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Deletion of pancreas-specific miR-216a reduces beta-cell mass and inhibits pancreatic cancer progression in mice

Summary miRNAs have crucial functions in many biological processes and are candidate biomarkers of disease. Here, we show that miR-216a is a conserved, pancreas-specific miRNA with important roles in pancreatic islet and acinar cells. Deletion of miR-216a in mice leads to a reduction in islet size, β-cell mass, and insulin levels. Single-cell RNA sequencing reveals a subpopulation of β-cells with upregulated acinar cell markers under a high-fat diet. miR-216a is induced by TGF-β signaling, and inhibition of miR-216a increases apoptosis and decreases cell proliferation in pancreatic cells. Deletion of miR-216a in the pancreatic cancer-prone mouse line KrasG12D;Ptf1aCreER reduces the propensity of pancreatic cancer precursor lesions. Notably, circulating miR-216a levels are elevated in both mice and humans with pancreatic cancer. Collectively, our study gives insights into how β-cell mass and acinar cell growth are modulated by a pancreas-specific miRNA and also suggests miR-216a as a potential biomarker for diagnosis of pancreatic diseases.


In brief
Erener et al. identify miR-216a as a pancreas-specific miRNA affecting pancreatic pathologies. Under a high-fat diet, deletion of miR-216a reduces beta cell mass and induces acinar gene expression. In cancer-prone mice, miR-216a deletion decreases pancreatic neoplasia frequency. Circulating miR-216a levels are elevated in mice and humans with pancreatic cancer.

INTRODUCTION
Perturbations in the function of pancreatic endocrine and exocrine cells leads to the development of life-burdening and/ or life-threatening diseases such as diabetes and pancreatic cancer. Although diabetes is characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both, pancreatic ductal adenocarcinoma (PDAC), a major form of pancreatic cancer in humans, results from the abnormal proliferation of the exocrine cells in response to oncogenic mutations. 1,2 To date many protein coding genes necessary for pancreas development and function have been identified, but the role of the majority of small RNAs is still unclear.
MicroRNAs (miRNAs) are short ($21-22 nt long) non-coding RNAs that have emerged in the past two decades as buffers of signaling pathways to maintain normal tissue development and function. 3 Mature miRNAs function as evolutionarily conserved post-transcriptional gene regulators that mainly decrease the stability or inhibit translation of messenger RNAs (mRNAs) through binding to complementary sequences. 4 A single miRNA can affect the regulation of hundreds of genes with multiple targets within cellular networks that enable modulation of entire pathways in the context of an individual biological process. 5,6 In the pancreas, conditional deletion of the miRNA processing endonuclease Dicer at the onset of pancreatic development (e9.5) using a Pdx1-Cre strain results in defects in all pancreatic lineages with dramatic reduction in the ventral pancreas as well as a reduction in the overall epithelial contribution to the dorsal pancreas at e18.5. 7 Postnatal Dicer ablation in b-cells using the conditional RIP2-Cre or Pdx1-CreER strain impairs islet Figure 1. miR-216a is enriched in pancreatic islets and is highly conserved among various species (A) miRNA profiling of adult human islets compared with human embryonic stem cells. miRNAs with greater than 3-fold increase in the islets are shown in magenta. Arrow points to miR-216a. n = 3 human islet donors. Data represent mean log 2 signals ± SEM (from human islets). (B) Equal amounts of RNA from various human tissues (each a pool of three tissue donors) was reverse-transcribed, and miR-216a expression was determined using qRT-PCR. Threshold cycle 33 (Ct = 33) was arbitrarily set as 1.
(C) Human embryonic stem cells (ESCs) were differentiated to pancreatic endocrine cells for the indicated days, and miR-216a expression was measured using qRT-PCR and expressed relative to levels in undifferentiated ESCs. In ESCs, Ct = 27. (D) Fetal and adult human pancreata were probed with DIG-labeled miR-216a and scrambled control miRNA probes at the indicated gestational weeks. Purple color indicates presence of miRNA expression. Scale bar, 100 mm. (E) Comparison of mature miR-216a sequences in different species. architecture, insulin secretion, and b-cell mass, while deletion of Dicer in acinar cells using Mist1-CreERT mice promotes epithelial to mesenchymal transition accompanied by acinar-to-ductal metaplasia. [8][9][10][11] Although previous findings demonstrated important roles for Dicer in the pancreas, in vivo studies investigating the role of individual miRNAs in both endocrine and exocrine function are limited. Furthermore, the majority of investigated miRNAs do not have pancreas-specific expression.
