Abstract
Background
Our recent studies have demonstrated the crucial involvement of FOXA2 in the development of human pancreas. Reduction of FOXA2 expression during the differentiation of induced pluripotent stem cells (iPSCs) into pancreatic islets has been found to reduce α-and β-cell masses. However, the extent to which such changes are linked to alterations in the expression profile of long non-coding RNAs (lncRNAs) remains unraveled.
Methods
Here, we employed our recently established FOXA2-deficient iPSCs (FOXA2−/− iPSCs) to investigate changes in lncRNA profiles and their correlation with dysregulated mRNAs during the pancreatic progenitor (PP) and pancreatic islet stages. Furthermore, we constructed co-expression networks linking significantly downregulated lncRNAs with differentially expressed pancreatic mRNAs.
Results
Our results showed that 442 lncRNAs were downregulated, and 114 lncRNAs were upregulated in PPs lacking FOXA2 compared to controls. Similarly, 177 lncRNAs were downregulated, and 59 lncRNAs were upregulated in islet cells lacking FOXA2 compared to controls. At both stages, we observed a strong correlation between lncRNAs and several crucial pancreatic genes and TFs during pancreatic differentiation. Correlation analysis revealed 12 DE-lncRNAs that strongly correlated with key downregulated pancreatic genes in both PPs and islet cell stages. Selected DE-lncRNAs were validated using RT-qPCR.
Conclusions
Our data indicate that the observed defects in pancreatic islet development due to the FOXA2 loss is associated with significant alterations in the expression profile of lncRNAs. Therefore, our findings provide novel insights into the role of lncRNA and mRNA networks in regulating pancreatic islet development, which warrants further investigations.
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Introduction
FOXA2 has distinct functions in various tissues. In pancreatic development, FOXA2 is expressed early on, starting from the endoderm stage, with its protein level increasing during endocrine specification [1, 2]. On the other hand, exocrine and ductal cell express FOXA2 at low levels [1]. We have recently reported that FOXA2 plays critical roles in human pancreatic and hepatic development, using human induced pluripotent stem cells (iPSCs) [3, 4]. FOXA2 is known to regulate the expression of multiple transcription factors (TFs) that control pancreatic endocrine cell fate and insulin secretion [5, 6]. Recent genomic studies have shown that FOXA2-bound enhancers in humans are associated with type 2 diabetes (T2D) risk alleles [7]. Additionally, a recent study reported a patient with diabetes caused by a heterozygous missense variant in FOXA2 [8]. These findings highlight the potential contribution of FOXA2 defects to the development of diabetes and its crucial role in pancreatic endocrine differentiation.
Earlier studies have revealed that noncoding RNAs (ncRNAs) have regulatory roles both at the transcriptional and posttranscriptional levels [9, 10]. The long ncRNAs (lncRNAs) and epigenetic modifications are a crucial part of the transcriptional mechanisms that control cell specification and development [11]. LncRNAs play a significant role in regulating the expression of target genes, making them essential epigenetic modulators in various cell types [12]. These lncRNAs are distributed in the genome in a way that allows them to co-regulate the targeted protein-coding regions they regulate [13,14,15]. The cell-type specific expression pattern of lncRNAs suggests their potential role as mediators of lineage specification [16, 17]. Understanding the regulatory role of lncRNAs during pancreatic development is crucial in decoding the regulatory network controlling pancreatic islet development [18, 19]. Several specific lncRNAs in pancreatic islets have been mapped close to TFs that regulate pancreatic embryogenesis and β-cell development. Over 1000 lncRNAs have been identified in human pancreatic islets through integrative epigenetic analysis, indicating their importance in the endocrine and β-cell differentiation program during pancreatic development [20]. Various TFs, such as PDX1, NKX6.1, NKX2.2, PAX6, and GATA6, that regulate pancreatic islet development have been identified to be regulated by lncRNAs such as PLUTO, MALAT1, ROIT, Gm10451, HI-LNC15, Paupar, PAX6-AS, and GATA6-AS [14, 21,22,23,24,25,26,27]. Given the lack of functional information about lncRNAs, stage-specific analyses throughout development are essential [11], especially considering the identification of lncRNAs specific to α-and β-cells in human islets [28]. Differential expression of lncRNAs has been reported in mouse islets with both type 1 diabetes (T1D) and type 2 diabetes (T2D) [29, 30], as well as in the islets of patients with T2D [20, 22]. These findings support the potential for lncRNAs to play a vital role in the development and functionality of pancreatic β-cells, and suggest that they may serve as biomarkers for early diagnosis, as has been reported in blood samples from T1D patients [31].
