The miRNAs miR-211-5p and miR-204-5p modulate ER stress in human beta cells

in Journal of Molecular Endocrinology

Correspondence should be addressed to D L Eizirik: deizirik@ulb.ac.be
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miRNAs are a class of small non-coding RNAs that regulate gene expression. Type 1 diabetes is an autoimmune disease characterized by insulitis (islets inflammation) and pancreatic beta cell destruction. The pro-inflammatory cytokines interleukin 1 beta (IL1B) and interferon gamma (IFNG) are released during insulitis and trigger endoplasmic reticulum (ER) stress and expression of pro-apoptotic members of the BCL2 protein family in beta cells, thus contributing to their death. The nature of miRNAs that regulate ER stress and beta cell apoptosis remains to be elucidated. We have performed a global miRNA expression profile on cytokine-treated human islets and observed a marked downregulation of miR-211-5p. By real-time PCR and Western blot analysis, we confirmed cytokine-induced changes in the expression of miR-211-5p and the closely related miR-204-5p and downstream ER stress related genes in human beta cells. Blocking of endogenous miRNA-211-5p and miR-204-5p by the same inhibitor (it is not possible to block separately these two miRs) increased human beta cell apoptosis, as measured by Hoechst/propidium Iodide staining and by determination of cleaved caspase-3 activation. Interestingly, miRs-211-5p and 204-5p regulate the expression of several ER stress markers downstream of PERK, particularly the pro-apoptotic protein DDIT3 (also known as CHOP). Blocking CHOP expression by a specific siRNA partially prevented the increased apoptosis observed following miR-211-5p/miR-204-5p inhibition. These observations identify a novel crosstalk between miRNAs, ER stress and beta cell apoptosis in early type 1 diabetes.