Tissue-specific patterns of gene expression play fundamental roles in tissue development and function. 12 Although the majority of miRNAs are ubiquitously expressed, some miRNAs exhibit tissue-specific or developmental stage-specific expression patterns and contribute to maintaining normal tissue identity and function. 13,14 Tissue-specific miRNAs are also found in mouse and human plasma and serum at surprisingly high concentrations. 15 In the present study, we hypothesized that pancreas-enriched miRNAs might have critical roles for endocrine and exocrine cell function and serve as biomarkers for diagnosing pancreatic diseases. We find that miR-216a is a pancreas-specific miRNA and contributes to b-cell and acinar cell function by modulating cell growth. We further show that circulating miR-216a levels are altered in both mice and humans with pancreatic tumors. Our data unveil the potential of tissue-specific miRNAs to contribute to disease mechanisms and to serve as biomarkers.

RESULTS
To identify the miRNAs that are enriched in pancreatic islets, we performed miRNA profiling from adult human islets and compared it with embryonic stem cells (ESCs). We identified nine miRNAs that showed greater than 3-fold expression in human islets compared with ESCs ( Figure 1A). Among the nine miRNAs identified, only miR-216a showed a pancreas-specific expression pattern ( Figure 1B), while the other miRNAs were ubiquitously expressed in the analyzed tissues ( Figure S1A). qRT-PCR analysis of miR-216a levels in multiple tissues showed that miR-216a was expressed in human islets and whole pancreas to comparable levels ( Figure S1B). To examine whether miR-216a levels are changed during endocrine cell development, we differentiated human ESCs to pancreatic-like cells. When comparing sequential time points, the single largest increase in levels of miR-216a occurred on day 14 ( Figure 1C) of the differentiation protocol, which marks the generation of PDX1 + /NKX6.1 + pancreatic endocrine progenitor cells. 16 miR-216a levels further increased during differentiation, reaching the highest levels at the final stage of differentiation (days [26][27][28][29][30][31][32][33], correlating with the presence of pancreatic endocrine cells. To further investigate miR-216a expression during development, we performed in situ hybridization with human fetal pancreatic tissue using DIG-labeled miR-216a-LNA probes. There was faint staining in the pancreatic tissue at the gestational age 8 weeks, 4 days ( Figure 1D; Figure S2), during the time when insulin and glucagon double-positive cells emerge. 17 However, we detected strong miR-216a staining in the branching pancreatic epithelium that harbor PDX1 + /NKX6.1 + pancreatic endocrine progenitor cells. 17 Analysis of human adult pancreas also revealed strong pancreatic miR-216a staining with further enrichment in pancreatic islets ( Figure 1D; Figure S2). To further investigate the specificity of miR-216a for pancreatic tissue, we performed in situ hybridization on kidney capsule grafts obtained from mice and rats implanted with pancreatic progenitor cells that had developed to endocrine cells. 18 There was strong reactivity for miR-216a in the grafts, whereas neighboring kidney sections had no detectable staining ( Figure S1C). We next examined the miR-216a sequence across diverse species and found it to be highly conserved ( Figure 1E), suggestive of a functional importance. A thorough tissue expression analysis from C57BL/6 mice showed that miR-216a levels were specifically expressed in the pancreas with further enrichment in pancreatic islets ( Figure 1F). To further investigate the expression of miR-216a in ductal cells, we differentiated mouse exocrine cells into ductal cells and quantified miR-216a expression by qRT-PCR. Expression of miR-216a was significantly downregulated as the cells differentiated into ductal cells ( Figures 1G and 1H). Overall, these data suggest a role for miR-216a miRNA in pancreatic acinar cell and islet development and/or function.
To explore the role of miR-216a in pancreatic islet function in vivo, we generated miR-216a knockout (KO) mice in which the precursor sequence of miR-216a (pre-miR-216) was deleted by homologous recombination. 19 miR-216a KO mice were viable, and qRT-PCR analysis from isolated islets of miR-216a KO mice confirmed miR-216a was efficiently deleted ( Figure S3A). Body weight, fasting blood glucose levels, and glucose tolerance were comparable between miR-216a KO and wild-type (WT) littermate controls when fed with a chow diet (Figures S3B-S3G). Pancreas weight and pancreatic cell size were unchanged ( Figures S3H and S3I). To investigate the effect of miR-216a deletion on endocrine pancreas, we isolated islets from miR-216a KO mice and WT littermates and analyzed islet function. During glucose-stimulated insulin secretion (GSIS) assays, miR-216a KO islets secreted less insulin at basal glucose concentration (2.8 mM) (Figure 2A). To determine whether the observed effects on insulin secretion were cell autonomous and direct, we transfected rat INS-1 cells with miR-216a mimetics and performed GSIS assays. Inhibition of miR-216a did not change insulin secretion at low or high glucose concentrations ( Figure S2J). Analysis of isolated islets revealed that miR-216a KO were smaller than the WT islets ( Figure 2B), with miR-216a KO mice lacking bigger islets and trending toward having an increased number of smaller islets ( Figures 2C and 2D). Immunostaining of pancreas from adult mice with insulin and glucagon antibodies demonstrated that b-cell mass was significantly reduced in the miR-216a KO mice compared with controls, while a-cell mass was unchanged (Figures 2E-2G). Islet circularity and the location of a-cells were unchanged ( Figures S3K and S3L). Consistent with the isolated islet data, the area of islets determined by synaptophysin immunostaining was significantly smaller in miR-216a KO mice ( Figures  2E and 2H), and miR-216a KO pancreatic sections harbored less of the larger islets, compared with WT controls ( Figure 2I). In 1-day-old miR-216a KO pups and WT littermates, b-cell and a-cell area were not different ( Figures S4A-S4C). Similarly, average islet size was comparable, with no statistical difference in various islet size groups ( Figures S4A, S4D, and S4E). Islet circularity and peripheral a-cells were also comparable ( Figures  S4F and S4G), suggesting a postnatal role for miR-216a in regulating b-cell mass and islet size.
