rawdata.zip---Long Intergenic Non-Coding RNA-p21 Regulates pancreatic β-cell Function through the Micro RNA-335-3p/Insulin Like Growth Factor 1 Axis in Type 2 Diabetes Mellitus

Fig1. sh-LINC-p21 attenuates high glucose-induced cellular injury and dysfunction. (A) The silencing efficiency of sh-LINC-p21#1, sh-LINC-p21#2, and sh-LINC-p21#3 were determined by qRT-PCR in high glucose-treated MIN6 cells. (B) CCK-8 cell proliferation tests were used to measure cell proliferation. (C) Apoptosis analysis of MIN6 cells after staining with Annexin-V and PI using flow cytometer. (D) Western blot analysis of apoptosis-associated proteins. (E) Representative Western blots of iNOS protein. (F) GSIS assay was used to detect the effect of sh-LINC-p21 on insulin secretion in MIN-6 cell line.. *At P Fig2. Silencing of LINC-p21 attenuates GSIS dysfunction in islets of HFD mice. (A) After LINC-p21 knockdown, HFD mice exhibited weight loss while diabetic (HFD+sh-NC) mice exhibited weight gain. (B) H&E staining under light microscope observation of normal and diabetic mice islets (magnification 200×, scale bar = 100 μm). (C) TUNEL staining of isolated islets following 48 h of culture is shown in representative photos (magnification 200×, scale bar = 100 μm). (D, E) Insulin resistance (ITT) and glucose tolerance (GTT) were assessed in mice. (F) Examples of two cross-reactive antibodies with anti-insulin and anti-glucagon antibodies on mouse pancreatic tissues using double indirect immunofluorescence. *At P Fig3. LINC-p21 directly interacted with miR-335-3p. (A) The combining sites between LINC-p21 and miR-335-3p, and the miRNA levels were determined using RT-qPCR. (B) The relative luciferase activities of luciferase reporters containing WT or Mut LINC-p21 were assayed after co-transfection with miR-335-3p mimics or NC mimics. (C) Effects of LINC-p21 knockdown on the expression of miR-335-3p. (D) miR-335-3p concentrations in serum from patients with T2DM and healthy controls. (E) The effect of high glucose treatment on the expression of miR-335-3p in MIN6 cells was detected by RT-qPCR. *At P Fig4. Identification of IGF-1 as a direct target of miR-335-3p. (A) Bioinformatics analysis was used to estimate the miR-335-3p target genes and the expression levels of the top 10 genes were detected by RT-qPCR in MIN6 cells, and IGF-1 was the most prominently elevated expression among them. (B) After 48 h, MIN6 cells co-transfected with miR-335-3p or miR-control mimic and either the control luciferase reporter plasmid or the reporter plasmids expressing IGF-1 WT 3'-UTR or IGF-1 MUT 3'-UTR showed luciferase activity. (C) In MIN6 cells transfected with miR-335-3p mimic or miR mimic, the protein expression of IGF-1 was examined by Western blot analysis (miR-NC). (D) Levels of IGF-1 in serum from T2DM patients and healthy controls. (E) Detection of IGF-1 expression in high glucose treated MIN6 cells by RT-qPCR. *At P Fig5. Silencing LINC-p21 alleviated β-cells dysfunction through miR-335-3p/IGF-1 axis. (A) The rescue experiments about sh-LINC-p21, anti-miR-335-3p and sh-IGF-1 for cell proliferation. (B) The rescue experiments about sh-LINC-p21, anti-miR-335-3p and sh-IGF-1 for cell apoptosis. (C) The rescue experiments about sh-LINC-p21, anti-miR-335-3p and sh-IGF-1 for apoptosis-related proteins. (D) The rescue experiments about sh-LINC-p21, anti-miR-335-3p and sh-IGF-1 for iNOS protein levels. (E) The rescue experiments about sh-LINC-p21, anti-miR-335-3p and sh-IGF-1 for GSIS in MIN6 cells. *At P Fig6. Silencing LINC-p21 alleviated β-cells dysfunction through miR-335-3p/IGF-1 axis in vivo. (A) Body weight of the HFD-induced diabetic mice and non-diabetic control mice at 7 weeks. (B) HE staining pictures of pancreatic islet tissues after transfection (magnification 200×, scale bar = 100 μm). (C) The proportion of TUNEL-positive apoptotic cells in pancreatic islets and TUNEL labeling of isolated islets after 48 h of culturing are shown in representative photos (magnification 200×, scale bar = 100 μm). GTT (D) and ITT (E) in control and diabetic mice. (F) Double immunofluorescent staining for islet hormones: glucagon and insulin, in pancreas islets of transfected mice. *At P FigS1. Enhanced expression of LINC-p21 in T2DM. (A) Differential expression of LINC-p21 in serum between patients with T2DM and control subjects. (B) High glucose treatment increased the expression of LINC-p21 in MIN6 cells. *At P