rawdata.zip---<strong>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</strong>

<strong>Fig1. sh-LINC-p21 attenuates high glucose-induced cellular injury and dysfunction.</strong> <strong>(A)</strong> 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. <strong>(B)</strong> CCK-8 cell proliferation tests were used to measure cell proliferation. <strong>(C)</strong> Apoptosis analysis of MIN6 cells after staining with Annexin-V and PI using flow cytometer. <strong>(D)</strong> Western blot analysis of apoptosis-associated proteins. <strong>(E)</strong> Representative Western blots of iNOS protein. <strong>(F)</strong> GSIS assay was used to detect the effect of sh-LINC-p21 on insulin secretion in MIN-6 cell line.. *At P &lt; 0.05, significant differences from the control groups are displayed. Each experiment was carried out in triplicate. <strong>Fig2. Silencing of LINC-p21 attenuates GSIS dysfunction in islets of HFD mice. (A) </strong>After LINC-p21 knockdown, HFD mice exhibited weight loss while diabetic (HFD+sh-NC) mice exhibited weight gain. <strong>(B)</strong> H&amp;E staining under light microscope observation of normal and diabetic mice islets (magnification 200×, scale bar = 100 μm). <strong>(C)</strong> TUNEL staining of isolated islets following 48 h of culture is shown in representative photos (magnification 200×, scale bar = 100 μm). <strong>(D, E)</strong> Insulin resistance (ITT) and glucose tolerance (GTT) were assessed in mice. <strong>(F)</strong> Examples of two cross-reactive antibodies with anti-insulin and anti-glucagon antibodies on mouse pancreatic tissues using double indirect immunofluorescence. *At P &lt; 0.05, significant differences from the control groups are displayed. Each experiment was carried out in triplicate. <strong>Fig3. LINC-p21 directly interacted with miR-335-3p.</strong> <strong>(A)</strong> The combining sites between LINC-p21 and miR-335-3p, and the miRNA levels were determined using RT-qPCR. <strong>(B)</strong> 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. <strong>(C)</strong> Effects of LINC-p21 knockdown on the expression of miR-335-3p. <strong>(D)</strong> miR-335-3p concentrations in serum from patients with T2DM and healthy controls. <strong>(E) </strong>The effect of high glucose treatment on the expression of miR-335-3p in MIN6 cells was detected by RT-qPCR. *At P &lt; 0.05, significant differences from the control groups are displayed. Each experiment was carried out in triplicate. <strong>Fig4.</strong> <strong>Identification of IGF-1 as a direct target of miR-335-3p. (A) </strong>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. <strong>(B)</strong> 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. <strong>(C)</strong> 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). <strong>(D)</strong> 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 &lt; 0.05, significant differences from the control groups are displayed. Each experiment was carried out in triplicate. <strong>Fig5.</strong> <strong>Silencing LINC-p21 alleviated β-cells dysfunction through miR-335-3p/IGF-1 axis. (A) </strong>The rescue experiments about sh-LINC-p21, anti-miR-335-3p and sh-IGF-1 for cell proliferation. <strong>(B) </strong>The rescue experiments about sh-LINC-p21, anti-miR-335-3p and sh-IGF-1 for cell apoptosis.<strong> (C) </strong>The rescue experiments about sh-LINC-p21, anti-miR-335-3p and sh-IGF-1 for apoptosis-related proteins.<strong> (D) </strong>The rescue experiments about sh-LINC-p21, anti-miR-335-3p and sh-IGF-1 for iNOS protein levels.<strong> (E) </strong>The rescue experiments about sh-LINC-p21, anti-miR-335-3p and sh-IGF-1 for GSIS in MIN6 cells. *At <em>P</em> &lt; 0.05, significant differences from the control groups are displayed. Each experiment was carried out in triplicate. <strong>Fig6. Silencing LINC-p21 alleviated β-cells dysfunction through miR-335-3p/IGF-1 axis </strong><em><strong>in vivo</strong></em><strong>. (A) </strong>Body weight of the HFD-induced diabetic mice and non-diabetic control mice at 7 weeks.<strong> (B)</strong> HE staining pictures of pancreatic islet tissues after transfection (magnification 200×, scale bar = 100 μm). <strong>(C)</strong> 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 <strong>(D)</strong> and ITT <strong>(E)</strong> in control and diabetic mice. <strong>(F)</strong> Double immunofluorescent staining for islet hormones: glucagon and insulin, in pancreas islets of transfected mice. *At <em>P</em> &lt; 0.05, significant differences from the control groups are displayed. Each experiment was carried out in triplicate. <strong>FigS1. Enhanced expression of LINC-p21 in T2DM. (A)</strong> Differential expression of LINC-p21 in serum between patients with T2DM and control subjects. <strong>(B)</strong> High glucose treatment increased the expression of LINC-p21 in MIN6 cells. *At P &lt; 0.05, significant differences from the control groups are displayed. Each experiment was carried out in triplicate.