Regulation of islet size is complex and involves cellular processes such as fusion, fission, growth, and migration. 20 Cell migration is critical for both islet formation and the movement of islets away from ducts. 21,22 To test whether miR-216a alters cell migration and/or proliferation, we transfected PDAC PANC-1 cells that have very low miR-216a levels (not shown) and migration ability with control (ctrl) or miR-216a mimetics and performed a migration assay using transwell chambers. Quantification of the cells that traversed the Boyden chambers demonstrated that miR-216a more than doubled the number of migrating cells compared with untransfected and control miRNA-transfected wells ( Figure 3A). We next assessed the migration ability of miR-216a KO islet cells ex vivo. We coated cell culture plates with the matrix secreted by 804G cells and monitored the spreading of islets by light micro-scopy. Five days post-seeding, more WT islets spread compared with miR-216a KO islets, while miR-216a KO islets had more defined borders, suggestive of less spreading ( Figure 3B). To further explore the possibility that miR-216a KO alters islet migration, we quantified the distance of islets from the ducts in both WT and KO pancreata. Islets from miR-216a KO mice were closer to the ducts compared with WT islets (p = 0.058) ( Figure 3C). TGF-b signaling regulates pancreatic epithelium branching and cell migration to form islet clusters. 23 It has been previously shown that miR-216a expression is regulated by TGF-b signaling and alters expression of Pten and Smad7. 24,25 To investigate the potential signaling pathways regulating miR-216a expression in the pancreas, we treated EndoC-bH1 cells, which have high endogenous miR-216a levels, with a TGF-b agonist TGF-b1 and the inhibitor SB431542 and measured miR-216a levels by qPCR. TGF-b1 treatment significantly increased miR-216a expression and inhibition of TGF signaling with SB431542 significantly decreased miR-216a levels ( Figures 3D and 3E). We next examined mRNA levels of potential miR-216a target genes in the pancreatic islets of WT and miR-216a KO mice. qRT-PCR analysis of islets of WT and miR-216a KO mice indicated that Smad7 expression was significantly upregulated in the miR-216a KO islets, whereas Pten, Becn1, and Ybx1 levels were unchanged ( Figure 3F). We also examined expression of key hormones and transcription factors regulating endocrine cell identity. Insulin, Glucagon, Pdx1, and Nkx6.1 levels were not changed in islets from miR-216a KO mice ( Figure S5). TGF-b signaling and Smad7 are known regulators of cell migration, and their mode of action is thought to be via altering the expression of genes involved in regulating extracellular matrix (ECM) composition. 26 We analyzed the levels of ECM genes in miR-216a transfected cells using an ECM gene array. miR-216a altered the expression of 12 of the 87 genes analyzed ( Figure 3G). Levels of genes involved in basement membrane, such as Col4a2, Ecm1, Fbln1, Lama3, Lamb2, and Lamb3, were upregulated in the miR-216a transfected cells, suggesting that TGF-b-induced miR-216a can increase cell migration by altering the ECM integrity of basement membrane. We next investigated whether inhibition of miR-216a can affect cell proliferation and cell death by transfecting INS-1E b-cells with miR-216a and a control scrambled miRNA inhibitor. INS-1E b-cells have higher levels of miR-216a compared with other rodent b-cell lines such as MIN6 (not shown) and thus were chosen for miR-216a inhibition experiments. Inhibition of miR-216a significantly reduced cell proliferation compared with control miRNA inhibitors ( Figure 3H). Analysis of cell death using live cell imaging indicated that inhibition of miR-216a in the presence of cytotoxic factors (TNF-a, IFN-g, IL-1b) increased the rate of apoptosis ( Figure 3I). Similarly, inhibition of miR-216a in mouse islets increased expression of the pro-apoptotic gene Bad ( Figure 3J).
