LncRNA Malat1 regulates iPSC-derived β-cell differentiation by targeting the miR-15b-5p/Ihh axis

Background: Differentiation of induced pluripotent stem cell (iPSC)-derived β-like cells is a novel strategy for treatment of type 1 diabetes. Elucidation of the regulatory mechanisms of long noncoding RNAs (lncRNAs) in β-like cells derived from iPSCs is important for understanding the development of the pancreas and pancreatic β-cells and may improve the quality of β-like cells for stem cell therapy. Methods: : β-like cells were derived from iPSCs in a three-step protocol. RNA sequencing and bioinformatics analysis were carried out to screen the differentially expressed lncRNAs and identify the putative target genes separately. LncRNA Malat1 was chosen for further research. Series of loss and gain of functions experiments were performed to study the biological function of this lncRNA. Quantitative real-time PCR (qRT-PCR), Western blot analysis and immunofluorescence (IF) staining were carried out to separately detect the functions of pancreatic β-cells at the mRNA and protein levels. Cytoplasmic and nuclear RNA fractionation and fluorescence in situ hybridization (FISH) were used to determine the subcellar location of lncRNA Malat1 in β-like cells. Flow cytometry and enzyme-linked immunosorbent assays (ELISAs) were performed to examine the differentiation and insulin secretion of β-like cells after stimulation with different glucose concentrations. Structural interactions between lncRNA Malat1 and miR-15b-5p and between miR-15b-5p and Ihh were detected by dual luciferase reporter assays (LRAs). Results: : We found that the expression of lncRNA Malat1 declined during differentiation, and overexpression of this lncRNA notably impaired the differentiation and maturation of β-like cells derived from iPSCs in vitro and in vivo . Localized to the cytoplasm, lncRNA Malat1 could function as a competing endogenous RNA (ceRNA) of miR-15b-5p to regulate the expression of Ihh according to bioinformatics prediction, mechanistic analysis and downstream experiments. Conclusion: This study established an unreported regulatory network of lncRNA Malat1 and the miR-15b-5p/ Ihh axis during the differentiation of iPSCs into β-like cells. In addition to acting as an oncogene promoting tumorigenesis, lncRNA Malat1 may be an effective and novel target for treatment of diabetes in the future.