Supplementary Materials

    • Supplementary Figure 1. Cytokines regulation of miR-211-5p and CHOP expression in human islets. Whole human islets were left untreated or treated with IL1B+IFNG for 48h. Expression of miR-211-5p (A) was assayed by RT-PCR and normalized by the small nucleolar RNA miR-RD61 (p-value = 0.0818 between untreated and treated). Expression of CHOP (B) was assayed by RT-PCR and normalized by the housekeeping gene ACTB (p-value = 0.1107 between untreated and treated). The results are shown as individual experiments before and after cytokines treatment; n=4 independent experiments.
    • Supplementary Figure 2. Thapsigargin co-regulates miR-211-5p and CHOP expression in human EndoC-βH1 cells. Human insulin-producing EndoC-βH1 cells (A and B) were left untreated or treated with the ER stressor thapsigargin for 24h. Expression of miR-211-5p (A) was assayed by RT-PCR and normalized by two different small nucleolar RNAs (miR-U6 and miR-RD61). Expression CHOP (B) was assayed by RT-PCR and normalized by the housekeeping gene ACTB. The results are shown as individual experiments before and after thapsigargin treatment; n=5 independent experiments. * p<0.05 versus untreated cells; paired Student’s t-test.
    • Supplementary Figure 3. miR-211-5p inhibition exacerbates cytokine-induced activation of cleaved caspase-3 in EndoC-βH1 cells. Human insulin-producing EndoC-βH1 cells were transfected with siControl (CTRL) (A-D and I-L) or an anti-miRNA targeting miR-211-5p (anti-miR-211) (E-H and M-P) for 8h. After 24h of recovery, cells were left untreated (A-H) or treated with IL1B+IFNG (I-P) for 48h. After cytokine treatment, cells were fixed and used for histological studies. Fluorescent microscopy analysis of insulin (A, E, I, and M in green) and cleaved caspase-3 (B, F, J and N in red) shows the presence of double-positive cells for insulin and cleaved caspase-3 (D, H, L and P merged panels in yellow). Hoechst staining (C, G, K and O in blue) shows the presence of nuclear condensation in the apoptotic cleaved-caspase-3 positive cells. Double-positive cells for insulin and cleaved caspase-3 are indicated by the arrows (A, B, D, E, F, H, I, J, L, M, N and P). The quantification of cleaved caspase-3 positive cells is shown in Fig. 3E in the main text; n=4 independent experiments.
    • Supplementary Figure 4. miR-211-5p inhibition exacerbates cytokine-induced activation of cleaved caspase-3 in in dispersed human islet cells. Human dispersed islets cells were transfected with siControl (CTRL) (A-D and I-L) or an anti-miRNA targeting miR-211-5p (anti-miR-211) (E-H and M-P) for 8h. After 48h of recovery, cells were left untreated (A-H) or treated with IL1B+IFNG (I-P) for 48h. After cytokine treatment, cells were fixed and used for histological studies. Fluorescent microscopy analysis of insulin (A, E, I, and M in green) and cleaved caspase-3 (B, F, J and N in red) shows the presence of double-positive cells for insulin and cleaved caspase-3 (H, L And P merged panels in yellow). Hoechst staining (C, G, K and O in blue) is shown. Double-positive cells for insulin and cleaved caspase-3 are indicated by the arrows (E, F, H, I, J, L, M, N and P); n=2 independent experiments.
    • Supplementary Figure 5. miR-211-5p inhibition does not affect expression of ER stress markers in an ATF6- and IRE1α-dependent manner. Human insulin-producing EndoC-βH1 cells (A-D) were transfected with siControl (CTRL) or with an anti-miRNA targeting miR-211-5p (anti-miR-211) for 8h. After 24h of recovery, the cells were left untreated or treated with IL1B+IFNG for 48h as indicated. Expression of BIP (A) and XBP1 spliced (B) (XBP1s) were assayed by RT-PCR and normalized by the housekeeping gene ACTB. BIP and tubulin expression were evaluated by western blot after 48h of cytokine treatment; one representative blot of 5 independent experiments is shown (C). The optical density quantification of BIP (example shown in C) was quantified and corrected by tubulin expression (D). In (A, B and D) results were normalized against the highest value in each independent experiment, considered as 1. Data shown are mean ± SEM of 5-6 independent experiments. * p<0.05, ** p<0.01 and *** p<0.001 versus siCTRL untreated cells; ANOVA followed by Bonferroni’s correction. The order of the bands in the blot image (C) is the same as in the quantification histogram (D): from left to right are respectively untreated cells transfected with a control siRNA, untreated cells transfected with miR-211-5p inhibitor, treated cells transfected with a control siRNA and lastly treated cells transfected with miR-211-5p inhibitor. Full-length blots are presented in Supplementary Figure 10.(PDF 146 KB)
    • Supplementary Figure 6. miR-211-5p inhibition exacerbates apoptosis and increases mRNA expression of ER stress markers in EndoC-βH1 cells in a PERK-dependent manner. Human insulin-producing EndoC-βH1 cells (A-F) were transfected with siControl (CTRL) or an anti-miRNA targeting miR-211-5p (anti-miR-211) for 8h. After 24h of recovery, the cells were left untreated or treated with the ER stressor thapsigargin for 24h as indicated. Apoptosis was evaluated by propidium iodide / Hoechst staining after 24h of thapsigargin treatment (A). Expression of PERK (B), EIF2A (C), ATF4 (D), ATF3 (E) and CHOP (F) were assayed by RT-PCR and normalized by the housekeeping gene ACTB. The results are represented as box plots, indicating lower quartile, median, and higher quartile, with whiskers representing the range of the remaining data points (A). In (B-F) results were normalized against the highest value in each independent experiment, considered as 1. Data shown are mean ± SEM of 6 independent experiments. *** p<0.001 versus siCTRL untreated cells; $ p<0.05, $$ p<0.01 and $$$ p<0.001 versus siCTRL thapsigargin-treated cells; §§§ p<0.001 as indicated by bars; ANOVA followed by Bonferroni’s correction.
    • Supplementary Figure 7. miR-211-5p inhibition increases expression of pro-apoptotic BCL2 family members in human EndoC-βH1 cells. Human insulin-producing EndoC-βH1 cells were transfected with siControl (CTRL), siCHOP, an anti-miRNA targeting miR-211-5p (anti-miR-211) or co-transfected with both siCHOP+anti-miR-211 (A-C) for 8h. After 24h of recovery, cells were left untreated or treated with IL1B+IFNG for 48h as indicated. Expression of c-JUN (A), DP5 (B) and PUMA (C) were assayed by RT-PCR and normalized by the housekeeping gene ACTB. In (A-C) results were normalized against the highest value in each independent experiment, considered as 1. Data shown are mean ± SEM of 6 independent experiments. * p<0.05, ** p<0.01 and *** p<0.001 versus siCTRL untreated cells; $$ p<0.01 and $$$ p<0.001 versus siCTRL cytokine-treated cells; §§ p<0.01 as indicated by bars; ANOVA followed by Bonferroni’s correction.
    • Supplementary Figure 8. Full unedited blots for Figure 3. Top blot showing Cleaved Caspase 3 bands, bottom blots showing Tubulin bands. Cropped images presented in Figure 3c of the main paper. Used Marker is PageRule Protein Ladder, # 26616 Thermo Scientific.
    • Supplementary Figure 9. Full unedited blots for Figure 5. (A) Top blots showing PERK bands (left) and the relative Tubulin bands (right). (B) Middle blots showing p-eIF2α and eIF2α bands (left) and the relative Tubulin bands (right). (C) Bottom blots showing ATF4 and ATF3 bands (left) and the relative Tubulin bands (right). Cropped images presented respectively in Figure 5A, 5C and 5D of the main paper. Used Marker is PageRule Protein Ladder, # 26616 Thermo Scientific.
    • Supplementary Figure 10. Full unedited blots for Supplementary Figure 5. Top blot showing BIP bands, bottom blots showing Tubulin bands. Cropped images presented in Supplementary Figure 5C. Used Marker is PageRule Protein Ladder, # 26616 Thermo Scientific.
    • Supplementary Table 1 – Characteristics of the human islet donors
    • Supplementary Table 2 – Antibodies used in the study
    • Supplementary Table 3 – Primers sequences

 

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