PTEN knockout leads to premature senescence of human endometrial stromal cells

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One of the defense mechanisms against neoplastic transformation of cells in response to oncogenic stimuli is cellular senescence. However, the ability of cells to activate this defense reaction depends on their nature and is not inherent in all cell types. Within the present study, we investigated reaction of human endometrial stromal cells (EnSC) towards classical oncogenic stimulus – PTEN inactivation. By using CRISPR/Cas9 genome editing technology, we generated EnSC line with PTEN knockout. We showed that reduced PTEN expression results in proliferation loss, cell hypertrophy, accumulation of lipofuscin and disturbed redox balance. Together these data favors senescence induction in PTEN-knockout EnSC. While studying the molecular mechanisms, we established the key role of the PI3K/AKT signaling pathway in the implementation of the EnSC senescence program under conditions of PTEN knockout. Inhibiting this signaling pathway by LY294002 prevented both the phenotypic manifestations of premature senescence and cell cycle arrest in PTEN-knockout EnSC. Thus, the development of premature senescence in response to reduced expression of the oncosuppressor PTEN can be considered as a protective mechanism that prevents malignant transformation of EnSC.

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Sobre autores

P. Parfenova

Institute of Cytology RAS

Email: borodkina618@gmail.com

Группа механизмов клеточного старения

Rússia, St. Petersburg, 194064

P. Deryabin

Institute of Cytology RAS

Email: borodkina618@gmail.com

Группа механизмов клеточного старения

Rússia, St. Petersburg, 194064

D. Pozdnyakov

Institute of Cytology RAS

Email: borodkina618@gmail.com

Группа механизмов клеточного старения

Rússia, St. Petersburg, 194064

A. Borodkina

Institute of Cytology RAS

Autor responsável pela correspondência
Email: borodkina618@gmail.com

Группа механизмов клеточного старения

Rússia, St. Petersburg, 194064

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2. Fig. 1. Confirmation of the effectiveness of the PTEN gene knockout system. K – control eSCs with nonsense guideRNA; 1–3 – eSCs carrying a knockout system with three different guideRNAs; a – PTEN gene mRNA expression; PTEN expression values are normalized to the expression level of the reference gene GAPDH. The average values and their standard deviations are presented (n = 3, n is the number of reaction repetitions); ** – differences from control are significant at – p < 0.01 compared to control, ANOVA test with Tukey’s correction; b – PTEN protein expression levels detected using specific antibodies; on the right are the piers. masses; GAPDH was used as a loading control.

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3. Fig. 2. A decrease in PTEN expression leads to the initiation of premature aging in eSCs. K – control eSCs with nonsense guideRNA; a – growth curves; b – average cell size determined by direct light scattering (SL); c – intensity of autofluorescence (AF) of cells, reflecting the accumulation of lipofuscin granules; d – levels of phosphorylation of proteins p53 (pp53) and Rb (pRb), as well as protein expression p21Waf1/Cip1 (p21), detected using specific antibodies; molecular weights are indicated on the right; GAPDH was used as a loading control; e, f – respectively, quantitative assessment of cytochemical staining SA-β-Gal and micrographs; a–c – mean values and standard deviations from three repeat measurements are presented; e – coloring of 100 cells was assessed; *** – differences from control are significant at p < 0.001 (Welch’s t-test).

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4. Fig. 3. Changes in redox balance and mitochondrial functioning in control eSCs with nonsense guideRNA (K) and in eSCs with PTEN gene knockout; a – levels of intracellular ROS determined by fluorescence intensity (FI) of the DCF dye; b – levels of mitochondrial ROS determined by the IF of the DHR123 dye; c – change in mitochondrial membrane potential determined by the IF of the JC-1 dye. Mean values and their standard deviations from triplicate measurements are presented; differences from control are significant at p < 0.001 (***) or p < 0.01 (**); Welch's t-test.

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5. Fig. 4. Activity of the PI3K/AKT signaling pathway in PTEN-regulated induction of eSC aging and its modulation by the LY inhibitor in control eSCs with nonsense guideRNA (K) and in eSCs with PTEN gene knockout (Nok.). Cells with a knockout of the PTEN gene were treated with LY at concentrations of 5 μM (LY 5), 10 μM (LY 10), or 20 μM (LY 20) for 6 days; a – levels of phosphorylation of AKT and Rb, as well as expression of the p21Waf1/Cip1 (p21) protein, detected using specific antibodies; molecular weights are indicated on the right; GAPDH was used as a loading control; b – growth curves; c – average cell size determined by direct light scattering (SL); d – autofluorescence intensity (AF) of cells, reflecting the accumulation of lipofuscin granules. Mean values and their standard deviations from triplicate measurements are presented; differences from the control are significant at p < 0.001 (***) or p < 0.01(**), ANOVA test corrected for multiple comparisons according to Tukey.

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6. Fig. 5. Inhibition of AKT prevents the increase in SA-β-Gal activity in eSCs by knockout of the PTEN gene. Shown are control eSCs with nonsense guideRNA (K), eSCs with PTEN gene knockout (Nok.), and eSCs with PTEN gene knockout treated with 20 μM LY (Nok. + LY20); a, b – microphotographs of eSCs and quantitative assessment of cytochemical staining SA-β-Gal, respectively; 6 days after adding the inhibitor. The average values and their standard deviations are presented (the color of 100 cells was assessed); differences from control are significant at p < 0.001 (***); ANOVA test with Tukey's correction for multiple comparisons.

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