Down-regulation of the autophagy gene, ATG7, protects bone marrow-derived mesenchymal stem cells from stressful conditions

Abstract

Background: Mesenchymal stem cells (MSCs) are valuable for cell-based therapy. However, their application is limited owing to their low survival rate when exposed to stressful conditions. Autophagy, the process by which cells recycle the cytoplasm and dispose of defective organelles, is activated by stress stimuli to adapt, tolerate adverse conditions, or trigger the apoptotic machinery. This study aimed to determine whether regulation of autophagy would affect the survival of MSCs under stress conditions.

Methods: Autophagy was induced in bone marrow-derived MSCs (BM-MSCs) by rapamycin, and was inhibited via shRNA-mediated knockdown of the autophagy specific gene, ATG7. ATG7 expression in BM-MSCs was evaluated by reverse transcription polymerase chain reaction (RT-PCR), western blot, and quantitative PCR (qPCR). Cells were then exposed to harsh microenvironments, and a water-soluble tetrazolium salt (WST)-1 assay was performed to determine the cytotoxic effects of the stressful conditions on cells.

Results: Of 4 specific ATG7-inhibitor clones analyzed, only shRNA clone 3 decreased ATG7 expression. Under normal conditions, the induction of autophagy slightly increased the viability of MSCs while autophagy inhibition decreased their viability. However, under stressful conditions such as hypoxia, serum deprivation, and oxidative stress, the induction of autophagy resulted in cell death, while its inhibition potentiated MSCs to withstand the stress conditions. The viability of autophagy-suppressed MSCs was significantly higher than that of relevant controls (P<0.05, P<0.01 and P<0.001).

Conclusion: Autophagy modulation in MSCs can be proposed as a new strategy to improve their survival rate in stressful microenvironments.

Keywords: Autophagy; Cell survival; Mesenchymal stem cells; Oxidative stress; shRNA.

Fig. 1. Confirmation of autophagy suppression via ATG7 gene silencing using shRNA. (A) Fluorescence microscopic observation 48 hours after transfection of hBM-MSCs with ATG7 shRNA clone 3 containing a GFP vector. Intrinsic GFP fluorescence and green cytoplasm in MSC-shRNA 3 confirmed the transfection. (B) RT-PCR analysis of transfected hBM-MSCs for selection of the most suppressive vector. Following transfection of hBM-MSCs with ATG7 shRNA suppression vectors, ATG7 expression was analyzed by RT-PCR. ATG7 was effectively downregulated in MSC-shRNA 3 cells. shRNA clone 3 was selected as a proper vector for autophagy suppression in hBM-MSCs. Expression of β-actin was also evaluated for normalization. (C) qPCR of ATG7 demonstrated that ATG7 was expressed at a very low level in MSC-shRNA 3 compared with MSC-shRNA Cont. (Data represented Mean±SD, ^a)P<0.001).

Fig. 2. Confirmation of autophagy induction with rapamycin. (A) Western blot analysis of 24 hour-rapamycin-treated hBM-MSCs exposed to 10, 200, and 500 nM rapamycin. LC3-I induction and conversion of LC3-I to LC3-II were determined by western blot analysis. Expression of LC3-II and its significantly increased level compared to LC3-I in MSCs-500 nM Rapa indicated induction of autophagy in these cells. β-actin served as the loading control. (B) Microscopic observation of transfected hBM-MSCs before and after rapamycin treatment. hBM-MSCs were transfected with GFP-LC3 containing a GFP vector (MSC-LC3), and 48 hours later, MSC-LC3 was treated with 500 nM rapamycin for 24 hours (MSC-LC3-Rapa). Fluorescence microscopic observations indicated that MSC-LC3-Rapa manifested green dots (punctate signal) (B.I), which confirmed effective autophagy induction in MSC-LC3-Rapa compared to MSC-LC3 (B.II). Autophagy was observed in MSC-shRNA Cont-Rapa cells, which were transfected with a negative control shRNA vector and treated with rapamycin (shiny green dots) (B.III). Very few green dots were detected in MSC-shRNA 3-Rapa cells, which were treated with rapamycin after suppression of autophagy (B.IV). (C) Western blot analysis of shRNA 3-transfected hBM-MSCs before and after rapamycin treatment. hBM-MSCs were treated with 500 nM rapamycin for 24 hours following transfection with ATG7-shRNA clone 3 (ATG7 suppressive vector) and the negative control shRNA, and subjected to western blot analysis.

Fig. 3. WST-1 assay to detect the effects of autophagy on hBM-MSC survival. (A) Survival rates of MSC-shRNA3 (autophagy-suppressed MSCs), MSC-Rapa (autophagy-induced MSCs) and the relevant control groups (MSC-shRNA Cont and MSC-Cont) were assayed at daily intervals during a five-day treatment. (B) Both autophagy modulated and control hBM-MSCs were exposed to different H₂O₂ concentrations for 1 hour. The viability of MSC-shRNA 3 was higher than MSC-Rapa and controls (MSC-Cont and MSC-shRNA Cont) at 2-4 mM H₂O₂. (C) After exposing the experimental groups to serum deprivation for different time durations, we found that the suppression of autophagy in MSC-shRNA 3 renders them more resistant to severe SD stress compared to other groups. (D) Following 8 and 24-hour-duration of hypoxia, ATG7-shRNA knockdown protected the hBM-MSCs from cell death compared to other groups. This demonstrated that autophagy knockdown enhances survival of hBM-MSCs under severe persistent stress conditions (Data represented Mean±SD; ^a)P<0.05, ^b)P<0.01 and ^c)P<0.001).