Previous studies have shown that FOXA2 expression is regulated by various lncRNAs, including lncRNA-NEF [32, 33], lncRNA-FTX [34], lncRNA-HOTAIR [35], and lncRNA-Falcor [36], with their overexpression leading to the activation of FOXA2 expression and their inhibition resulting in reduced FOXA2 expression. A previous study demonstrated that during human endoderm differentiation, FOXA2 is activated by lncRNA DEANR1 (LINC00261) [37], which has been shown to be essential for generation of insulin + cells from hESCs [38]. Interestingly, a regulatory feedback loop has been identified between FOXA2 and its associated lncRNAs, with studies in lung tissue indicating that FOXA2 can either activate or repress its regulatory lncRNAs. FOXA2 binds to the promotor of lncRNA-Falcor, leading to its repression [36], while it activates the expression of lncRNA-NEF, a direct downstream target of FOXA2 [33]. The function of lncRNAs during pancreatic lineage specification is not fully understood. Our recent studies revealed that the expression of several genes involved in the development and function of pancreatic islet cells is dysregulated by FOXA2 deficiency [3, 39]. Therefore, in the current study, we used established FOXA2−/ −iPSC lines to investigate the effect of FOXA2 loss on the lncRNA profiles in the pancreatic progenitors (PPs) and pancreatic islets derived from hiPSCs.
Materials and methods
Differentiation of iPSCs into pancreatic progenitors and pancreatic islets
Two different FOXA2 knockout iPSC lines (FOXA2−/−iPSCs) recently established in our lab were used in this study [3]. Both FOXA2−/−iPSC lines and their isogenic controls (WT-iPSCs), were differentiated into PPs and pancreatic islets using our modified stepwise differentiation protocol [3] as illustrated in Supplementary Fig. 1A.
RNA extraction and qPCR analysis
RNeasy Plus Mini Kit (QIAGEN) used for total RNA extraction following the manufacturer's instructions. The RNA was reversely transcribed using superscript IV, First-Strand Synthesis System (Thermo Fisher Scientific). The quantity and integrity of RNA quantity were assessed using Agilent Bioanalyzer 2100 (Agilent Technologies). The sequences of selected lncRNA primers listed in Supplementary Table 1. The amplification was detected using Quant Studio 7 system (Applied Biosystems) using GoTaq qPCR Master Mix (Promega) and GAPDH as an internal control. Fold change 2-ΔΔ Ct used to present the expression level of performed lncRNAs.
Total RNA library preparation and sequencing
RNA was extracted using Direct-zol RNA extraction kit (Zymo Research) from two biological replicates for each sample of cells at PPs and pancreatic islets stages of differentiation. mRNA was captured from 1 μg of total RNA using NEBNext (Poly A) mRNA magnetic isolation kit (NEB, E7490) according to the manufacturer’s instructions. NEBNext ultra directional RNA library prep kit (NEB, E7420L) used to NEBNext ultra directional RNA library prep kit (NEB, E7420L) used to prepare RNA-seq libraries which is sequenced on an Illumina Hiseq 4000 system. The initial processing of the raw data involved basic trimming and quality control, which was carried out using Illumina BCL2Fastq Conversion Software v2.20.
Total RNA‑Seq data and bioinformatics analysis
Pair-end FASTQ files were subsequently aligned to the GRCh38 reference genome using built-in module and default settings in CLC genomics workbench v21.0.5. Normalized expression data (TPM, transcript per million) were then subjected to differential expression analysis using twofold change (Log2 FC 1) and < 0.05 p-value cut-off. Transcripts with raw expression values < 1.0 TPM were excluded from the analysis. Differential expression analysis and hierarchical clustering were conducted using AltAnalyze v.2.1.3 as described before [40, 41].
Correlation analysis between lncRNA and differentially expressed genes (DEGs)
To construct networks between differentially expressed genes (DEGs) and corresponding DE-lncRNAs in the context of FOXA2 loss, we first identified the list of DEGs essential for PP development, and the corresponding DE-lncRNAs using transcriptome analysis of iPSC-derived PPs (stage 4 of differentiation), and iPSC-derived pancreatic islets (stage 7 of differentiation) from FOXA2−/− iPSCs and WT controls. We subsequently assessed the relevance of identified networks to normal pancreatic cell development, by retrieving the mRNA and lncRNA expression data from 305 normal pancreatic tissue samples from the Genotype-Tissue Expression (GTEx) portal (https://gtexportal.org/home/). Correlation between the identified DE-lncRNAs-DEGs based on the in vitro iPSC-derived PPs, and iPSC-derived pancreatic islets were subsequently validated in the GTEx pancreatic dataset. Pearson correlation analysis was performed on the expression values of DE-lncRNA and DEG pairs using IBM SPSS statistics v26. We selected the co-expressed pairs (LncRNA-DEG) with a Pearson correlation coefficient ≥ 0.3 to establish and draw the network using Cytoscape software (National Resource for Network Biology), as described before [42, 43].
Statistical analysis
Statistical analysis was performed using unpaired two-tailed student’s t-test by Prism 8 software, with data represented as mean ± standard deviation (SD).
