miR-375 Promotes Pancreatic Differentiation In Vitro by Affecting Different Target Genes at Different Stages

Abstract

Human embryonic stem cells (hESCs) possess the ability to differentiate into insulin-producing cells (IPCs), which can be used to treat type I diabetes. miR-375 is an essential transcriptional regulator in the development and maturation of the pancreas. In this study, we optimized a protocol to differentiate hESCs into IPCs and successfully obtained IPCs. Then, we performed overexpression and inhibition experiments of miR-375 on cells at different stages of differentiation and performed RNA-seq. The results showed that the expression of miR-375 fluctuated during hESC differentiation and was affected by miR-375 mimics and inhibitors. miR-375 influences global gene expression and the target genes of miR-375. The overexpression of miR-375 can cause changes in multiple signaling pathways during pancreatic development. miR-375 is a major participant in the differentiation of pancreatic β-cells through different target genes at different stages. This study provides new ideas for further investigation of how microRNAs affect cell fate and gene transcription.

Figure 1

A four-stage protocol for differentiation of human embryonic stem cells to insulin-producing cells. (a) Flowchart of the cell differentiation process. Differentiation is divided into four stages: definitive endoderm (stage I), gastrulation (stage II), endocrine precursor (stage III), and pancreatic endocrine cell (stage IV). (b) The expression of marker genes at each stage of the differentiation process. ∗∗∗ indicates P < 0.001, ∗∗ indicates P < 0.01, and ∗ indicates P < 0.05. (c) Immunofluorescence detection of PDX-1 (samples collected on D13) and INSULIN (samples collected on D21). Scale bar =100 μm.

Figure 2

The effects of miR-375 overexpression and inhibition during differentiation of human embryonic stem cells to insulin-producing cells. (a) Illustration of the transfection process. We used liposomes to perform transfection of miR-375 mimics and miR-375 inhibitors at the beginning of each phase (D0, D4, D7, D11, D14, and D18). Samples were collected at the end of each stage (D3, D6, D10, D13, D17, and D21). (b) The effect of the miR-375 mimic and miR-375 inhibition on transcription factors. (c) Western blotting detection and grayscale analysis were used to determine the protein expression of PDX-1 (D13) and INSULIN (D21) after miR-375 overexpression and inhibition.

Figure 3

Overall description of differentially expressed genes (DEGs) in the four differentiation stages for control vs. miR-375 overexpression (OE) and control vs. miR-375 inhibition. (a) Heatmap showing the expression of DEGs in the miR-375OE group and the miR-375 inhibition group compared with the control group in different stages of differentiation. Hierarchical clustering was used to cluster gene expression patterns, and its expression level was standardized to TPM. (b) Venn diagram showing the distribution of DEGs in the four stages of differentiation in OE vs. control and bar plot showing the top 45 pathways of KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment, presented in descending order by gene number. (c) Venn diagram showing the distribution of DEGs in the four stages of differentiation for IE vs. control and bar plot showing the top 15 pathways of three GO (gene ontology) terms: BP (biological process), CC (cellular component), and MF (molecular function).

Figure 4

Overall description of 272 possible miR-375 target genes for the four differentiation stages in control vs. OE and control vs. IE. (a) Heatmap showing the expression levels of 272 possible miR-375 target genes in the four differentiation stages in the OE, IE, and control groups. Hierarchical clustering of gene expression patterns was performed, and its expression level was standardized to TPM (transcripts per kilobase of exon model per million mapped reads). (b) Dot plot showing the KEGG enrichment pathways of 272 possible target genes. The abscissa represents the gene enrichment ratio, the bubble size represents the number of enriched genes in this pathway, and the color represents the q value (adjusted P value). (c) Trend analysis of target gene expression (10 trends) by STEM (Short Time-series Expression Miner). Colored blocks: significantly enriched trend (P < 0.05).

Figure 5

Interaction of genes related to pancreatic differentiation. (a) The number of differential genes obtained by comparing miR-375 with its corresponding control group after overexpression at different stages. (b) The number of genes that overexpressed miR-375 has changed significantly compared with its corresponding control group in different signaling pathways. Blue represents decreased expression, and red represents increased expression. (c) The heatmap shows the expression of important transcription factors in different pathways in the OE group and the control group.

Figure 6

Overexpression of miR-375 can lead to changes in multiple signaling pathways during pancreatic development. (a) The line chart shows the expression level of some differentially expressed genes occupying a central position in protein-protein interaction networks. The red line represents the control group and the green line represents the overexpression group, the abscissa represents the differentiation time, and the ordinate represents the expression of the gene in different differentiation stages (normalized to TPM). (b) The protein-protein interaction network obtained from the STRING database. Each circle represents a gene, and each edge represents a possible interaction between two genes. The size of the circular area represents the importance of the gene in the network. The highlighted genes (black characters on a yellow background) play an important role in the network and need to be analyzed individually and are plotted in (a).