Contribution of the intronic mir-338-3p and its Hosting Gene AATK to Compensatory β-cell Mass Expansion

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1 Contribution of the intronic mir-338-3p and its Hosting Gene AATK to Compensatory β-cell Mass Expansion Cécile Jacovetti 1, Veronica Jimenez 2, Eduard Ayuso 2#, Ross Laybutt 3, Marie-Line Peyot 4, Marc Prentki 4, Fatima Bosch 2 and Romano Regazzi 1 1 Department of Fundamental Neurosciences, University of Lausanne, Lausanne, Switzerland. 2 Center of Animal Biotechnology and Gene Therapy and Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas, Barcelona, Spain. # Present address: INSERM UMR189, CHU de Nantes, France and Atlantic Gene Therapies, Nantes, France 3 Diabetes and Obesity Research Program, Garvan Institute of Medical Research, St. Vincent s Hospital, Sydney, New South Wales, Australia. 4 Montreal Diabetes Research Center and CRCHUM, Montreal, QC, Canada and Departments of Nutrition, Biochemistry and Molecular Medicine, University of Montreal, QC, Canada. Supplemental information

2 Supplemental Figure 1. Jacovetti et al. A B C Normalized luciferase activity RIP- sponge RIP-miR-338-3p sponge Ki67 + cells (%) RIP- sponge RIP-miR-338-3p sponge Supplemental Figure 1. Effect of beta-cell specific blockade of mir-338-3p activity using a mirna sponge. A, MIN6B1 cells were transiently cotransfected with a Renilla luciferase reporter construct containing a binding site for mir-338-3p (mir-338-3p sensor) and mir-338-3p or control sponges driven by the Rat Insulin Promoter (RIP). Renilla luciferase activity was measured 3 days later and was normalized to a plasmid leading to constitutive expression of Firefly luciferase. The mir-338-3p sponge increases the expression of the sensor by relieving the repression exerted by the mirna. B, C, MIN6B1 cells were transfected either with RIP- sponge or RIP-miR-338-3p sponge for three days. A, Proliferating cells were scored using Ki67 labeling. B, Cell death was assessed by counting pyknotic cell nuclei in the absence (-) or presence (+) of cytokine mix for 24h. Values are mean ± SD of 3 to 4 different experiments (p<.5). Cell death (%) Cytokines + RIP- sponge - + RIP- sponge RIP-miR-338-3p sponge

3 Supplemental Figure 2. Jacovetti et al. GFP Glucagon Merge Supplemental Figure 2. The RIP-sponge mir-338-3p is not expressed in glucagon-secreting α-cells. Mice were injected intraductally with viral genomes of the AAV8-control sponge or AAV8-miR-338-3p sponge. Both expression cassettes encode also the GFP reporter gene and are driven by the rat insulin promoter. Animals were analyzed 1 month after injection. Immunohistochemical analysis of GFP (green) and glucagon (red) abundance in islets. None of the α-cells express the construct.

4 Supplemental Figure 3. Jacovetti et al. Chromosome 1: 18,786,271-18,822,529 reverse strand mir-338-3p 3 UTR Reverse strand Intron kb 5 Supplemental Figure 3. Schematic overview of the intronic mir-338-3p encoded from the hosting gene AATK. The AATK gene is located on chromosome 1 in rat. mir-338-3p (the mature form is represented in red) is transcribed from the seventh intron.

5 Supplemental Figure 4. Jacovetti et al. A B AATK expression (% of control) Estradiol AATK Tubulin AATK/Tubulin Estradiol C AATK expression (% of control) Exendin-4 D AATK Tubulin AATK/Tubulin p=.6 Exendin-4 Supplemental Figure 4. Down-regulation of AATK mrna and protein levels in INS832/13 cells. INS832/13 cells were treated for 48h with 1nM of estradiol (A, B) or 1nM of the GLP1 analogue exendin-4 (C, D). (A, C) AATK expression was measured by qrt PCR and normalized to 18S, n=3. (B, D) The cells were homogenized and the lysates analyzed by Western blotting with an antibody against AATK. The figure shows representative blots and band quantification from 3 to 4 independent experiments (means ± SD). Protein levels were normalized to tubulin. Results are expressed as percentage of control or as fold change over control. Significantly from control condition (p<.5).