Many miRNAs regulated by metabolic stressors coordinate the adaptive responses of islet cells to metabolic stresses during obesity and diabetes. 27 We sought to investigate whether deletion of miR-216a would change insulin secretion when challenged with metabolic overload. We first explored miR-216a levels in the islets of leptin KO (LepKO) rats, a model of obesity with disturbed glucose and energy metabolism and larger islets. 28 miR-216a levels were significantly higher in the LepKO rat islets, whereas levels of another well-studied islet enriched miRNA, miR-375, were unchanged ( Figure 4A). Notably, miR-216a levels were also reported to be increased in human islets from subjects with T2D. 29 When placed on a 60% high-fat diet (HFD) for 8 weeks, fasting body weight and blood glucose levels were comparable between miR-216a KO mice and littermate WT controls ( Figures S6A and S6B). However, an oral glucose tolerance test performed 2 weeks after HFD revealed that miR-216a KO mice secreted less insulin in response to glucose administration despite no significant differences in blood glucose levels ( Figures 4B and 4C). Insulin tolerance tests indicated similar insulin sensitivity of miR-216a KO and WT mice ( Figure S6C), suggesting compromised islet function in miR-216a KO mice under HFD. Consistent with the reduced insulin secretion from miR-216a KO mice in vivo, pancreata from miR-216a KO mice had significantly reduced b-cell mass compared with controls after HFD feeding ( Figures 4D and 4E). Furthermore, miR-216a KO mice had decreased circulating miR-375 and proinsulin levels compared with WT after 8 weeks of HFD feeding ( Figures 4F  and 4G). The degree of cell proliferation was comparable in the WT and miR-216a KO b-cells as assessed by PCNA immunostaining ( Figure 4H), and miR-216a KO mice fed the HFD also had significantly smaller islets ( Figure 4I), with increased frequency of smaller islets and decreased occurrence of larger islets ( Figure 4J). Overall, these data indicate that under metabolically stressed condition of 60% HFD, miR-216a KO mice secrete lower insulin during a glucose challenge and have decreased islet size with reduced b-cell mass and proinsulin protein levels.
For an unbiased investigation of islet dysfunction, we performed single-cell RNA sequencing (RNA-seq) analysis from the islets of WT and miR-216a KO mice exposed to 60% HFD for 9 weeks. Cell counts and viability assessments prior to sequencing did not indicate any differences between WT and KO islet cell preparations (Table S1). After sequencing, we assigned cell clusters to specific cell types for further analysis on the basis of differential expression of canonical genes (Tables  S2 and S3 clusters. The proportion of cells in each cluster were similar, and all clusters were represented in both genotypes ( Figure 4K). b-cells were restricted to those that had statistically significantly upregulated expression of Ins1, Ins2, and Iapp genes ( Figure 4L). Many clusters had no (7 of 30 clusters) or fewer than 10 (15 of 30 clusters) differentially expressed genes between genotypes (Table S4), suggesting that overall, most pancreatic cells were similar between genotypes. There appeared to be two major subpopulations of b-cells ( Figures 4K and 4L); one population contained clusters with upregulation of genes typical for mature b-cells, such as Unc3 and Mafa (b.1; Table S3), whereas the other population (b.2) lacked these genes and had higher expression of Cpa1, a canonical acinar gene, as well as being closer to the acinar population in local UMAP (uniform manifold approximation and projection) space ( Figure 4L) compared with cluster b.1. There were no or few differentially expressed genes in the b.1 cluster between the genotypes, but many genes were differentially expressed in the b.2 cluster when comparing WT and KO cells ( Figure 4M; Table S4). Similarly, many genes were differentially expressed in the acinar cell cluster (Figure S6D; Table S4). To further investigate the differentially expressed genes, we performed gene set enrichment analysis for the two most different clusters, b.2 and acinar cells ( Figure 4N; Figure S6E). For the b.2 cluster, many genes related to digestion were upregulated, suggesting that islets from KO mice had more characteristics of acinar-like cells than those of WT mice. Additionally, the TGF-b receptor signaling pathway was downregulated in KO cells compared with WT. Many pathways that were significantly different between WT and KO cells in the b.2 cluster, including ''activation of matrix metalloproteinases,'' ''digestion,'' and ''collagen degradation,'' were also different in the acinar cell clusters. Interestingly, Gcg was statistically significantly upregulated in many of the KO clusters.
