scholarly journals LncRNA MALAT1 Regulates iPSC-derived Β-cell Differentiation by Targeting the miR-15b-5p/Ihh Axis

2020 ◽  
Author(s):  
Yao Wang ◽  
Xinxing Lin ◽  
Jin Lv ◽  
Jiachen Zhu ◽  
Haowen Fan ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Cell Therapy ◽  
Target Genes ◽  
Differentially Expressed ◽  
Luciferase Reporter ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Lncrna Malat1

Abstract Background: iPSCs-derived β-like cell differentiation provides a novel strategy for type 1 diabetes treatment. Clarifying the regulatory mechanisms of lncRNAs in β-like cells derived from induced pluripotent stem cells (iPSCs) is not only significant for understanding the development of pancreas or pancreatic β cells, but also helpful for improving the quality of β-like cells for stem cell therapy.Methods: β-like cells derived from iPSCs followed a three-step protocol. RNA-sequencing was carried out to screen the differentially expressed lncRNAs which was probably involved in the differentiation of pancreatic β cells. Bioinformatics was performed to analyze the putative target genes of significantly differentially expressed lncRNAs. LncRNA Malat1 was chosen for further research. Lentivirus victor, siRNA victor, antagomir victor and mimic victor were constructed for overexpression of lncRNA Malat1, knockdown of lncRNA Malat1, knockdown of miR-15b-5p and overexpression of miR-15b-5p respectively. Quantitative Real-Time PCR (qRT-PCR), Western Blot and Immunofluorescence (IF) staining were carried out to detect the functions of pancreatic β cells at mRNA and protein level separately. Cytoplasmic and nuclear RNA fractionation and Fluorescence in situ hybridization (FISH) were to ventilate the subcellar location of lncRNA Malat1 in β-like cells. Flow cytometry and ELISA were performed to examine differentiation efficiency and function of insulin secretion from β-like cells after being stimulated with different concentrations of glucose. Structural interactions between lncRNA Malat1 and miR-15b-5p and between miR-15b-5p and Ihh were detected by Dual luciferase reporter assay (LRA).Results: We found that expression of lncRNA Malat1 was on the decline during the differentiation and overexpression of this lncRNA obviously 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 the bioinformatic prediction, mechanistic analysis and downstream experiments.Conclusion: This study built an unreported regulatory network of lncRNA Malat1 and miR-15b-5p/Ihh axis during differentiation of iPSCs into β-like cells. Except for acting as a proverbial oncogene promoting tumorigenesis, lncRNA Malat1 may provide effective and novel molecule for diabetes cell therapy in the future.

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

2021 ◽  
Author(s):  
Yao Wang ◽  
Xinxing Lin ◽  
Jin Lv ◽  
Jiachen Zhu ◽  
Haowen Fan ◽  
...  
Keyword(s):  
Stem Cell ◽  
Target Genes ◽  
Induced Pluripotent Stem Cell ◽  
Luciferase Reporter ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Protein Levels ◽  
Lncrna Malat1

Abstract 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.

scholarly journals Targeted delivery of antisense oligonucleotides to pancreatic β-cells

2018 ◽  
Vol 4 (10) ◽  
pp. eaat3386 ◽  
Author(s):  
C. Ämmälä ◽  
W. J. Drury ◽  
L. Knerr ◽  
I. Ahlstedt ◽  
P. Stillemark-Billton ◽  
...  
Keyword(s):  
Targeted Delivery ◽  
Target Genes ◽  
Cell Types ◽  
Cell Type Specificity ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Glucagon Like Peptide 1 ◽  
Novel Therapeutic

Antisense oligonucleotide (ASO) silencing of the expression of disease-associated genes is an attractive novel therapeutic approach, but treatments are limited by the ability to deliver ASOs to cells and tissues. Following systemic administration, ASOs preferentially accumulate in liver and kidney. Among the cell types refractory to ASO uptake is the pancreatic insulin-secreting β-cell. Here, we show that conjugation of ASOs to a ligand of the glucagon-like peptide-1 receptor (GLP1R) can productively deliver ASO cargo to pancreatic β-cells both in vitro and in vivo. Ligand-conjugated ASOs silenced target genes in pancreatic islets at doses that did not affect target gene expression in liver or other tissues, indicating enhanced tissue and cell type specificity. This finding has potential to broaden the use of ASO technology, opening up novel therapeutic opportunities, and presents an innovative approach for targeted delivery of ASOs to additional cell types.

