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Identification of a microRNA (miR-663a) induced by ER stress and its target gene PLOD3 by a combined microRNome and proteome approach

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Abstract

Introduction

MicroRNAs (miRs) regulate gene expression to support important physiological functions. Significant evidences suggest that miRs play a crucial role in many pathological events and in the cell response to various stresses.

Methods

With the aim to identify new miRs induced by perturbation of intracellular calcium homeostasis, we analysed miR expression profiles of thapsigargin (TG)-treated cells by microarray. In order to identify miR-663a-regulated genes, we evaluated proteomic changes in miR-663a-overexpressing cells by two-dimensional differential in-gel electrophoresis coupled to mass spectrometric identification of the differentially represented proteins. Microarray and proteomic analyses were supported by biochemical validation.

Results

Results of microarray revealed 24 differentially expressed miRs; among them, miR-663a turned out to be by ER stress and under the control of the PERK pathway of the unfolded protein response. Proteomic analysis revealed that PLOD3, which is the gene encoding for collagen-modifying lysyl hydroxylase 3 (LH3), is regulated by miR-663a. Luciferase reporter assays demonstrated that miR-663a indeed reduces LH3 expression by targeting to 3′-UTR of PLOD3 mRNA. Interestingly, miR-663a inhibition of LH3 expression generates reduced extracellular accumulation of type IV collagen, thus suggesting the involvement of miR-663a in modulating collagen 4 secretion in physiological conditions and in response to ER stress.

Conclusion

The finding of the ER stress-induced PERK-miR-663a pathway may have important implications in the understanding of the molecular mechanisms underlying the function of this miR in normal and/or pathological conditions.

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Acknowledgments

This work was supported by Università degli Studi di Salerno (FARB2013 and FARB2014 to Paolo Remondelli).

The authors thank V. A. Lasorsa for help with the graphical representation of the differential proteomic data.

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Authors and Affiliations

  1. Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, 84034, Fisciano, Salerno, Italy

    Giuseppina Amodio, Ornella Moltedo & Silvia Franceschelli

  2. Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, 80131, Naples, Italy

    Emanuele Sasso & Nicola Zambrano

  3. CEINGE Biotecnologie Avanzate, 80145, Naples, Italy

    Emanuele Sasso & Nicola Zambrano

  4. Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147, Naples, Italy

    Chiara D’Ambrosio & Andrea Scaloni

  5. Dipartimento di Medicina, Chirurgia e Odontoiatria “Scuola Medica Salernitana”, Università degli Studi di Salerno, 84034, Baronissi, Salerno, Italy

    Paolo Remondelli

  6. Dipartimento di Medicina e Chirurgia, Università degli Studi di Salerno, Via Salvador Allende, I-84034, Baronissi, Salerno, Italy

    Paolo Remondelli

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  1. Giuseppina Amodio
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  2. Emanuele Sasso
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Fig. S1

Activation of UPR markers following TG treatment in Huh7 cells. XBP1 splicing assay and RT-PCR of Bip/Grp78 and Chop mRNAs performed on Huh7 cells at different time points of TG treatment (300 nM). GAPDH was used as control of RNA recovery. (GIF 18 kb)

High Resolution Image (TIF 191 kb)

Fig. S2

qRT-PCR of miR-663a in Huh7 cells transfected with the pre-miR-663a vector. Relative expression levels of miR-663a in Huh7 cells transfected with the pre-miR-663a (pmiR-663a) or with the control vector (pmiR empty) at 48 h of expression. Fold change were calculated from 2-ΔΔCT formula. Each bar represents the mean ± SD of three independent replicates. (GIF 2 kb)

High Resolution Image (TIF 625 kb)

Fig. S3

2D-map of the proteins down-represented after miR-663a expression. The Figure shows a representative (master) gel of the 2D-DIGE experiment from Huh7 cells transfected with the pre-miR663a or with the control vector. The down-represented spots are indicated by a yellow contour. The extremes of the pH gradient used for first-dimension are reported at the top of the figure (GIF 23 kb)

High Resolution Image (TIF 2212 kb)

Fig. S4

Activation of UPR markers following TG treatment in Hek 293 cells. XBP1 splicing assay and RT-PCR of Bip/Grp78 and Chop mRNAs performed on Hek 293 cells in control condition (0) or after 8 h of TG treatment (300 nM). GAPDH served as control of RNA recovery (GIF 3 kb)

High Resolution Image (TIF 191 kb)

Fig. S5

Collagen IV and LH3 immunofluorescence in miR-663a overexpressing and TG-treated Huh7 cells. Huh7 cells seeded on glass coverslips were transfected with the GFP-expressing negative control vector (mock) or with the pre-miR-663a vector (pmiR-663a) and untreated (Untreated) or treated with 300 nM TG for 8 h (TG). Forty-eight h after transfection, coverslips were processed for immunofluorescence with collagen IV (COL IV) or LH3-specific antibodies (LH3). The smaller magnification insets shows the GFP fluorescence of transfected cells as grayscale mode (GIF 24 kb)

High Resolution Image (TIF 5353 kb)

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Amodio, G., Sasso, E., D’Ambrosio, C. et al. Identification of a microRNA (miR-663a) induced by ER stress and its target gene PLOD3 by a combined microRNome and proteome approach. Cell Biol Toxicol 32, 285–303 (2016). https://doi.org/10.1007/s10565-016-9335-z

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