Results
Generation of pancreatic progenitors and islets from FOXA2 –/– iPSCs and wild-type iPSCs
To evaluate the influence of FOXA2 deficiency on lncRNA expression profiles in PPs and pancreatic islets, we differentiated FOXA2–/– iPSC lines into PPs and pancreatic islets following our previously described protocol [3] (Supplementary Fig. 1A). Lack of FOXA2 resulted in a significant decrease in the expression levels of the crucial pancreatic progenitor markers, PDX1 and NKX6.1 (Supplementary Fig. 1B), as recently reported in our published article [3]. The absence of FOXA2 resulted in a substantial decrease in the expression levels of endocrine progenitor markers, including NGN3 and NKX2.2 (Supplementary Fig. 1C). In addition, there was an almost complete loss of insulin (INS) and glucagon (GCG) expression indicating a reduction in the masses of β-cells and α-cells (Supplementary Fig. 1D). The complete loss of FOXA2 protein was confirmed through western blotting, as we previously reported [3, 39]. These findings validate our previously published results regarding the impact of FOXA2 absence on islet development [3, 39].
Characterization of lncRNA profiles in FOXA2 knockout iPSC-derived pancreatic progenitors
To assess the impact of FOXA2 loss on the expression profile of lncRNAs in PPs, we conducted RNA-Seq analysis on FOXA2–/–PPs and WT-PPs. We identified a total of 826 DE-lncRNAs in FOXA2–/– PPs compared to WT-PPs, with 442 significantly downregulated (Log2 FC < − 1.0, p < 0.05) and 114 significantly upregulated (Log2 FC > 1.0, p < 0.05) DE-lncRNAs (Fig. 1A and Supplementary Table 2). Figure 1B presents the volcano plot of the DE-lncRNAs in FOXA2–/– PPs versus WT-PPs. The expression of the top 5 upregulated and top 5 downregulated lncRNA transcripts illustrated in Fig. 1C and D, with LINC02864 being the most significantly downregulated and AL009031.1 being the most significantly upregulated lncRNA transcripts in FOXA2–/– PPs compared to WT-PPs.
Differentially expressed lncRNAs in pancreatic progenitors (PPs) derived from FOXA2−/− iPSCs compared to those derived from WT-iPSCs. A Hierarchical clustering of the captured lncRNAs in the pancreatic progenitors (PPs) from FOXA2 knocked out iPSCs (FOXA2−/− iPSCs) and WT-iPSCs. Each column represents one differentiation experiment, and each row represents the lncRNA transcript. The expression level of each transcript (log2) is depicted according to the color scale. B Volcano plot depicting upregulated (red) and downregulated (blue) lncRNAs (p < 0.05, Log2 FC > 1). Graphs represent the expression of the top 5 downregulated (C) and upregulated (D) lncRNA transcripts
Correlations between DE-lncRNAs and DE-mRNAs in iPSC-derived pancreatic progenitors
Next, we sought to explore the potential function of the identified DE-lncRNAs in this study. To accomplish this, we conducted a correlation analysis to establish links between these DE-lncRNAs and selected DEGs (Supplementary Table 3) that are known to be essential for PPs, as reported in our recent study [39]. Our analysis identified significant correlation between numerous DEGs and lncRNAs, based on the FOXA2–/–PPs and WT-PPs in vitro models. To assess the relationship between the identified DEGs and lncRNAs from the in vitro model in normal pancreatic cells, we subsequently obtained mRNA and lncRNA expression data from an online database of 305 pancreatic tissues (https://gtexportal.org/home/). Our analysis revealed significant correlations between the downregulated DE-lncRNAs and key pancreatic genes that were also downregulated, based on the FOXA2–/–PPs and WT-PPs in vitro models. These genes include PDX1, NKX6.1, FOXA2, RFX6, GATA6, GATA4, PTF1A, NEUROD1, NKX2.2, INSM1, FEV, DALL4, CPA2, ONECUT1, MNX1, GLIS3, PROX1, TCF7L2, HES6, NR5A2, PCSK1, HNF4G, CHGA, CHGB, GP2, and GCK (Fig. 2A). DE-lncRNAs and DEGs with Pearson correlation coefficients > 0.3 were selected and the DE-lncRNA-mRNA correlation network analysis in PPs was constructed (Fig. 2A). We excluded 191 downregulated DE-lncRNAs, with weak correlations (less than 0.3) to any of the selected DEGs. Out of the strongly correlated downregulated 195 DE-lncRNAs, 93 DE-lncRNAs were strongly correlated (> 0.3) with FOXA2 and were enlisted in Table 1 with their correlated genes (Fig. 2B). Furthermore, we identified 169, 155, 148, 133, 128, 127, 126, 119, 117, and 110 DE-lncRNAs that strongly correlated with TCF7L2, GLIS3, PROX1, MNX1, PDX1, HNF1B, DALL4, ONECUT1, NKX6.1, and GATA6, respectively (Fig. 2B). PDX1 and NKX6.1 are known as the main TFs that mark the pancreatic precursors of β-cells. In our analysis, we identified 81 DE-lncRNAs that were commonly correlated with FOXA2, PDX1, and NKX6.1 in normal pancreatic cells (Fig. 2C, Supplementary Fig. 2, Supplementary Table 4). The most downregulated DE-lncRNAs of this common list were MEG3, H19, ZNF667-AS1, AC013275.1, LINC00543, LINC00261, MIR7-3HG, AC097639.1, PRDM16-DT, LINC02381, LINC01963, AL662797.2, LINC00511, AP000345.2, GPRC5D-AS1, NRAV, and MNX1-AS1 (Supplementary Fig. 2). We noticed that the top correlated DE-lncRNAs were significantly downregulated and were linked to FOXA2. It is intriguing to note that the results obtained when we integrated the DE-lncRNAs and DEGs of our RNA-Seq analysis were consistent with those obtained from normal pancreatic tissues, as shown in Supplementary Fig. 3.