6 Supplemental Figure 5. Jacovetti et al. A B C Normalized luciferase activity ns mir-338-3p mir-338-3p Cell death (%) Vehicle Cytokines Vehicle Tnfrsf1b Ki67 + cells (%) Vehicle ns Tnfrsf1b 3'UTR 3'UTR Tnfrsf1b Supplemental Figure 5. Tnfrsf1b is a direct target of mir-338-3p. (A) INS832/13 cells were co-transfected with a Firefly luciferase construct, a Renilla luciferase reporter plasmid containing a 3 UTR control or the 3 UTR sequence of rat Tnfrsf1b and a control oligonucleotide or a mir-338-3p mimic. Renilla luciferase activity was measured 3 days later and was normalized to Firefly luciferase activity generated from a constitutive promoter to correct for potential differences in transfection efficiency. (B) Cell death was assessed by counting the fraction of INS832/13 cells displaying pyknotic-nuclei upon exposure for 24h to vehicle (water) or to 1 nm soluble Tnfrsf1b in the presence (+) or absence (-) of a mix of pro-inflammatory cytokines (1 ng/ml TNF-α,.1 ng/ml IL-1β, 3 ng/ml IFN-γ). (C) Cell proliferation was assessed by scoring Ki67-positive INS832/13 cells in the presence or absence of 1 nm Tnfrsf1b. Values represent the mean ± SD of 3 independent experiments. p<.5.

7 Supplemental Figure 6. Jacovetti et al. TOTAL AKT pakt-thr 48 P-AKT/AKT total anti-control anti-338-3p Supplemental Figure 6. Down-regulation of mir-338-3p activates the AKT pathway. INS832/13 cells were transfected with a control anti-mirna or with anti-mir-338-3p. Two days later, the cells were homogenized and the lysates analyzed by Western blotting with antibodies against total AKT or pakt-thr 48. The figure shows representative blots and band quantification from 3 independent experiments (means ± SD). Protein levels were normalized to total AKT and expressed as fold change over control. Significantly different from control condition (p<.5).

8 Supplemental Table 1. Jacovetti et al. Supplemental Table 1. Characteristics of human islet donors. F, female; M, male; BMI, Body mass index.

9 Supplemental Table 2. Jacovetti et al. Mice group Phenotype Weight (g) Glycaemia (mmol/l) db/ db/ db/ db/ Pre-diabetic db/db db/db db/db db/db db/db Supplemental Table 2. Characteristics of pre-diabetic db/db mice and agematched controls. Weights and glycaemia of each of the 6-week-old prediabetic db/db mice from which pancreatic islet RNA was isolated are shown.

10 Supplemental Table 3. Jacovetti et al. Mice group Weight before diet (g) Weight at 6.5 weeks (g) Glyaeamia at 6.5 weeks (mmol/l) Weight at sacrifice (g) Glyaeamia at sacrifice (mmol/l) Insulinemia at sacrifice (mmol/l) Normal diet High-fat diet Supplemental Table 3. Characteristics of high-fat diet fed mice and age-matched controls. Weights and glycaemia of mice fed a normal or high-fat diet from which pancreatic islet RNA was isolated are reported.

11 Supplemental Table 4. Jacovetti et al. Term (KEGG analysis) Targets P-Value Target genes PATHWAYS IN CANCER E-5 ARNT, CDK6, ETS1, FGF2, FGF9, FGFR2, FOS, FZD3, HGF, HIF1A, LAMC2, PTCH1, SMO, TPM3, WNT5A, XIAP REGULATION OF ACTIN CYTOSKELETON E-4 ARPC1B, FGD1, FGF2, FGF9, FGFR2, GNG12, ITGA5, ITGB3, NCKAP1L, PPP1CB, VAV3 MAPK SIGNALING PATHWAY E-3 CACNB4, FGF2, FGF9, FGFR2, FOS, GNG12, MAP3K3, MAP4K3, NTRK2, PLAG3 FOCAL ADHESION E-3 HGF, ITGA5, ITGB3, KDR, LAMC2, PPP1CB, THBS1, VAV3, XIAP NEUROTROPHIN SIGNALING PATHWAY E-3 CAMK2A, CAMK2G, FRS2, MAP3K3, NTRK2, YWHAB, YWHAH CYTOKINE-CYTOKINE RECEPTOR INTERACTION E-2 ACVR1, CCL21, CNTFR, HGF, KDR, TNFRSF1B, TNFSF11 OOCYTE MEIOSIS E-3 CAMK2A, CAMK2G, PPP1CB, SLK, YWHAB, YWHAH HYPERTROPHIC CARDIOMYOPATHY E-3 CACNB4, DES, ITGA5, ITGB3, TPM3, TTN DILATED CARDIOMYOPATHY 6 3.8E-3 CACNB4, DES, ITGA5, ITGB3, TPM3, TTN CELL ADHESION MOLECULES E-2 CADM1, CD8B, ICOS, NLGN2, NRXN1, NRXN3 AXON GUIDANCE E-2 NRP1, SEMA3F, SEMA4F, SEMA6A, SEMA6D, SRGAP3 Supplemental Table 4. Pathway analysis of the predicted targets of mir-338-3p. We performed a KEGG analysis of the predicted target genes. The table shows the pathways displaying a significant enrichment in mir-338-3p targets and the list of the genes predicted to be regulated by the mirna.