YY1-MIR372-SQSTM1 regulatory axis in autophagy

Abstract

Autophagy is a self-proteolytic process that degrades intracellular material to enable cellular survival under unfavorable conditions. However, how autophagy is activated in human carcinogenesis remains largely unknown. Herein we report an epigenetic regulation of autophagy in human cancer cells. YY1 (YY1 transcription factor) is a well-known epigenetic regulator and is upregulated in many cancers. We found that YY1 knockdown inhibited cell viability and autophagy flux through downregulating SQSTM1 (sequestosome 1). YY1 regulated SQSTM1 expression through the epigenetic modulation of the transcription of MIR372 (microRNA 372) which was found to target SQSTM1 directly. During nutrient starvation, YY1 was stimulated to promote SQSTM1 expression and subsequent autophagy activation by suppressing MIR372 expression. Similar to YY1 depletion, MIR372 overexpression blocked autophagy activation and inhibited in vivo tumor growth. SQSTM1 upregulation and competent autophagy flux thus contributed to the oncogenic function of YY1. YY1-promoted SQSTM1 upregulation might be a useful histological marker for cancer detection and a potential target for drug development.

Keywords: MIR372; SQSTM1; YY1; autophagy; epigenetics.

Figure 1. YY1 is implicated in autophagy. (A) YY1 expression in breast cancer cells and MCF-10A cells was determined by western blot analysis. (B) YY1 expression in human mammary tissues was determined by immunohistochemistry staining. (C) The Fisher exact test was used for statistical analysis of staining results (P = 0.046). (D) After 72 h of transfection, cell viability of MCF7 or T47D cells transfected with or without YY1 siRNAs was determined with the MTS assay. The asterisks indicate significant statistic difference (P < 0.05). (E) In vivo growth of MCF cells was evaluated by tumorigenecity assay in nude mice. The curve of tumor volumes and the weight of the tumor were shown in the left and right panels, respectively. The asterisk indicates significant statistic difference (P < 0.05). (F) After 72 h of transfection, LC3 expression in MCF7 and T47D cells transfected with or without YY1 siRNAs was explored by western blot analysis. The values in the blot were relative quantification of the indicated band/GAPDH. (G) The effect of different autophagy inhibitors (5 mM 3-MA and 50 nM BafA1 for 24 h before lysate preparation) on YY1 siRNA-induced LC3-II accumulation in MCF7 cells was analyzed by western blot analysis. Different exposure times of LC3 were used to show clear LC3 in control (less LC3, longer exposure) or BafA1 treated cells (more LC3, shorter exposure). (H) mRFP-GFP-LC3 distribution in MCF7 and T47D cells transfected with mRFP-GFP-LC3 and YY1 or control siRNAs were analyzed by confocal microscopy. All experiments were repeated 3 times and the representative results were shown. The bottom panel indicates that quantification of LC3 puncta numbers. The asterisks indicate significant statistic difference (P < 0.05).

Figure 2. YY1 regulates SQSTM1 expression. (A) The expression of SQSTM1 in MCF7 and T47D cells transfected with or without YY1 siRNAs was explored by western blot analysis after 72 h of transfection. (B) The expression of SQSTM1 in MCF10A cells transfected with YY1 expression vector was explored by western blot analysis. (C and D) SQSTM1 expression in primary mammary tissues was analyzed as in Figure 1B and C (Chi-Square test, P < 0.001). (E and F) The correlation between YY1 and SQSTM1 expression was analyzed by the Chi-Square test (P = 0.012). The representative photographs are shown in (E).

Figure 3. Neither protein stability nor mRNA level of SQSTM1 was affected by YY1. The effect of YY1 siRNA on the expression of SQSTM1 in cells treated with the autophagy inhibitor 3-MA (5mM) (A) or the proteasome inhibitor MG132 (20 µM) (B) 24 h before cells were harvested was analyzed by western blot analysis. (C) The viability of MCF7 cells transfected with exogenous SQSTM1 and YY1 siRNA was determined by the MTS assay after 72 h of transfection. (D) mRFP-GFP-LC3 distribution in MCF7 cells as in (C) was analyzed by confocal microscopy after 72 h of transfection. (E) Exogenous SQSTM1 and LC3 expression in MCF7 cells as in (C and D) was measured by western blotting after 72 h of transfection. (F) The expression of SQSTM1 in MCF7 cells treated with or without YY1 siRNA was determined by real-time RT-PCR after 72 h of transfection. (G) The expression of SQSTM1 in MCF10A cells transfected with or without the YY1 expression-vector was determined by real-time RT-PCR after 72 h of transfection. The asterisks indicate significant statistic difference (P < 0.05).