Co-expression network analysis of downregulated lncRNAs and DEGs in pancreatic progenitors derived from FOXA2−/− iPSCs. The correlation analysis between the downregulated DE-lncRNAs and previously identified DEGs in our previous study of the pancreatic progenitor (PP) stage after its incorporation with the online database of 305 pancreatic tissues (A). The number of correlated lncRNAs with each specific DEG is presented in the graph (B), and (C) shows the commonly correlated lncRNAs with FOXA2, PDX1, and NKX6.1
The network analysis of the upregulated DE-lncRNAs was conducted by examining their correlation with selected upregulated genes identified in FOXA2−/− PPs. These DEGs include APOC2, GCGR, HKDC1, SLC2A3, ABCC2, SLC2A2, APOC1, CEBPA, GCKR, ANXA1, WNT5A, ABCA1, BMP2, SLC16A1, ABCA7, and SLC3A2 (Supplementary Table 3). Incorporating these results with lncRNA and mRNA expression data from 305 pancreatic tissue database revealed that only a limited number of upregulated DE-lncRNAs exhibited a strong correlation with the selected upregulated genes, as depicted in the constructed network (Supplementary Fig. 4 and Table 1). Among the correlated lncRNAs, those demonstrating the highest levels of upregulation included AC104958.2, AC008264.2, DBH-AS1, AC005261.4, AC092535.5, and AC025857.2.
Characterization of lncRNA profiles in FOXA2 knockout iPSC-derived pancreatic islets
In order to investigate the impact of FOXA2 deletion on the expression of lncRNAs in pancreatic islets, we extracted the DE-lncRNAs and DEGs from RNA-seq data. This data was obtained by comparing the pancreatic islets derived from FOXA2–/–iPSCs and WT-iPSCs. Our transcriptomic analysis revealed a total of 1031 significantly downregulated DEGs (Log2 FC < -1.0, p < 0.05), and 734 significantly upregulated DEGs (Log2 FC > 1.0, p < 0.05) in pancreatic islets derived from FOXA2–/– iPSCs compared to the WT controls. The relevant DEGs selected for the correlation study are listed in Supplementary Table 3. Furthermore, our RNA-Seq data revealed 517 DE-lncRNAs in FOXA2–/–pancreatic islets compared to WT-pancreatic islets. Among those DE-lncRNAs, 177 were significantly downregulated (Log2 FC < − 1.0, p < 0.05) and 59 were significantly upregulated (Log2 FC > 1.0, p < 0.05) (Fig. 3A, B and Supplementary Table 5). The expression of the top 5 downregulated and top 5 upregulated DE-lncRNA transcripts in pancreatic islets derived from FOXA2–/– iPSCs compared to the WT controls are shown in Fig. 3C and D. AC129926.1 was the top downregulated and AC087477.4 was the top upregulated DE-lncRNA in FOXA2–/– pancreatic islets compared to WT-pancreatic islets.
Differentially expressed lncRNAs in pancreatic islets derived from FOXA2−/− iPSCs compared to those derived from WT-iPSCs. A Hierarchical clustering of captured lncRNAs in the pancreatic islets derived from FOXA2−/− iPSCs and WT-iPSCs. Each column represents one differentiation experiment, and each row represents the lncRNA transcript. The expression level of each transcript (log2) is depicted according to the color scale. B Volcano plot depicting upregulated (red) and downregulated (blue) lncRNAs (p < 0.05, Log2 FC > 1). Graphs represent the expression of the top 5 downregulated (C) and upregulated (D) lncRNA transcripts
Correlations between DE-lncRNAs and DE-mRNAs in iPSC-derived pancreatic islets
To identify potential interaction, we constructed a co-expression network through correlation analysis between the DE-lncRNAs and key DE-mRNAs in iPSC-derived pancreatic islets. Our transcriptome analysis showed that mRNA expression of several pancreatic genes related to the development and function of pancreatic islet cells was significantly downregulated in iPSC-derived pancreatic islets lacking FOXA2 compared to WT controls (Supplementary Table 3). We selected key downregulated DE-mRNAs that are known to play an essential role in β-cell development and function, including