As miR-216a is expressed in both endocrine and exocrine pancreas, we sought to perform a global analysis to further explore signaling pathways targeted by miR-216a in the whole pancreas. We performed RNA-seq from the pancreata of 1day-old WT and miR-216a KO mice. The RNA integrity numbers (RINs) obtained from the pancreata of miR-216a KO mice were all above 8 and suitable for RNA-seq (not shown). We examined the top 50 most abundant genes expressed in WT and miR-216a KO pancreata and observed the expected abundance for genes encoding amylase, trypsin, and insulin, but they were not significantly different between WT and miR-216a KO mice ( Figure 5A). Differential gene expression analysis from all the transcripts using an adjusted p value < 0.05 revealed that 409 genes were differentially expressed between WT and KO pancreata (Table  S5). To identify more globally the affected biological processes  in the miR-216a KO pancreata, we next performed a Gene Ontology (GO) analysis and found cell cycle to be most significantly altered ( Figure 5B). Similarly, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis indicated that cell cycle, DNA replication, and repair pathways were statistically different in the miR-216a KO versus WT pancreata ( Figure 5C). Next, we compared the expression of genes involved in cell cycle pathways between WT and miR-216a KO pancreata and observed that many cyclin-dependent kinases (e.g., Cdk4, Cdk1) and cyclins (e.g., Ccne1) as well as DNA replication genes (e.g., Pcna, Mcm2, Mcm5) were decreased in the pancreas of miR-216a KO mice ( Figure 5D). This was consistent with our observation that inhibition of miR-216a decreases cell proliferation in INS-1E b-cells ( Figure 3H) and the presence of reduced b-cell mass and islet size in the miR-216a KO mice. A map of the cell cycle pathway with genes significantly regulated in miR-216a KO pancreata is shown in (Figure S7). To investigate the effect of miR-216a on cell proliferation in cells with exocrine origin, we transfected human PANC-1 cells with the miR-216a mimetic and another miRNA, miR-217. Similar to INS1-1E b-cells, miR-216a transfection significantly increased the number of cells ( Figure 5E), and decreased the rate of apoptosis (Figure 5F), thus confirming the role of miR-216a in regulating cell proliferation in another pancreatic cell type. We next investigated whether the absence of miR-216a alters the progression of a pancreatic pathology related to cell cycle/ proliferation in acinar cells. Oncogenic KRAS can induce PDAC precursor lesions from pancreatic acinar cells. 30,31 A number of studies have shown that PDAC develops from abnormally proliferating cells in the precursor lesions termed pancreatic intraepithelial neoplasia (PanIN), the most common precursor lesions observed in humans. 32 A mutation in KRAS oncogene is currently considered as the initiating factor in pancreatic cancer. 33 Expression of constitutively active Kras G12D allele in mice induces PanINs and, after a significant latency period, PDAC. 34 We crossed miR-216a KO mice with the pancreatic cancerprone Kras G12D ;Ptf1a CreER mice ( Figure 6A) and analyzed the frequency of PanINs in the pancreata of offspring. As expected, (G) Total RNA was isolated from the plasma of LSL KrasG12D;Ptf1a-Creex1 mice at the indicated weeks. miR-216a levels were measured using qRT-PCR and normalized to miR-93-5p levels. n = 3-6 mice.
A two-way ANOVA (F) or one-way ANOVA (G and H) with Bonferroni's multiple-comparison posttest was performed to assess significance. *p < 0.05. Data represent mean ± SEM.
Cell Reports Medicine 2, 100434, November 16, 2021 9 Article ll OPEN ACCESS H&E staining from the pancreata of Kras G12D ;Ptf1a CreER ; miR-216a (+/+) mice revealed widespread lesions with columnar to cuboidal cells with varying degrees of cytological and architectural atypia ( Figure 6B). In contrast, the pancreata of mice lacking miR-216a had limited neoplastic lesions. Further histological analysis with Alcian blue staining confirmed the histological characteristic of high acidic mucin content of PanINs in the WT mice, whereas miR-216a KO pancreata had decreased PanIN frequency ( Figure 6C). In Kras G12D ;Ptf1a CreER ;miR-216a (À/À) mice, there was more than 2-fold reduction in the Alcian blue + area compared to Kras G12D ;Ptf1a CreER ;miR-216a (+/+) mice ( Figure 6D). We assessed acinar-to-ductal metaplasia by immunostaining the pancreas using the acinar and ductal cell markers Cpa1 and Sox9, respectively. Pancreata from the Kras G12D ; Ptf1a CreER ;miR-216a (+/+) mice had increased Sox9 and reduced CpaI expression indicative of ductal metaplasia, with widespread acinar cell loss. Pancreata from Kras G12D ; Ptf1a CreER ;miR-216a (À/À) mice maintained abundant acinar cell content with little ductal-like cells ( Figure 6E). These data indicate that acinar cells from the miR-216a (À/À) mice have less propensity than miR-216a (+/+) mice to form PanINs and acinarto-ductal metaplasia in response to oncogenic KRAS. Finally, to explore whether circulating miR-216a levels are changed in diabetes and pancreatic cancer, we used STZinduced diabetes and LSL-Kras G12D ;Ptf1a-Cre ex1 pancreatic cancer-prone mouse models, respectively. Plasma miR-216a levels were increased by 1 day post-STZ, reaching significant levels 7 days post-injection ( Figure 6F). In the pancreatic cancer mouse model, we detected a significant increase in plasma miR-216a levels as the mice developed tumors, reaching significance at 36 weeks of age ( Figure 6G). To investigate the relevance in humans, we measured miR-216a levels from the sera of patients with type 2 diabetes (T2D), chronic pancreatitis (CP), PDAC, or PDAC with metastasis (mPDAC). Age and gender information of the donors is provided in Table S6. miR-216a levels were not elevated in T2D patients ( Figure 6H). Yet there was a trend toward an increase in miR-216a levels in all exocrine pancreas-related diseases, with a significant increase in sera from patients with PDAC with metastasis. These data suggest a clinical opportunity to use circulating miR-216a as a biomarker for exocrine-related pancreatic diseases.

