-embedded, and sections were immunolabelled with an anti-KI67 antibody visualized with a Cy3 (red) conjugated secondary antibody. Nuclei were labelled with DAPI (blue). (A) Quantitative analysis of the total number of KI67+ cells relative to the total amount of cells present (KI67+/DAPI, expressed as a percentage) for control and N2-M1 treatments. Data are expressed as means ± SEM where n=3 for each experimental group. *p<0.05 vs. control-EGFP treatment. (B, C)
Representative images denoting proliferation (KI67+) for (B) control and (C) N2-M1 treatment groups. For each set, the top panel of images shows staining for KI67 (red), DAPI (blue), and an overlay of images. Scale bars correspond to 25 micrometers. The bottom panels show magnified images corresponding to the boxed section in the above image. Arrows indicate a KI67 positive cell.
Figure 3.20. A.
B.
glucagon (Figure 3.22A) mRNA with qRT-PCR showed no changes in the N2-M1 treatment group when compared to the control group. Immunofluorescence staining techniques were employed for insulin (Figure 3.21B, C), glucagon (Figure 3.22B, C), somatostatin (Figure 3.23A, B), and pancreatic polypeptide (Figure 3.23C, D) expressing endocrine cells, all of which exhibited similar populations with menin overexpression compared to the control groups.
To elucidate by what means MEN1 overexpression increases cellular proliferation, we analyzed the effect of N2-M1 transfection on cell cycle regulators similar to section 3.4. In this study, analysis of p18, p21, and p27 mRNA showed no significant change with
MEN1 overexpression (Supplementary Figure 5B).
To further explore a function of menin within islet cell development and differentiation, various transcription factors which are critical for endocrine development were chosen and analyzed by qRT-PCR using PDX1, SOX9, NGN3, NKX2.2, and NKX6.1 probes (Figure 3.24). Analysis of these results indicated consistent mRNA levels for PDX1 and
NGN3. Examination of SOX9 revealed a significant 4.5 fold increase in SOX9 mRNA expression with overexpression of menin. NKX2.2 analysis displayed an increasing trend with N2-M1 transfection where p=0.0527 and NKX6.1 mRNA expression also displayed an increasing trend but was highly variable.