FOXA2, TRPM4, MAPK3, PDX1, HES, ABCG1, DLL4, STX1A, NKX6-1, PLCB4, CAMK2B, ALDH1A3, RFX6, UCN3, CACNA1A, ABCC8, DLL1, PCLO, INSM1, FFAR1, CHGA, SUSD4, KLF4, NEUROD1, ADCY7, CHGB, NKX2-2, ARX, PTF1A, GCG, HES6, SHH, PRKCG, INS, ADCY1, PPY, and IAPP. We subsequently performed correlation analysis on those identified downregulated DE-lncRNAs and downregulated DE-mRNAs in normal pancreatic cells from GTEx data as well as in iPSC-derived pancreatic islets. Interactions with Pearson correlation coefficients > 0.3 were selected and the co-expression network analysis was constructed (Fig. 4A; Supplementary Fig. 5). The analysis revealed 86 downregulated DE-lncRNAs with a strong correlation (> 0.3) to the selected downregulated DE-mRNAs. Out of these DE-lncRNAs, 42 DE-lncRNAs showed a strong correlation with FOXA2, listed with its correlated mRNAs in Table 2. The commonly correlated downregulated DE-lncRNAs with FOXA2 and other DEGs are shown in Fig. 4B and Table 2. The analysis revealed that ZNF667-AS1, AL035661.1, AL390719.2, AC091563.1, AC090510.3, MEG3, LINC00261, U73166.1, AC097639.1, MNX1-AS2, and H19 were the top downregulated DE-lncRNAs with a strong correlation to FOXA2 (Supplementary Fig. 6). There were 65, 64, 50, 50, 50, 48, 46, and 45, DE-lncRNAs correlated with TRPM4, MAPK3, PDX1, HES6, ABCG1, DLL4, STX1A, and NKX6.1 (Fig. 4B). As previously known, FOXA2 commonly regulates PDX1, NKX6.1, NKX2.2 and ABCC8 TFs. We identified 23 lncRNAs that are commonly and strongly correlated to FOXA2, PDX1, NKX6.1 and ABCC8, with the lowest log2 fold expression for ZNF667-AS1, AL390719.2, AC091563.1, AC090510.3, and AC097639.1, as shown in Fig. 4C and Supplementary Fig. 6 and Supplementary Table 6.
Co-expression network analysis of downregulated lncRNAs and DEGs in pancreatic islets derived from FOXA2.−/−iPSCs. The correlation analysis between the downregulated DE-lncRNAs and DEGs in the pancreatic islets (A). The number of lncRNAs correlated with each specific DEG is presented in graph (B)
Similarly, we conducted a correlation analysis to establish connections between the upregulated DE-lncRNAs to the selected upregulated DEGs identified in FOXA2−/− pancreatic islets. These DEGs included ATP1B1, ATP1B2, FXYD2, CPA4, PLA2G2A, KCNQ1, SST, HHEX, ABCC2, BMP4, WNT6, and MYC (Supplementary Table 3). Their incorporation with the database of 305 pancreatic tissue profiles resulted in identification of 12 upregulated DE-lncRNAs with Pearson correlation coefficients > 0.3, as enlisted in Table 2. Among them, the highly upregulated lncRNAs were B3GALT5-AS1, AC087477.2, AC027031.2, DNM3OS, and AC124067.4 (Supplementary Fig. 7 and Table 2). Using the identical analysis approach on our RNA-Seq data, we observed that the upregulated DE-mRNAs exhibited associations with the same DE-lncRNAs identified in pancreatic tissues (Supplementary Fig. 8).
Identifications of commonly DE-lncRNAs in pancreatic progenitors and islets derived from FOXA2 −/− iPSCs
Next, we aimed to identify commonly dysregulated lncRNAs in both PPs and pancreatic islets. We identified 35 lncRNAs that were downregulated in PPs and pancreatic islets lacking FOXA2 compared to WT controls (Fig. 5A, B; Table 3). Of those downregulated DE-lncRNAs, only 12 lncRNAs had a strong Pearson correlation > 0.3 with FOXA2 including MEG3, H19, ZNF667-AS1, LINC00543, LINC00261, AC097639.1, AL035661.1, SLC25A25-AS1, U73166.1, ZNF790-AS1, MNX1-AS2, and AC091563.1 (Fig. 5C). Furthermore, we showed the global correlation between these 12 lncRNAs and the specific mRNAs in the PPs and pancreatic islets in normal pancreatic cells from GTEx data (Fig. 5D, E). Independent networks for each lncRNA were constructed to highlight its linked DEGs in the PPs and pancreatic islets (Fig. 6). Multiple DEGs were commonly correlated to a specific lncRNA in both stages (Fig. 6). Furthermore, we identified 8 lncRNAs that were upregulated in PPs and pancreatic islets, including AC108865.2, PANCR, AP001528.1, LINC02732, LINC01357, AC111000.4, AP000547.3, and AC140479.5 (Table 3). The correlation analysis revealed that only AC111000.4 had Pearson correlation coefficients > 0.3, especially with ABCC2 TF.
