We compared the global miRNA expression profile of pancreatic islets of 4- and 8-week-old NOD mice. In agreement with our previous findings (11
), the islets of 8-week-old mice displayed an increase in the expression of miR-21, mir-34a, and miR-146a/b (Supplementary Table 1
). In addition, we detected significant alterations in the level of several other miRNAs, including miR-142-3p, miR-142-5p, miR-150, miR-155, and miR-216a, which are almost undetectable in the islets of 4-week-old NOD mice (Supplementary Fig. 2 and 3
). Because these miRNAs are expected to be particularly abundant in immune cells (28
), their expression was evaluated in islets of 14-week-old, prediabetic NOD mice, which were cultured to allow for the escaping of the infiltrated lymphocytes (29
). These miRNAs were 100- to 1,000-fold more abundant in the immune cells that had escaped the islets than in islet cells (Supplementary Fig. 3
). In situ hybridization combined with immunofluorescence staining for insulin and CD8a (the hybridization and the immunostaining were performed on sequential sections of the very same pancreases), confirmed that most of the miR-142-3p is expressed in immune cells and not in insulin-producing cells (Supplementary Fig. 4
, top panels
). We concluded that the increase in the latter miRNA in the islet preparations of aging NOD animals likely reflected mainly the presence of immune cells; for this reason, this group of miRNAs was not investigated any further in this study.
Among the several other miRNAs that were differentially expressed between 4- and 8-week-old animals, we noticed the members of the miR-29 family (miR-29a/b/c) that are among the most abundant miRNAs present in normal β-cells (30
) (Supplementary Table 1
). These miRNAs have been shown to downregulate the expression of the β-cell disallowed gene, monocarboxylase transporter 1 (31
) and to contribute to insulin resistance in insulin target tissues in GK rats (33
). Analysis by qPCR confirmed upregulation of miR-29a/b/c in the islets of 8-week-old mice and revealed a further increase in islet preparations of normoglycemic 13- to 14-week-old mice (). The level of expression of miR-29a/b/c in lymphocytes escaping the islets of NOD mice was comparable to the level detected in the islets of 4-week-old mice (). Moreover, islets of 14-week-old mice incubated for 24 h to permit the escape of infiltrating lymphocytes retained strongly elevated levels of miR-29a/b/c (). In agreement with these observations, in situ hybridization of NOD pancreas at different ages, combined with an immunofluorescence staining for insulin on close by sections, confirmed that the bulk of miR-29a (Supplementary Fig. 4, lower panels
, and Supplementary Fig. 5
) and miR-29b (not shown) is concentrated in insulin-producing cells and not in infiltrating immune cells.
FIG. 1. The expression of the members of the miR-29 family increases with age in the pancreatic islets of NOD mice. A: Pancreatic islets were isolated from female NOD mice of different ages. Only animals displaying blood glucose levels within the normal range (more ...)
Taken together, these findings indicate that the rise of miR-29a/b/c reflects changes occurring in β-cells and is not merely the result of the presence of immune cells in the islets. The observed increase in miR-29 expression is also not the result of mice aging. Indeed, miR-29a/b/c levels were not significantly different between the islets of 6- and 12-week-old C57BL/6 mice (data not shown), and no significant changes were observed in the islets of 13-week-old NOD-SCID mice that do not develop type 1 diabetes. In contrast, proinsulin 2-deficient mice, which display accelerated insulitis and diabetes (34
), showed a dramatic rise in miR-29a/b/c expression already at age 8 weeks (Supplementary Fig. 6
Incubation of MIN6 cells (), mouse islets (), or dissociated human islet cells () in the presence of Il-1β (0.1 ng/mL; 50 units/mL), TNF-α (10 ng/mL; 500 unit/mL), and IFN-γ (30 ng/mL; 50 units/mL) led to a twofold increase in the expression of the three miR-29 family members, suggesting that at least part of the observed induction occurring during early stages of diabetes development in NOD mice was linked to the chronic exposure of β-cells to these proinflammatory cytokines.
FIG. 2. Proinflammatory cytokines increase miR-29 expression. A: MIN6 cells were incubated for 30 h in the presence or absence of a cytokine mixture (Cyt Mix) of IL-1β (0.1 ng/mL; 50 units/mL), plus TNF-α (10 ng/mL; 500 units/mL) and IFN-γ (more ...)
To test the effect of miR-29a/b/c overexpression on pancreatic β-cell functions, oligonucleotides mimicking the mature sequence of each of the miR-29 family members were transiently transfected into MIN6 cells. This transfection resulted in a cellular content of the respective miRNA, which was comparable to that observed in the islets of 8-week-old NOD mice (Supplementary Fig. 7
). We first assessed the effect of alterations in the level of the three miRNAs on insulin biosynthesis. Overexpression of miR-29 family members led to a small decrease in proinsulin mRNA levels but did not significantly modify total insulin content (). We then investigated the effects on insulin release. The rise of miR-29a/b/c in MIN6 cells did not affect basal insulin secretion (). In contrast, glucose-induced insulin release was decreased by 20–40%. A similar reduction in insulin secretion elicited in the presence of glucose was detected upon overexpression of miR-29 family members in primary mouse islet cells () and in primary human islet cells (Supplementary Fig. 8
FIG. 3. miR-29 overexpression does not change insulin content. A: MIN6 were transfected with a control RNA duplex or with duplexes corresponding to the mature forms of miR-29a, miR-29b, and miR-29c. Proinsulin mRNA levels were measured 2 days later by qPCR. Results (more ...)
