A long non-coding RNA that harbors a SNP associated with type 2 diabetes regulates the expression of TGM2 gene in pancreatic beta cells

Figures

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  Figure 1. An exonic SNP in LncTGM2 is associated with T2D risk. The graph shows the percent of T2D in two independent cohorts segregated according to their rs2076380 genotype.

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  Figure 2. LncTGM2 co-expresses with TGM2 and regulates its transcriptional activity in pancreatic beta cells. (A) LncTGM2 was silenced in the EndoC-βH1 cell line using a siRNA, and (B) EndoC-βH1 cells were exposed to palmitate (0.5 mM) for 4 or 8h. LncTGM2 and TGM2 expression was assessed by RT-qPCR and normalized by the reference gene RPLP0. The results are means ± S.E.M. of 3-4 independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001 by Student’s t-test. (C) RT-qPCR analysis of LncTGM2, MEG3 (as nuclear marker) and RPLP0 (as cytoplasmic marker) in nuclear and cytoplasmic fractions in EndoC-βH1 cells. (D) HEK-293 cells were transfected with a control vector (ovCTRL) or a vector overexpressing LncTGM2 (ovLncTGM2), and co-transfected with a TGM2 promoter luciferase reporter construct plus a pRL-CMV plasmid (used as internal control). After 48h of recovery, bioluminescence was measured.

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  Figure 3. The T2D-associated risk allele in LncTGM2 disrupts its secondary structure impacting on its stability, and correlates with decreased expression of TGM2 in beta cells. (A) In silico prediction of the secondary structure of LncTGM2 harboring each allele for rs2076380; T2D protective allele (G) or T2D risk allele (A). (B) Electrophoretic mobility profiles of in vitro-transcribed LncTGM2 molecule harboring the T2D protective allele (rs2076380-G) or the risk allele (rs2076380-A). (C) EndoC-βH1 cells were transfected with overexpression plasmids of LncTGM2 harboring the protective (ovLncTGM2-G) or risk allele (ovLncTGM2-A) for T2D, and mRNA levels of LncTGM2 and TGM2 were determined by RT-qPCR and normalized to RPLP0. The results are means ± S.E.M. of 3 independent experiments; *p < 0.05 by Student’s t-test. (D) EndoC-βH1 cells were transfected with LncTGM2 overexpression plasmids harboring the protective (ovLncTGM2-G) or risk allele (ovLncTGM2-A) for T2D. EndoC-βH1 cells were exposed to Actinomycin D (ActD) (5 μg/ml) for 2, 4 or 6h and LncTGM2 mRNA level was determined by RT-qPCR. The results are means ± S.E.M. of 3 independent experiments. *p < 0.05 ovLncTGM2-G vs. ovLncTGM2-A at the…

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  Figure 4. LncTGM2 downregulation affects glucose-stimulated insulin secretion. LncTGM2 was silenced using a siRNA and EndoC-βH1 cells were exposed to 0 or 20 mmol/l glucose. Insulin release was determined by ELISA. The results are means ± S.E.M. of 4 independent experiments; *p<0.05 by Student’s t-test.