Identification of commonly downregulated lncRNAs in pancreatic progenitors and islets. A A Venn diagram showing the number of lncRNAs that are downregulated in pancreatic progenitor (PPs) and pancreatic islets stages. B Heatmaps showing the expression of the commonly downregulated lncRNAs in PPs and pancreatic islets stages. C The commonly downregulated lncRNAs in both stages showed a strong correlation with FOXA2. Constructed networks to explore the global correlation of these FOXA2-strongly correlated lncRNAs with other DEGs in PPs (D) and pancreatic islet stage (E)
Individual networks for each commonly downregulated lncRNA exhibited strong correlation to FOXA2. The networks were constructed for MEG3, H19, ZNF667-AS1, LINC00543, LINC00261, AC097639.1, AL035661.1, SLC25A25-AS1, U73166.1, ZNF790-AS1, MNX1-AS2, and AC091563.1. The curved lines represent the transcription factors, which are commonly correlated to the lncRNA in both stages of PPs and pancreatic islets
Validation of the dysregulated lncRNAs in iPSC-derived pancreatic progenitors and islets
To validate the DE-lncRNAs, we performed RT-qPCR on pancreatic progenitors and pancreatic islets. RT-qPCR analysis showed that the expression of lncRNA transcripts, including MEG3, H19, LINC00261, MIR7-3HG, and KCNQ1OT1 was significantly downregulated in PPs derived from FOXA2−/−iPSCs compared to WT controls (Fig. 7A). Furthermore, at the islet stage, the expression of H19, LINC00261, MIR7-3HG, and KCNQ1OT1 was significantly downregulated in pancreatic islets lacking FOXA2 compared to WT controls (Fig. 7B).
RT-qPCR validation for DE-lncRNAs crucial for pancreatic development and function. A RT-qPCR analysis for validation of selected DE-lncRNAs in iPSC-derived pancreatic progenitors (PPs), including MEG3, H19, LINC00261, MIR7-3HG, and KCNQ1OT1 for pancreatic progenitors. B RT-qPCR analysis for validation of selected DE-lncRNAs in iPSC-derived pancreatic islet stage, including H19, LINC00261, MIR7-3HG, and KCNQ1OT1. Data are represented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001
Discussion
Differentiation of hPSCs into pancreatic islets is controlled by the expression of key genes and TFs that are specific for each stage during pancreatic development (as reviewed in [2]. Our recent study demonstrated that the absence of FOXA2 in iPSCs results in impaired differentiation into pancreatic islets, as evidenced by a notable decrease in the expression of pancreatic developmental genes [3]. Furthermore, we found that those downregulated genes are targets for several upregulated miRNAs in PPs lacking FOXA2 [39]. In this study, we employed the same iPSC model to examine the effect of FOXA2 depletion on the lncRNA profile at pancreatic progenitor and pancreatic islet stages. Our findings revealed that the alterations in the mRNA profiles linked to FOXA2 were accompanied with significant alterations in the expression of lncRNAs at both stages.
By analyzing RNA-seq results from PPs and pancreatic islets derived from WT-iPSCs and FOXA2−/−iPSCs, we observed a decrease in the expression of critical pancreatic genes involved in the development and function of pancreatic islets, such as PDX1, NKX6.1, NEUROG3, NEUROD1, NKX2.2, INS, GCG, and others [39]. We conducted a network analysis combining these downregulated pancreatic genes with DE-lncRNAs. This analysis revealed that 195 and 86 DE-lncRNAs in the PPs and pancreatic islets, respectively, were strongly correlated with genes known to regulate the development and function of pancreatic islets. Moreover, we identified 12 DE-lncRNAs that exhibited decreased expression in both PPs and pancreatic islets derived from FOXA2−/−iPSCs. These DE-lncRNAs include MEG3, H19, ZNF667-AS1, LINC00543, LINC00261, AC097639.1, AL035661.1, SLC25A25-AS1, U73166.1, ZNF790-AS1, MNX1-AS2, and AC091563.1. Some of those lncRNAs play important role in regulating the development and/or function of pancreatic islets. For example, MEG3 plays a crucial role in controlling pancreatic β-cell mass and regulating the expression of Pdx1, Mafa, and Ins2, which are vital for pancreas development and insulin secretion [44, 45]. MEG3 binding has been observed in the promotor region of FOXA2, and its depletion has been linked to reduced FOXA2 expression [46]. MEG3 enhancer has been found to bind by FOXA2, PDX1, and NKX2.2 in human β-cells [47]. Moreover, the maternally expressed H19 lncRNA has been shown to promote β-cell development by its close association with the insulin-like growth factor 2 (Igf2) locus [48, 49]. Intriguingly, our data confirmed this strong correlation, as IGF2-AS was significantly downregulated, particularly in the PPs stage. FOXA2 has been found to interact with the H19 E1 enhancer, regulating its expression, particularly in the early developmental stage of fetal liver [50]. Additionally, we found that LINC00261 was significantly downregulated and displayed a robust correlation with several pancreatic genes, such as FOXA2, PDX1, NKX6.1, TCF7L2, MNX1, GLIS3, CACNA1A, and PLCB4. It has been previously reported in several studies that LINC00261 positively regulates FOXA2 [36, 37, 51], and the loss of LINC00261 during pancreatic endocrine differentiation has been found to reduce the percentage of the generated insulin-secreting cells [38]. Moreover, we noticed a significant correlation between FOXA2 and SLC25A25-AS1 lncRNA. Downregulation of SLC25A25-AS1 has been linked to epithelial mesenchymal transition (EMT), resulting in the acquisition of mesenchymal characteristics [52], suggesting a possible epigenetic modulation of EMT-related lncRNAs by FOXA2. Another lncRNA that showed a strong correlation with FOXA2 and was downregulated in our study is SLC25A3, which its suppression has been reported to contribute to diabetes development by reducing ATP levels [53]. Furthermore, we identified MNX1-AS2, ZNF667-AS1 (MORT) and ZNF790-AS1, which were strongly correlated with FOXA2. MNX1 and ZNF are essential for pancreatic development [2, 54]. We also found that several downregulated lncRNAs, such as LINC00543, AC097639.1, AL035661.1, U73166.1, and AC091563.1, have not been investigated in the context of pancreatic development and their correlation with FOXA2 should be explored in future studies. Taken together, these findings suggest that lncRNAs may play a role in regulating the expression of key genes involved in pancreatic development, including FOXA2 and vice versa.