scholarly journals Circular RNA circSDHC serves as a sponge for miR-127-3p to promote the proliferation and metastasis of renal cell carcinoma via the CDKN3/E2F1 axis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Junjie Cen ◽  
Yanping Liang ◽  
Yong Huang ◽  
Yihui Pan ◽  
Guannan Shu ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Target Genes ◽  
Expression Patterns ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Proliferation And Metastasis

Abstract Background There is increasing evidence that circular RNAs (circRNAs) have significant regulatory roles in cancer development and progression; however, the expression patterns and biological functions of circRNAs in renal cell carcinoma (RCC) remain largely elusive. Method Bioinformatics methods were applied to screen for circRNAs differentially expressed in RCC. Analysis of online circRNAs microarray datasets and our own patient cohort indicated that circSDHC (hsa_circ_0015004) had a potential oncogenic role in RCC. Subsequently, circSDHC expression was measured in RCC tissues and cell lines by qPCR assay, and the prognostic value of circSDHC evaluated. Further, a series of functional in vitro and in vivo experiments were conducted to assess the effects of circSDHC on RCC proliferation and metastasis. RNA pull-down assay, luciferase reporter and fluorescent in situ hybridization assays were used to confirm the interactions between circSDHC, miR-127-3p and its target genes. Results Clinically, high circSDHC expression was correlated with advanced TNM stage and poor survival in patients with RCC. Further, circSDHC promoted tumor cell proliferation and invasion, both in vivo and in vitro. Analysis of the mechanism underlying the effects of circSDHC in RCC demonstrated that it binds competitively to miR-127-3p and prevents its suppression of a downstream gene, CDKN3, and the E2F1 pathway, thereby leading to RCC malignant progression. Furthermore, knockdown of circSDHC caused decreased CDKN3 expression and E2F1 pathway inhibition, which could be rescued by treatment with an miR-127-3p inhibitor. Conclusion Our data indicates, for the first time, an essential role for the circSDHC/miR-127-3p/CDKN3/E2F1 axis in RCC progression. Thus, circSDHC has potential to be a new therapeutic target in patients with RCC.

MiR-30a-5p Downregulation Induces Neuronal Autophagy by Activating AMPK After 2856MHz Microwave Radiation

2021 ◽  
Author(s):  
Yanhui Hao ◽  
Wenchao Li ◽  
Hui Wang ◽  
Jing Zhang ◽  
Haoyu Wang ◽  
...  
Keyword(s):  
Microwave Radiation ◽  
Hippocampal Neurons ◽  
Target Genes ◽  
Neuronal Damage ◽  
Luciferase Reporter ◽  
Potential Health ◽  
Downstream Target ◽  
Neuronal Autophagy

Abstract Background With the development of science and technology, microwaves are being widely used. More and more attention has been paid to the potential health hazards of microwave exposure. The regulation of miR-30a-5p (miR-30a) on autophagy is involved in the pathophysiological process of many diseases. Our previous study found that 30 mW/cm2 microwave radiation could reduce miR-30a expression and activate neuronal autophagy in rat hippocampus. However, the roles played by miR-30a in microwave-induced neuronal autophagy and related mechanisms remain largely unexplored. Results In the present study, we established neuronal damage models by exposing rat hippocampal neurons and rat adrenal pheochromocytoma (PC12) cell-derived neuron-like cells to 30 mW/cm2 microwave, which resulted in miR-30a downregulation and autophagy activation in vivo and in vitro. Bioinformatics analysis was conducted, and Beclin1, Prkaa2, Irs1, Pik3r2, Rras2, Ddit4, Gabarapl2 and autophagy-related gene 12 (Atg12) were identified as potential downstream target genes of miR-30a involved in regulating autophagy. Based on our previous findings that microwave radiation can cause a neuronal energy metabolism disorder, Prkaa2, encoding adenosine 5’-monophosphate-activated protein kinase α2 (AMPKα2, an important catalytic subunit of energy sensor AMPK), was selected for further analysis. Dual-luciferase reporter assay results showed that Prkaa2 is a downstream target gene of miR-30a. Microwave radiation increased the expression and phosphorylation (Thr172) of AMPKα both in vivo and in vitro. Moreover, the transduction of cells with miR-30a mimics suppressed AMPKα2 expression, inhibited AMPKα (Thr172) phosphorylation and reduced autophagy flux in neuron-like cells. Importantly, miR-30a mimics abolished microwave-activated autophagy and inhibited microwave-induced AMPKα (Thr172) phosphorylation. Conclusions AMPKα2 was a newly founded downstream gene of miR-30a involved in autophagy regulation, and miR-30a downregulation after microwave radiation could promote neuronal autophagy by increasing AMPKα2 expression and activating AMPK signaling.