DISCUSSION
We predicted that pancreas-enriched miRNAs likely have specific, critical roles for pancreas function and hence sought to identify miRNAs enriched in pancreatic cells. Using human and mouse tissue panels, fetal and adult human pancreas and human endocrine cell transplant models, we demonstrated that miR-216a is specific to pancreatic tissue. It was highly expressed in the islets and acinar cells, with little expression in the ducts. Therefore, a whole-body miR-216a KO mouse was used to investigate the role of miR-216a in both endocrine and exocrine compartments in vivo. Islets isolated from the miR-216a KO mice were smaller and secreted less insulin than control islets. Similarly, analysis of pancreatic tissue confirmed the presence of smaller islets and reduced b-cell mass in the miR-216a KO adult mice. Interestingly, pancreata from the miR-216a KO newborn mice had a trend toward increased islet size. Islet growth is size dependent during development, with preferential expansion of smaller islets and fission of large interconnected islet-like structures occurring most actively at approximately 3 weeks of age at the time of weaning. 20 The ROCK-nmMyoII pathway was shown to coordinate epithelial cell morphogenesis by narrowing the apical surface of the cell, basalward cell movement and cellrear detachment from the apical lumen, enabling clustering as nascent islets under the basement membrane. 35 More recently, it was shown that as endocrine progenitors differentiate, they migrate in cohesion and form bud-like islet precursors. 36 As we observed a major difference in islet size in the miR-216a KO mice postnatally, our data support a postnatal role for miR-216a in islet cell expansion and migration. Indeed, we showed miR-216a increases cell migration and proliferation in vitro. Islets from miR-216a KO mice were also closer to the ducts compared with WT islets. Further studies are required to examine the spatiotemporal expression of miR-216a within the epithelial plexus.
Levels of miR-216a were regulated by TGF-b signaling and deletion of miR-216a led to an increase in Smad7 levels in the miR-216a KO islets. This result is in alignment with published data showing that forced expression of miR-216a increases epithelial-to-mesenchymal transition (EMT) and migration with metastatic ability in epithelial hepatic cellular carcinoma (HCC) cells by targeting Pten and Smad7. 25 Although we did not observe any differences in Pten, Becn1, and Ybx1 mRNA levels, we found an increase in Smad7 levels in islets of miR-216a KO mice. We also observed changes in the expression of genes involved in EMT and migration in MIN6 and PANC-1 cells. Lack of Pten, Becn1, and Ybx1 regulation by miR-216a in our study could be explained by differences in target gene selection under physiological versus overexpression conditions. Alternatively, the selection of studied cell types (pancreatic islets versus renal mesangial cells or endothelial cells) could lead to differences in miRNA targets. miRNAs can mediate various stress responses in diseases. 27 Although miR-216a KO mice had smaller islets and reduced b-cell mass, it was insufficient to cause glucose intolerance even when placed on 60% HFD, suggesting absence of miR-216a did not affect glucose homeostasis in vivo under metabolic stress. In rodents, hyperglycemia is reported to develop when $80% of b-cell mass is lost. 37 The extent of b-cell loss in patients with overt T2D is estimated at $65%. 38 Type 1 diabetes is diagnosed when patients lose the majority of b-cells (>70%). 39 The degree of b-cell mass reduction in the KO mice was not sufficient to significantly impair glucose tolerance. However, miR-216a KO mice had smaller islets, reduced b-cell mass, and diminished insulin secretion. A recent study showed that miR-216a-targeting theranostic nanoparticles could promote proliferation of insulinsecreting cells in vivo. 40 These results are in line with our data and provide additional evidence for a role of miR-216a in regulating b-cell mass. Single-cell RNA sequencing from the islets of HFD fed WT and KO mice revealed no major differences in the proportions of different cell types. However, interestingly, we observed a subpopulation of b-cells with upregulated acinar cell markers in the KO islets, suggesting a dysfunctional b-cell subpopulation may contribute to the islet dysfunction in the KO mice. Analysis of RNA-seq results from the pancreata indicated that cell cycle pathways were the most significantly enriched GO term in the miR-216a KO mice. Similarly, DNA replication, nuclear division, and cell proliferation were among the most altered biological processes. As miRNAs affect the expression of multiple genes, identifying signaling pathways targeted by miRNAs rather than minor changes in individual genes is more meaningful to interpret the role of miRNAs in the cellular and physiological context. By opting to isolate pancreatic RNA from 1-day-old pups, we focused on the earliest biological changes that occurred in the miR-216a KO mice postnatally, thereby revealing the potential causal mechanisms for the observed phenotype. On the basis of the expression of miR-216a in the acinar tissue as well as on the altered pathways in miR-216a KO pancreata, we hypothesized miR-216a may have a functional role in pancreatic cancer. Our data suggest that deletion of miR-216 in the presence of oncogenic signals reduces incidence of neoplastic lesions in pancreatic cancer. In contrast to our findings, ectopic miR-216 expression in PANC-1 cells inhibited tumor growth in mouse xenografts. 41,42 However, these studies are considerably different from ours. We induced pancreatic cancer in the pancreatic acinar cells in vivo by crossing the pancreatic cancer-prone mouse line Kras G12D with Ptf1 CreER mice. Thus, in our studies, miR-216a was fully absent, including during embryonic development. In addition, pancreatic cancer was induced in our studies in the acinar pancreatic tissue, whereas in the aforementioned studies, PANC-1 cells were implanted subcutaneously, such that the findings may not be directly comparable with ours. Engineered exosomes carrying short hairpin RNA specific to oncogenic Kras G12D targeted oncogenic KRAS with enhanced efficacy and suppressed cancer in multiple mouse models of pancreatic cancer. 43 It remains to be investigated whether inhibition of miR-216a either alone or in combination with KRAS can lead to a greater attenuation of tumor growth and an increase in overall survival in PDAC.