Several studies have reported that changes in the expression of lncRNAs are linked to pancreatic islet development and various types of diabetes. lncRNAs have been identified to play a role in the development of diabetes and may be potential biomarkers for the early detection of diabetes [55,56,57]. LINC00261 and MIR7-3HG have been shown to be highly expressed lncRNAs in hESC-derived PPs in recent profiling studies [38]. Our study further supports the correlation between FOXA2 and these lncRNAs by demonstrating that their expression levels were decreased upon FOXA2 deletion, indicating their significance in pancreatic progenitor and β-cell development. Moreover, our findings demonstrated that several lncRNAs, downregulated upon FOXA2 deletion are known to be linked to diabetes. For instance, MEG3 expression is strongly associated with diabetes, and its expression is downregulated in pancreatic islets of type 1 (T1D) and type 2 diabetes (T2D) in mouse and human [44, 58, 59]. Decreased expression of lncRNA H19 has also been noticed in diabetes and linked to its clinicopathological abnormalities [60, 61]. Downregulation of KCNQ1OT1 is associated with an increased risk of T2D [62, 63]. GAS5 lncRNA is decreased in the serum of patients with diabetes and individuals with low GAS5 levels are more susceptible to develop diabetes [64], as its reduction decreases the insulin secretion and increases the β-cell dysfunctionality [65]. Downregulation of lncRNA TUG1 expression alters insulin secretion and induces apoptosis in pancreatic β-cells [66]. MALAT1 downregulates the PDX1 expression and impairs the β-cell function [23], and the upregulation of MALAT1 has been reported in the blood of T2D patients [67, 68]. Linc13 is associated with T1D through its contribution in the inflammatory pathway activation and β-cell destruction [69]. Increased expression of lncRNA PVT1 activates the oxidative stress and β-cell apoptosis; however, its silencing enhances the insulin secretory capacity [70], which classify the PVT1 as a diabetes-associated lncRNA. A previous report has indicated that a heterozygous missense variant in FOXA2 can cause monogenic diabetes [8]. Also, the risk alleles for T2D have been found to be associated with the FOXA2 binding enhancer [7]. When take in conjunction with our recent findings demonstrating that deficiency of FOXA2 impairs pancreatic islet development and may lead to diabetes, these observations suggest the involvement of the alterations in lncRNA expression reported in this study may play a role in this effect.
To summarize, this study demonstrates that the deletion of FOXA2 disrupts the profiles of mRNAs and lncRNAs during pancreatic progenitor and β-cell stages of pancreatic development. Furthermore, this study revealed a strong association between lncRNAs and several critical pancreatic genes and TFs during pancreatic differentiation. The presented data suggest that the observed impairment in pancreatic islet development in the absence of FOXA2 is linked to significant changes in the expression profile of lncRNAs. Together with prior research, these findings suggest that dysregulated lncRNAs may play an essential role in the development and function of pancreatic islet cells and may contribute to the development of diabetes. Whether FOXA2 directly regulates the expression of the identified lncRNAs, or loss of FOXA2 and subsequent impairment of pancreatic development leads to deregulated lncRNA expression remains to be investigated. These findings provide a basis for further functional investigation into the newly identified lncRNAs to gain a better understanding of their role during pancreatic development.
Availability of data and materials
The data that support the findings of this study are available from the corresponding author upon reasonable request.
References
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Acknowledgements
We thank the Genomic Core members at QBRI for their assistance for technical support in RNA and miRNA sequencing.
Funding
This work was funded by grants from Qatar Biomedical Research Institute (QBRI) (Grant No. IGP3). Open Access funding provided by the Qatar National Library (QNL).
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A.K.E. performed the experiments. A.K.E. and E.M.A. analyzed the data and wrote the manuscript. N.M.A. analyzed the RNA-Seq data, performed correlation analysis, and revised the manuscript. E.M.A. conceived and designed this research, revised the manuscript, and obtained research funding. All authors reviewed and approved the final version of the manuscript.
