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. 2023 Dec;13(12):2263-2272.
doi: 10.1002/2211-5463.13724. Epub 2023 Nov 2.

ETV5 regulates proliferation and cell cycle genes in the INS-1 (832/13) cell line independently of the concentration of secreted insulin

Yael E Díaz-López  1   2 , Gloria Erandi Pérez-Figueroa  3 , Vicenta Cázares-Domínguez  2 , María E Frigolet  2 , Ruth Gutiérrez-Aguilar  1   2
Affiliations

ETV5 regulates proliferation and cell cycle genes in the INS-1 (832/13) cell line independently of the concentration of secreted insulin

Yael E Díaz-López et al. FEBS Open Bio. 2023 Dec.

Abstract

The transcription factor E-twenty-six variant 5 (ETV5) regulates acute insulin secretion. Adequate insulin secretion is dependent on pancreatic β-cell size and cell proliferation, but the effects of ETV5 on proliferation, cell number, and viability, as well as its relationship with insulin secretion, have not been established yet. Here, we partially silenced ETV5 in the INS-1 (832/13) cell line by siRNA transfection and then measured secreted insulin concentration at different time points, observing similar levels to control cells. After 72 h of ETV5 silencing, we observed decreased cell number and proliferation, without any change in viability or apoptosis. Thus, partial silencing of ETV5 modulates cell proliferation in INS-1 (832/13) independently of secreted insulin levels via upregulation of E2F1 and of inhibitors of the cyclin/CDKs complexes (p21Cdkn1a , p27Cdkn1b , and p57Cdkn1c ) and downregulation of cell cycle activators (PAK3 and FOS).

Keywords: E2F1; p27 Cdkn1b; E-twenty-six variant 5; INS-1 (832/13); insulin secretion; proliferation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Partial E‐twenty‐six variant 5 (ETV5) silencing provokes reduced cell number independently of secreted insulin concentration in INS‐1 (832/13) cell line. (a) mRNA ETV5 relative expression after control siRNA (siC) and ETV5 siRNA (siE) transfection assays at 24, 48, and 72 h post‐transfection. (b) Representative images (10×) of growing culture cells siC and siE transfection at 0, 24, 48, and 72 h post‐transfection. Scale bars: 100 μm. (c) Cell number in siC and siE treatments at 0, 24, 48, and 72 h post‐transfection. (d) Secreted insulin concentration normalized to total protein at different time points after siC or siE transfection: at 0 h (A), 24 h (B), from 24 to 48 h (C), from 48 to 72 h (D), and from 24 to 72 h (E). Two‐way ANOVA, *P < 0.05, **P < 0.001, ****P < 0.00001, n = 4. All results are presented as the mean ± SD.
Fig. 2
Fig. 2
E‐twenty‐six variant 5 (ETV5) silencing reduces cell proliferation in INS‐1 (832/13) cell line and does not affect viability and apoptosis. (a) Percentage of positive EdU proliferating cell (% ratio proliferating cells divided by the total cell number) at 0, 24, 48, and 72 h post‐transfection with siC or siE. Two‐way ANOVA, *P < 0.05, n = 3. All results are presented as the mean ± SD. (b) Representative images of cell proliferation determination by fluorescence microscopy (20×), DAPI labeled in blue, EdU labeled in red, after 0, 24, 48, and 72 h post‐transfection with siC or siE. Scale bars: 50 μm. (c) MTT (3‐(4,5‐dimethilthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay, after 0, 24, 48, and 72 h post‐transfection with siC or siE. One‐way ANOVA *P < 0.05. n = 4. (d) Viability percentage after 72 h of post‐transfection with siC or siE. One‐way ANOVA **P < 0.001, n = 3, (e) Percentage of apoptotic cells after 72 h of post‐transfection with siC or siE. One‐way ANOVA *P < 0.05, n = 3. For (d and e), gray columns represent control cells without transfection (untreated); white columns represent cells transfected with control siRNA (siC); black columns represent cells transfected with ETV5 siRNA (siE), and vertical lines columns represent control cells incubated 48 h in PBS. All results are presented as the mean ± SD.
Fig. 3
Fig. 3
E‐twenty‐six variant 5 (ETV5) expression and its target genes CCND2 (cyclin D2), S6K1, E2F1, p27 Cdkn1b , p21 Cdkn1a , p57 Cdkn1c , PAK3, and FOS. (a) mRNA relative gene expression in transfected cells with control siRNA (siC) and ETV5 siRNA (siE). Student's t‐test, **P < 0.001, ***P < 0.0001, n = 4. (b) Immunoblot for ETV5, E2F1, and β‐Actin, in presence (siC) or ETV5 partial absence (siE). (c) Protein levels analysis of ETV5 and E2F1 normalizing to β‐Actin. Student's t‐test, *P < 0.05, n = 3. (d) p27Cdkn1b immunodetection in control siRNA (siC) and ETV5 siRNA (siE) transfected cells. (e) Protein levels analysis of ETV5 and p27Cdkn1b normalized to β‐Actin. Student's t‐test, *P < 0.05, n = 3. All experiments were analyzed at 72 h post‐transfection with siRNA control or siRNA ETV5 (siC or siE). All results are presented as the mean ± SD.
Fig. 4
Fig. 4
E‐twenty‐six variant 5 (ETV5) partial silencing modulates cell proliferation in INS‐1 (832/13) without modifying apoptosis or viability. ETV5 regulates cell proliferation by increasing gene expression of inhibitors of the cyclin/CDKs complexes (p21 Cdkn1a , p27 Cdkn1b , and p57 Cdkn1c ) and E2F1, and downregulation of cell cycle activators (PAK3 and FOS).
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