Cancer is characterized by uncontrolled proliferation resulting from aberrant activity of cell cycle proteins, so many cell cycle regulators are important targets in cancer therapy. 44 A hallmark genetic event in PDAC is loss of the CDKN2A/2B tumor suppressor locus, which encodes Cdk4/6 inhibitors that are particularly important for KRAS driven tumors, such as PDAC. [45][46][47] Our RNA-seq analysis identified downregulation of many cell cycle and proliferation genes, such as Cdk1, Cdk4, and Pcna, in the miR-216a KO mice, and we found reduced PanIN formation in the presence of oncogenic KRAS, both of which suggest partial protection from pancreatic cancer initiation. TGF-b signaling pathway is another important and commonly deregulated signaling pathway in pancreatic carcinomas. 48 Activation of TGF-b leads to phosphorylation of Smad complexes to translocate to the nucleus and activate their target genes involved in cell cycle progression. 48 We showed that miR-216a levels are regulated by TGF-b signaling and miR-216a deletion increases levels of Smad7, which is a known inhibitor of cell cycle progression. Our results support a model whereby TGF-b-induced miR-216 directly reduces Smad7 levels and increases the expression of genes involved in cell cycle progression, which in the presence of oncogenic mutations such as KRAS give rise to PanIN lesions. Whether there are other direct miR-216a targets mitigating this effect needs further evaluation. RNA-interference-based therapies are becoming more attractive because of their stability and sequence specificity and the relative ease of synthesis. 49 miR-375 was the first reported islet-''specific'' miRNA with a role in maintaining normal aand b-cell mass. 50,51 Although we also observed an enrichment for miR-375 in human and mouse islets, it is also expressed at high levels in other tissues, and non-pancreatic roles for this miRNA have been identified, such as in the regulation of gut mucosal immunity and gastric tumorigenesis. 52,53 Specificity of miR-216a for the pancreas suggests clinical opportunities to exploit this miRNA as a biomarker in pancreatic pathologies. Indeed, in humans, we show a significant increase in serum miR-216a levels in patients with metastatic PDAC. Previously, Goodwin et al. 54 showed that circulating miR-216 levels are increased in a mouse model of acute pancreatitis. Given the pancreas specificity of miR-216a, it is not surprising for miR-216a to be elevated in pancreatic cell damage mouse models. Our human data also point to a trend toward elevated miR-216a levels in CP. Given the complexity of accessing pancreatic tissue for biopsy, a blood-based biomarker that can diagnose pancreatic pathologies would be of tremendous value for clinical use. At the very least, circulating miR-216a could be useful as a biomarker for the noninvasive monitoring of PDAC progression in mice.
In summary, our results reveal a pancreas-specific miRNA (miR-216a) with physiological roles in both endocrine and exocrine cells and reveal how dysregulation of miR-216a levels affects b-cell mass and pancreatic cancer initiation. Given the specificity of miR-216a for the pancreas and elevated blood levels in pancreatic diseases, our findings also provide biomarker opportunities to evaluate disease status or progression.