FIG. 4. miR-29 overexpression alters glucose-induced insulin secretion. A: MIN6 cells were transiently transfected with a control RNA duplex or with duplexes corresponding to the mature forms of miR-29a, miR-29b, and miR-29c. Two days later, the cells were incubated (more ...)
The transcription factor Onecut2 is a predicted target of miR-29a/b/c (http://www.targetscan.org
). We previously demonstrated that Onecut2 modulates the expression of granuphilin, a secretory granule protein acting as a potent inhibitor of insulin exocytosis (8
) that is expressed in all β-cells of nondamaged islets, irrespective of the age of NOD mice (data not shown). In agreement with the computational predictions, transfections under the conditions that led to miR-29a/b/c overexpression resulted in a decrease in the level of Onecut2 and a consequent rise in the expression of granuphilin () in MIN6 cells and in dissociated mouse islet cells, potentially explaining at least part of the effect of miR-29 family members on glucose-induced insulin release. In contrast, we were unable to detect significant changes in the level of Syntaxin 1a, a key component of the machinery of insulin exocytosis (36
), which is also identified by computational programs as a potential target of miR-29 ().
FIG. 5. Overexpression of miR-29 selectively alters the expression of proteins involved in insulin exocytosis. MIN6 cells (left panel) or dissociated mouse islet cells (right panel) were transiently transfected with RNA duplexes that allow for overexpression (more ...)
The effect of elevated levels of miR-29 family members on cell survival was next investigated. Overexpression of miR-29a/b/c in MIN6 cells (, left panel) and in mouse (, left panel) and human dissociated islet cells () led to a significant increase in cell death, as assessed by scoring the cells displaying pyknotic nuclei. Identical results were obtained when apoptosis was assessed by Annexin V labeling (, right panels). In contrast, the number of necrotic cells determined by propidium iodide labeling remained unchanged (data not shown). Pretreatment with anti–miR-29c significantly decreased, to control levels, the number of MIN6 cells undergoing apoptosis in the presence of IL-1β, TNF-α, and IFN-γ ().
FIG. 6. Overexpression of miR-29 increases the apoptosis of transformed and primary β-cells. A: MIN6 cells were transiently transfected with a control RNA duplex or with duplexes leading to miR-29a, miR-29b, and miR-29c overexpression. Three days later, (more ...)
We then attempted to elucidate the mechanisms through which a rise in the level of miR-29a/b/c can trigger β-cell death. Interestingly, Mcl1, an antiapoptotic protein belonging to the Bcl2 family and playing a key role in β-cell survival (38
), is a predicted target of miR-29a/b/c (26
). Indeed, we found that increased levels of miR-29a/b/c inhibited the expression by MIN6 cells of a luciferase construct containing the 3′UTR sequence of Mcl1 (). This effect was abrogated by mutation of the sequence recognized by these miRNAs, indicating a direct interaction with the 3′UTR of Mcl1. Western blot analysis confirmed a significant reduction of the level of Mcl1 in MIN6 cells and in mouse islet cells overexpressing the miR-29 family members (). No significant changes in MCL1 mRNA levels were detected in miR-29 overexpressing cells, indicating that the effect of the miRNAs is mainly post-transcriptional (Supplementary Fig. 9
FIG. 7. Mcl1 is a direct target of miR-29 family members. A: MIN6 cells were cotransfected with a Renilla luciferase construct and Firefly luciferase reporter plasmids containing the wild-type (wt) or a mutated (mut) sequence of the 3′UTR of mouse Mcl1 (more ...)
To demonstrate a direct link between the reduction of Mcl1 and the effect of miR-29 on β-cell apoptosis, MIN6 cells were transfected with an oligonucleotide designed to specifically protect the Mcl1 mRNA from miR-29 binding, without interfering with the regulation of other miR-29 targets (25
). The reduction in Mcl1 levels elicited by miR-29c overexpression or by proinflammatory cytokines was efficiently prevented by transfection of this target protector (Supplementary Fig. 10A and B
). As shown in , the cells expressing the Mcl1 target protector were resistant to apoptosis induced by miR-29 overexpression. Moreover, the cells transfected with the Mcl1 target protector were not sensitive to cytokine-induced apoptosis (), indicating that at least part of the deleterious effect of these inflammatory mediators on β-cell survival is caused by a miR-29–induced drop in the expression of the antiapoptotic Mc11 protein.
FIG. 8. miR-29– and cytokine-induced apoptosis is blocked by preventing binding to Mcl1. A: MIN6 cells were transfected with a control RNA duplex or with duplexes corresponding to the mature form of miR-29c, together with a scrambled miScript Target Protector (more ...)