Figure 4. YY1 regulates SQSTM1 protein expression through MIR372. (A) The expression of mature MIR372 in MCF7 cells treated with or without YY1 siRNA were determined by real-time RT-PCR after 72 h of transfection. (B) The expression of mature MIR372 in MCF10A cells transfected with or without the YY1 expression-vector was determined by real-time RT-PCR after 72 h of transfection. (C) The effect of MIR372 mimic (C) and MIR372 inhibitor (D) on the expression of SQSTM1 and LC3 was analyzed by western blotting after 72 h of transfection. (E) Cell viability of MCF7 cells transfected with YY1 siRNAs and MIR372 inhibitor was determined by the MTS assay after 72 h of transfection. (F) The effect of MIR372 on the expression of the luciferase reporter driven by the SQSTM1 3′ UTR (SQSTM1UTR) fragment with or without MIR372-binding sites was determined by the luciferase activity assay. The asterisks indicate significant statistic difference (P < 0.05).

Figure 5.MIR372 is a direct target of YY1. (A) There are 2 YY1-binding sites in the upstream of MIR372 transcription start site. (B) pri-MIR372 expression in MCF7 cells treated with or without YY1 siRNA was determined by real-time RT-PCR after 72 h of transfection. (C) pri-MIR372 expression in MCF10A cells transfected with or without the YY1 expression-vector was determined by real-time RT-PCR after 72 h of transfection. (D) The effect of YY1 on the expression of the luciferase reporter driven by the upstream sequence of MIR372 (MIR372p) with or without YY1-binding sites (M1: site 1 was mutated, M2: site 2 was mutated, 2M: both sites were mutated) were determined by the luciferase activity assay. (E) The binding of YY1 to the MIR372 gene was determined by ChIP assay. (F) Pri-MIR372 expression in MCF7 cells treated for 48 h with DMSO or 5–5-aza-2′-deoxycytidine (10 µM) was determined by real-time RT-PCR. G, DNA methylation status of the MIR372 promoter region in MCF7 cells transfected with or without YY1 siRNA were determined by MSP (G) and the methylated DNA IP assay (H) after 72 h of transfection. The asterisks indicate significant statistical difference (Student t test, P < 0.05).

Figure 6. YY1 downregulates MIR372 to promote SQSTM1 expression and autophagy activation upon nutrient starvation. (A) The time course of expression of SQSTM1, YY1 in MCF7 cells cultured with EBSS was measured by western blotting. The time course of expression of primary (B, Kruskal-Wallis test, P = 0.0337) and mature (C, Kruskal-Wallis test, P = 0.0187) MIR372 in MCF7 cells cultured with EBSS was determined by real-time RT-PCR. One-way ANOVA test (and the Kruskal-Wallis test) were used to calculate the statistical difference. (D) The effect of EBSS incubation on LC3 in MCF7 cells with or without MIR372 expression was analyzed by western blotting. (E) The effect of EBSS incubation on mRFP-GFP-LC3 distribution in MCF7 cells with or without MIR372 expression was analyzed by confocal microscopy after 48 h of mRFP-GFP-LC3 plasmid transfection. (F–H) The in vivo tumorigenecity of MCF7 cells with or without MIR372 expression was determined by nude mice xenograft assay. The asterisks indicate significant statistic difference (P < 0.05).

Figure 7. YY1 stimulates SQSTM1 expression to activate autophagy, through the inhibition of MIR372 expression. The autophagy regulator SQSTM1 is directly modulated by MIR372 which can be epigenetically downregulated by YY1. Under nutrient deprivation, YY1 is accumulated to stimulate SQSTM1 and promote autophagy activation.