Limitations of study
In this study, we investigated the function of miR-216a using whole-body miR-216a KO mice. Although miR-216a is expressed mainly in the pancreas, we cannot exclude the possibility that some of the effects we observed resulted from miR-216a deletion in other organs. The role of miR-216a in pancreatic cancer was studied in a mouse model in which a common Kras G12D mutation seen in pancreatic cancer was induced in acinar cells. Yet pancreatic cancer can originate in acinar or ductal cells, and pancreatic tumors accumulate other mutations such as TP53, CDKN2A, and SMAD4. It remains to be investigated how miR-216a affects tumor progression in tumors with ductal origin and in the presence of multiple driver mutations. We observed an increase in serum miR-216a levels in pancreatic pathologies, especially in pancreatic cancer. Further studies are required to evaluate the utility of miR-216a as a serum biomarker of pancreatic pathologies in larger cohorts and whether it can distinguish among different forms of pancreatic cancer and/or pancreatitis.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:
To differentiate CA1S human ES cells into pancreatic endocrine cells, we used our previously published 33-day, 7-stage differentiation protocol. 16,55 Briefly, CA1S cells were cultured in mTesR1 media (Stem Cell Technologies, Vancouver BC, Canada) on 1:30 Matrigel coated surfaces (Stem Cell Technologies). Upon $90%-95% confluency, hESCs were directed in MCDB 131 media through 7 stages of pancreatic development in the presence of growth factors and small molecules as described in detail before. 16

Mouse models
The miR-216a KO ES cell line was generated by the Wellcome Trust Sanger Institute 19  Isolation of pancreatic acinar cells and 3D-culture Primary pancreatic acinar cells were prepared as previously described. 66 In brief, the pancreas was removed, washed three times with ice-cold Hank's balanced salt solution (HBSS), minced to $1 mm and digested with collagenase I at 37 C with gentle shaking. The digested pancreatic pieces were washed twice with HBSS containing 5% FBS and then pipetted through 100 mm filters. The supernatant of the cell suspension containing acinar cells was added dropwise to HBSS containing 30% FBS. Acinar cells were then centrifuged and re-suspended in Waymouth media supplemented with 1% FBS, 0.1 mg ml À1 trypsin inhibitor, 1 mg ml À1 dexamethasone. For the 3D acinar culture, cell culture plates were coated with collagen I in Waymouth media. Isolated primary pancreatic acinar cells were added as a 1:1 mixture with collagen I/Waymouth media on the top of this layer. The cells were cultured in the presence of TGF-a (50 ng/mL). Further, Waymouth complete media with 10% FBS, 100 units/mL penicillin and 100 mg/mL streptomycin, 0.1 mg ml À1 trypsin inhibitor, 1 mg ml À1 dexamethasone was added on top of the cell/gel mixture, replaced on days 1 and 3. At days 1 and 5 formation of ducts were visualized under a microscope.
Cloning miRNA-GFP expression vectors were generated by initially creating a EGFP expression vector, pScamp EF1a-EGFP, which contains the 5 0 end of the human EF1A gene (a genomic PCR amplicon containing 1407 bp of proximal promoter, exon 1, intron 1, and the UTR of exon 2), followed by the EGFP ORF and SV40 poly(A) signal, all cloned into pBluescript. This was subsequently modified into a ''miRtron'' expression vector, pScamp EF1a-premir-216a-EGFP, by removing 450 bp of the middle EF1A intron with restriction endonucleases BfuAI and XhoI, and replacing it with a compatible BsmBI-SalI restriction fragment of a 575-bp amplicon* consisting of the full hsa-premir-216a sequence, flanked on either side by roughly 200 bp of endogenous genomic sequences. Cells transfected with this plasmid produce a chimeric EF1A-EGFP mRNA that gets translated into a fluorescent protein, while the excised intron is processed into mature hsa-miR-216a.* miRtron hsa-premir-216a cloning primers (BsmBI and XhoI 5 0 overhangs are underlined). Forward: 5 0 gggcacacaCGTCTCGACGCAGATTATACTTTTATGACATTACATGCAATATAGC 3 0 ; Reverse: 5 0 cacacaGTCGACCCAAGT AGCACTGAAGGAGCG 3 0 .

Pancreas immunohistochemistry and histology
Pancreata were fixed overnight in 4% paraformaldehyde (PFA) and then stored in 70% EtOH prior to paraffin embedding and sectioning (5 mm thickness; Wax-it Histology Services; Vancouver, BC). Immunofluorescent staining was performed as previously described. 67 Briefly, slides were deparaffinized in xylene and hydrated by graded ethanol washes. Slides were then washed in phosphate-buffered saline (PBS) followed by 10-15 min incubation in 10 mM sodium citrate/0.05% Tween 20, pH 6.0 at 95 C. Slides were incubated with DAKO Protein Block Serum-Free (Agilent Technologies, Inc, Santa Clara, CA) at room temperature for 10 minutes and then with the primary antibodies diluted in Dako Antibody Diluent (Agilent Technologies, Inc.) overnight at 4 C. The following primary antibodies were used: insulin (Cell Signaling, # 3014 at 1:200), glucagon (Sigma Aldrich, #G2654 at 1:1000), synaptophysin (Monosan, #MON9013 at 1:10), and PCNA (BD Transduction, #610665 at 1:100). The next day slides were serially washed with PBS and were incubated in appropriate Alexa Fluor 488 or 594 secondary antibodies (Thermo Fisher Scientific, Waltham, MA) diluted in Dako Antibody Diluent. Following serial PBS washes, slides were coverslipped with VECTASHIELD HardSet Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). Images were captured using a ImageXpress Micro TM Imaging System and analyzed using