Dysregulated lncRNAs Regulate Human Umbilical Cord Mesenchymal Stem Cells Differentiation into Insulin-Producing Cells by Forming a Regulatory Network with mRNAs

ObjectiveIn recent years, cell therapy has become a new research direction in the treatment of diabetes. However, the underlying molecular mechanisms of mesenchymal stem cells (MSCs) participate in such treatment has not been clarified. MethodsIn this study, human umbilical cord mesenchymal stem cells (HUC-MSCs) isolated from newborns were progressively induced into insulin-producing cells (IPCs) using small molecules. HUC-MSCs (S0) and four induced stage (S1-S4) samples were prepared. We then performed transcriptome sequencing experiments to obtain the dynamic expression profiles of both mRNAs and long noncoding RNAs (lncRNAs). ResultsWe found that the number of differentially expressed lncRNAs and mRNAs showed a decreasing trend during differentiation. Gene Ontology (GO) analysis showed that the target genes of differentially expressed lncRNAs were associated with translation, cell adhesion, and cell connection. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the NF-KB signaling pathway, MAPK signaling pathway, HIPPO signaling pathway, PI3K-Akt signaling pathway, and p53 signaling pathway were enriched in these differentially expressed lncRNA-targeting genes. We also found that the coexpression of the lncRNA: CTBP1-AS2 with the PROX1, and the lncRNAs AC009014.3 and GS1-72M22.1 with the mRNA JARID2 was related to the development of pancreatic beta cells. Moreover, the coexpression of the lncRNAs :XLOC_ 050969, LINC00883, XLOC_050981, XLOC_050925, MAP3K14- AS1, RP11-148K1.12, and CTD2020K17.3 with p53, regulated insulin secretion by pancreatic beta cells.ConclusionThis research revealed that HUC-MSCs combined with small molecule compounds were successfully induced into IPCs. Differentially expressed lncRNAs may regulate the insulin secretion of pancreatic beta cells by regulating multiple signaling pathways. The lncRNAs: AC009014.3,Gs1-72m21.1 and CTBP1-AS2 may be involved in the development of pancreatic beta cells, and the lncRNAs: XLOC_050969, LINC00883, XLOC_050981, XLOC_050925, MAP3K14-AS1, RP11-148K1.12, and CTD2020K17.3 may be involved in regulating the insulin secretion of pancreatic beta cells, thus providing a lncRNA catalog for future research regarding the mechanism of the transdifferentiation of HUC-MSCs into IPCs. It also provides a new theoretical basis for the transplantation of insulin-producing cells into diabetic patients in the future.

Specific Reprogramming of Alpha Cells to Insulin-Producing Cells by Short Glucagon Promoter-Driven Pdx1 and MafA

Endogenous reprogramming of pancreas-derived non-beta cells into insulin-producing cells is a promising approach to treat type 1 diabetes (T1D). One strategy that has yet to be explored is the specific delivery of insulin-producing essential genes, Pdx1 and MafA, to pancreatic alpha cells to reprogram the cells into insulin-producing cells in an adult pancreas. In this study, we utilized an alpha cell-specific glucagon (GCG) promoter to drive Pdx1 and MafA transcription factors to reprogram alpha cells to insulin-producing cells in chemically induced and autoimmune diabetic mice. Our results showed that a combination of a short glucagon-specific promoter with AAV serotype 8 can be used to successfully deliver Pdx1 and MafA into alpha cells in the mouse pancreas. Pdx1 and MafA expression specifically in alpha cells was also able to correct hyperglycemia in both induced and autoimmune diabetic mice. With this technology, targeted gene specificity and reprogramming were accomplished with an alpha-specific promotor combined with an AAV-specific serotype and provide an initial basis to develop a novel therapy for the treatment of T1D.