scholarly journals Protein Kinase CK2 Controls CaV2.1-Dependent Calcium Currents and Insulin Release in Pancreatic β-cells

2020 ◽  
Vol 21 (13) ◽  
pp. 4668
Author(s):  
Rebecca Scheuer ◽  
Stephan Ernst Philipp ◽  
Alexander Becker ◽  
Lisa Nalbach ◽  
Emmanuel Ampofo ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Calcium Currents ◽  
Insulin Biosynthesis ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Voltage Dependent ◽  
Regulatory Impact ◽  
Kinase Ck2

The regulation of insulin biosynthesis and secretion in pancreatic β-cells is essential for glucose homeostasis in humans. Previous findings point to the highly conserved, ubiquitously expressed serine/threonine kinase CK2 as having a negative regulatory impact on this regulation. In the cell culture model of rat pancreatic β-cells INS-1, insulin secretion is enhanced after CK2 inhibition. This enhancement is preceded by a rise in the cytosolic Ca2+ concentration. Here, we identified the serine residues S2362 and S2364 of the voltage-dependent calcium channel CaV2.1 as targets of CK2 phosphorylation. Furthermore, co-immunoprecipitation experiments revealed that CaV2.1 binds to CK2 in vitro and in vivo. CaV2.1 knockdown experiments showed that the increase in the intracellular Ca2+ concentration, followed by an enhanced insulin secretion upon CK2 inhibition, is due to a Ca2+ influx through CaV2.1 channels. In summary, our results point to a modulating role of CK2 in the CaV2.1-mediated exocytosis of insulin.

Pannexin-2-deficiency sensitizes pancreatic β-cells to cytokine-induced apoptosis in vitro and impairs glucose tolerance in vivo

2017 ◽  
Vol 448 ◽  
pp. 108-121 ◽  
Author(s):  
Lukas A. Berchtold ◽  
Michela Miani ◽  
Thi A. Diep ◽  
Andreas N. Madsen ◽  
Valentina Cigliola ◽  
...  
Keyword(s):  
Glucose Tolerance ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Induced Apoptosis

scholarly journals LukS-PV-Regulated MicroRNA-125a-3p Promotes THP-1 Macrophages Differentiation and Apoptosis by Down-Regulating NF1 and Bcl-2

2017 ◽  
Vol 44 (3) ◽  
pp. 1093-1105 ◽  
Author(s):  
Xiao-Xi Sun ◽  
Shan-Shan Zhang ◽  
Chun-Yang Dai ◽  
Jing Peng ◽  
Qing Pan ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Cellular Differentiation ◽  
Target Genes ◽  
B Cell Lymphoma ◽  
Neurofibromatosis Type ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Leukaemia Cell Line ◽  
Reporter Assays

Background/Aims: LukS-PV is a component of Panton-Valentine leukocidin (PVL). We have previously demonstrated that LukS-PV potently promoted differentiation and induced apoptosis in THP-1 cells. However, the precise mechanisms of these actions remain unknown. MicroRNAs (miRs) play important roles in cellular differentiation and apoptosis. This study aimed to investigate the role of miR-125a-3p in LukS-PV-regulated differentiation and apoptosis and its underlying mechanism in THP-1 cells. Methods: MicroRNA profiling analyses were conducted to determine differential miRNA expression levels in THP-1 cells treated with LukS-PV. Cell differentiation and apoptosis were measured in THP-1 cells by gain-of-function and loss-of-function experiments. Bioinformatics analysis and luciferase reporter assays were used to confirm the targets of miR-125a-3p. The effects of the miR-125a-3p targets on cellular differentiation were determined by knocking them down. Results: MiR-125a-3p was up-regulated after treating the human monocytic leukaemia cell line THP-1 with LukS-PV. In addition, miR-125a-3p positively regulated apoptosis and differentiation in THP-1 cells treated with LukS-PV. Concordantly, luciferase reporter assays confirmed that neurofibromatosis type 1 (NF1) and B-cell lymphoma 2 (Bcl-2) were direct target genes of miR-125a-3p. Moreover, NF1 knockdown in THP-1 cells significantly promoted differentiation in vitro. Finally, the extracellular signal-regulated kinase (ERK) pathway, a downstream target of NF1, was activated after NF1 knockdown. Conclusions: These findings confirm that miR-125a-3p is involved in LukS-PV-mediated cell differentiation and apoptosis in THP-1 cells.

Sign in / Sign up

Export Citation Format

Share Document