Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells

Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes

Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells

International audienceReprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adeno-viral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miR-NAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes

Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells

Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes

Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells

Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes

Expression levels of endothelial and mesenchymal markers in AR42J and B13 cells.

<p>Relative mRNA expression levels of the mesenchymal factors <i>Zeb1</i> (<b>A</b>) and <i>Zeb2</i> (<b>B</b>) and the endothelial marker <i>E-cad</i> (<b>C</b>) in AR42J and B13 cells. E-cadherin protein levels in AR42J and B13 cells (<b>D</b>). The results are depicted as means ± SEM. n = 3 wells per group. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.01, as determined by using Student’s <i>t</i> test to compare AR42J cells to B13 cells. A.U., arbitrary units.</p

Downregulation of exocrine markers after adenoviral transduction.

<p>Relative mRNA expression levels of the exocrine markers <i>Amy</i> (<b>A</b>), <i>Cela1</i> (<b>B</b>), <i>Cpa1</i> (<b>C</b>) and <i>Ptf1a</i> (<b>D</b>) at 4 days post-transduction with Ad-PNM or Ad-GFP vectors. The results are depicted as means ± SEM. n = 3 wells per group. *<i>p</i>< 0.05, **<i>p</i><0.01, ***<i>p</i><0.001, as determined by one-way ANOVA followed by a post hoc Dunnett’s post test. A.U., arbitrary units.</p

Expression levels of miRNAs of interest.

<p>Relative miRNA expression levels of miR-200c-3p (<b>A</b>), miR-141-3p (<b>B</b>), miR-325-3p (<b>C</b>), miR-2137 (<b>D</b>), miR-210-3p (<b>E</b>), miR-181a-5p (<b>F</b>), miR-204-5p (<b>G</b>), miR-455-3p (<b>H</b>), miR-137-3p (<b>I</b>), miR-135a-5p (<b>J</b>), miR-384-5p (<b>K</b>) in rat exocrine fractions, rat islets, AR42J cells, B13 cells and B13 transduced with Ad-GFP or Ad-PNM at 4 days post-transduction, quantified by individual qPCR assays. The results are depicted as means ± SEM. n = 4 for exocrine and islets controls, n = 3 for AR42J and B13 samples. ND, not detected. NA, not analyzed (because of 2 detected values and 2 non-detected values). *<i>p</i> < 0.05, **<i>p</i><0.01, ***<i>p</i><0.001, as determined by one-way ANOVA followed by a post hoc Tukey’s post test. A.U., arbitrary units.</p

miRNA expression profiles of AR42J and B13 cells that were either transduced or not transduced with adenoviral vectors.

<p>(<b>A</b>) Heatmap for all valid miRNAs detected in samples, calculated from pairwise Pearson distances. Green: lower Ct, Red: higher Ct (<b>B-C</b>). First plan of a principal component analysis (PCA) from data reduced, centered and classified by hierarchical cluster analysis. (<b>B</b>) and (<b>C</b>) represent data obtained from a projection of all samples, and (<b>D</b>) and (<b>E</b>) show only data from B13 cells that were transduced with either Ad-GFP or Ad-PNM. (<b>B</b>) and (<b>D</b>) represent a projection in individual space (samples), whereas (<b>C</b>) and (<b>E</b>) represent a projection in variable space (miRNAs).</p

Expression levels of β cell markers after Ad-PNM transduction in AR42J and B13 cells.

<p>Relative mRNA expression levels of mouse <i>Pdx1</i> (<b>A</b>), <i>Ngn3</i> (<b>B</b>) and <i>MafA</i> (<b>C</b>) and rat endocrine markers <i>Ins1</i> (<b>D</b>), <i>Ins2</i> (<b>E</b>), <i>IAPP</i> (<b>F</b>), <i>NeuroD1</i> (<b>G</b>) and <i>Pax4</i> (<b>H</b>) in AR42J and its subclone B13 at 4 days post-transduction. The results are depicted as means ± SEM. ND, not detected. n = 3 wells per group. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.01, as determined by using Student’s <i>t</i> test to compare AR42J cells transduced with Ad-PNM to B13 transduced with Ad-PNM. A.U., arbitrary units.</p

Expression levels of the insulin processing enzymes Glut 2 and Gck and insulin protein production in reprogrammed B13 cells.

<p>Relative mRNA expression levels of the insulin processing enzymes <i>Pcsk1</i> (<b>A</b>), <i>Pcsk2</i> (<b>B</b>) and <i>Cpe</i> (<b>C</b>), the glucose transporter <i>Glut2</i> (<b>D</b>) and glucokinase (<i>Gck</i>) (<b>E</b>). (<b>F</b>) Representative images of immunocytochemical detection of insulin (in red) and GFP (in green) protein production in reprogrammed B13 cells. Blue, Hoescht (nuclei). All of the analyses were performed at 4 days after transduction with Ad-PNM or Ad-GFP vectors. The results are depicted as means ± SEM. ND, not detected. n = 3 wells per group. **<i>p</i><0.01, ***<i>p</i><0.001, as determined by one-way ANOVA followed by a post hoc Dunnett’s post test. A.U., arbitrary units.</p