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Additional file 1: Supplementary Fig. 1.
Differentiation of FOXA2 knockout iPSCs into pancreatic islets. (A) A diagram showing the differentiation protocol used in this study. FOXA2-/- iPSCs and WT- 2 iPSCs were differentiated into pancreatic progenitors (PPs), endocrine progenitors (EPs), and pancreatic islet cells. The initial four stages of differentiation were generated using our protocol, while the subsequent stages of islet differentiation followed Rezania protocol. (B) In comparison to the WT control, the differentiation of FOXA2-/- iPSCs demonstrated a significant reduction in the expression of PP markers, including PDX1 and NKX6.1. (C) The Absence of FOXA2 resulted in a notable decrease in the expression of endocrine progenitor markers, NGN3 and NKX2.2. (D) Loss of FOXA2 led to a substantial reduction in the expression of insulin (INS) and glucagon (GCG), indicating a decline in both beta and alpha cell mass within the derived pancreatic islets. Supplementary Fig. 2. Heatmaps for the correlated downregulated lncRNAs in pancreatic progenitors. Separate heatmaps are displayed for the correlation of lncRNAs with FOXA2, PDX1, and NKX6.1, as well as a heatmap for lncRNAs that are commonly correlated with FOXA2, PDX1, and NKX6.1. Supplementary Fig. 3. Co-expression network analysis of downregulated lncRNAs and DEGs in pancreatic progenitors derived from FOXA2-/- iPSCs. A network showing the correlation analysis between the downregulated DE-lncRNAs and DEGs in the iPSC-derived pancreatic progenitors (PPs) lacking FOXA2. Supplementary Fig. 4. Strongly upregulated lncRNAs with a Pearson correlation coefficient (PC) > 0.3 in the pancreatic progenitors derived from iPSCs lacking FOXA2. The upregulated lncRNAs are presented in a heatmap (A), and their correlation with DEGs is depicted in a network using expression data from an online database of 305 pancreatic tissues (B) and data generated in this study from iPSC-derived pancreatic islets (C). Supplementary Fig. 5. Co-expression network analysis of downregulated lncRNAs and DE-mRNAsin pancreatic islets derived from FOXA2-/- iPSCs. The correlation analysis between the downregulated DE-lncRNAs and DE-mRNAs in iPSC-derived pancreatic islets lacking FOXA2. Supplementary Fig. 6. Heatmaps for the downregulated lncRNAs that are correlated in the pancreatic islets. Separate heatmaps are displayed for the correlation of lncRNAs with FOXA2, PDX1, NKX6.1, and ABCC8, as well as a heatmap for lncRNAs that are commonly correlated with FOXA2, PDX1, NKX6.1 and ABCC8. Supplementary Fig. 7. Strongly upregulated lncRNAs with a Pearson correlation coefficient (PC) > 0.3 in the pancreatic islets derived from iPSCs lacking FOXA2. The upregulated lncRNAs are presented in a heatmap (A), and their correlation with DEGs is depicted in a network (B). Supplementary Fig. 8. Co-expression network analysis of upregulated lncRNAs and DEmRNAs in pancreatic islets derived from FOXA2-/- iPSCs. The correlation analysis between the upregulated DE-lncRNAs and DE-mRNAs in iPSC-derived pancreatic islets lacking FOXA2.
Additional file 2: Supplementary Table 1.
List of primers used for qPCR validation of selected deferentially expressed lncRNAs in the pancreatic progenitors and islets derived from FOXA2-/- iPSCs in comparison to those derived from WT-iPSCs. Supplementary Table 2. Top dysregulated lncRNAs in pancreatic progenitors (PPs) derived from FOXA2-/- iPSCs in comparison to those derived from WT-iPSCs (p < 0.05). Supplementary Table 3. Selected differentially expressed genes (DEGs) to study its correlation to the lncRNAs. Supplementary Table 4. List of downregulated lncRNAs with common strong correlation to FOXA2, PDX1 and NKX6.1 with a Pearson correlation (PC) > 0.3 in the pancreatic progenitors (PPs) derived from FOXA2-/- iPSCs in comparison to those derived from WT-iPSCs (p < 0.05). Supplementary Table 5. Top dysregulated lncRNAs in pancreatic islets derived from FOXA2-/- iPSCs in comparison to those derived from WT-iPSCs (p < 0.05). Supplementary Table 6. List of downregulated lncRNAs with common strong correlation to FOXA2, PDX1, NKX6.1, and ABCC8 with a Pearson correlation (PC) > 0.3 in the pancreatic islets derived from FOXA2 -/- iPSCs in comparison to those derived from WT-iPSCs (p < 0.05).
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Elsayed, A.K., Alajez, N.M. & Abdelalim, E.M. Genome-wide differential expression profiling of long non-coding RNAs in FOXA2 knockout iPSC-derived pancreatic cells. Cell Commun Signal 21, 229 (2023). https://doi.org/10.1186/s12964-023-01212-2
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DOI: https://doi.org/10.1186/s12964-023-01212-2