Specific Reprogramming of Alpha Cells to Insulin-Producing Cells by Short Glucagon Promoter-Driven Pdx1 and MafA

Endogenous reprogramming of pancreas-derived non-beta cells into insulin-producing cells is a promising approach to treat type 1 diabetes (T1D). One strategy that has yet to be explored is the specific delivery of insulin-producing essential genes, Pdx1 and MafA, to pancreatic alpha cells to reprogram the cells into insulin-producing cells in an adult pancreas. In this study, we utilized an alpha cell-specific glucagon (GCG) promoter to drive Pdx1 and MafA transcription factors to reprogram alpha cells to insulin-producing cells in chemically induced and autoimmune diabetic mice. Our results showed that a combination of a short glucagon-specific promoter with AAV serotype 8 can be used to successfully deliver Pdx1 and MafA into alpha cells in the mouse pancreas. Pdx1 and MafA expression specifically in alpha cells was also able to correct hyperglycemia in both induced and autoimmune diabetic mice. With this technology, targeted gene specificity and reprogramming were accomplished with an alpha-specific promotor combined with an AAV-specific serotype and provide an initial basis to develop a novel therapy for the treatment of T1D.

Activation of a Ductal-to-Endocrine Transdifferentiation Transcriptional Program in the Pancreatic Cancer Cell Line PANC-1 Is Controlled by RAC1 and RAC1b through Antagonistic Regulation of Stemness Factors

Epithelial–mesenchymal transition (EMT) is a driving force for tumor growth, metastatic spread, therapy resistance, and the generation of cancer stem cells (CSCs). However, the regained stem cell character may also be exploited for therapeutic conversion of aggressive tumor cells to benign, highly differentiated cells. The PDAC-derived quasimesenchymal-type cell lines PANC-1 and MIA PaCa-2 have been successfully transdifferentiated to endocrine precursors or insulin-producing cells; however, the underlying mechanism of this increased plasticity remains elusive. Given its crucial role in normal pancreatic endocrine development and tumor progression, both of which involve EMT, we analyzed here the role of the small GTPase RAC1. Ectopic expression in PANC-1 cells of dominant negative or constitutively active mutants of RAC1 activation blocked or enhanced, respectively, the cytokine-induced activation of a ductal-to-endocrine transdifferentiation transcriptional program (deTDtP) as revealed by induction of the NEUROG3, INS, SLC2A2, and MAFA genes. Conversely, ectopic expression of RAC1b, a RAC1 splice isoform and functional antagonist of RAC1-driven EMT, decreased the deTDtP, while genetic knockout of RAC1b dramatically increased it. We further show that inhibition of RAC1 activation attenuated pluripotency marker expression and self-renewal ability, while depletion of RAC1b dramatically enhanced stemness features and clonogenic potential. Finally, rescue experiments involving pharmacological or RNA interference-mediated inhibition of RAC1 or RAC1b, respectively, confirmed that both RAC1 isoforms control the deTDtP in an opposite manner. We conclude that RAC1 and RAC1b antagonistically control growth factor-induced activation of an endocrine transcriptional program and the generation of CSCs in quasimesenchymal PDAC cells. Our results have clinical implications for PDAC patients, who in addition to eradication of tumor cells have a need for replacement of insulin-producing cells.

Specific Reprogramming of Alpha Cells to Insulin-Producing Cells by Short Glucagon Promoter-Driven Pdx1 and MafA

Endogenous reprogramming of pancreas-derived non-beta cells into insulin-producing cells is a promising approach to treat type 1 diabetes (T1D). One strategy that has yet to be explored is the specific delivery of insulin-producing essential genes, Pdx1 and MafA, to pancreatic alpha cells to reprogram the cells into insulin-producing cells in an adult pancreas. In this study, we utilized an alpha cell-specific glucagon (GCG) promoter to drive Pdx1 and MafA transcription factors to reprogram alpha cells to insulin-producing cells in chemically induced and autoimmune diabetic mice. Our results showed that a combination of a short glucagon-specific promoter with AAV serotype 8 can be used to successfully deliver Pdx1 and MafA into alpha cells in the mouse pancreas. Pdx1 and MafA expression specifically in alpha cells was also able to correct hyperglycemia in both induced and autoimmune diabetic mice. With this technology, targeted gene specificity and reprogramming were accomplished with an alpha-specific promotor combined with an AAV-specific serotype and provide an initial basis to develop a novel therapy for the treatment of T1D.