Research On The Epigenetic Regulatory Mechanisms Of YY1 Transcriptional Factor On Autophagy In Breast Cancer

BackgroundBC (Breast Cancer) has the highest morbidity in female malignancies all over the world. Autophagy is a self-proteolytic process that degrades intracellular material to enable cellular survival under unfavorable conditions. Increasing evidence in the recent years shows that aberrant autophagy is involved in carcinogenesis with mechanism largely unknown. Furthermore, YY1 (Yin Yang 1) transcription factor is a well-known epigenetic regulator and is upregulated in many cancers. However, the mechanism how YY1 regulates autophagy in BC cells has not been reported ever.ObjectiveOverexpression of YY1 is observed in various cancers. Recent study suggests that YY1 functions as an oncogene via cell cycle arrest, inhibition of apoptosis and cellular differentiation. However, it remains poorly understood how YY1 exhibits its effects on autophagy. We hope to elucidate whether YY1 is implicated in autophagy to achieve its biological function as a proto-oncogene in BC carcinogenesis and development through a series of experiments in vitro and in vivo.MethodsThe expression of YY1 in cell level and clinical tissue level was detected via Western Blot and Immunohistochemistry with the correlation of YY1 and SQSTM1 calculated. Detecting with Western Blot, fluorescence microscope and MTS in cells treated with proteasome inhibitor and different autophagy inhibitors after knockdown of YY1 in BC cell lines MCF7 and T47D, we confirmed that YY1 deletion blocked but not activated autophagy flux via the regulation of SQSTM1 post-transcriptionally. We analyzed the possible candidate miRNAs that target SQSTM1 through some biological informational software, detecting miRNA expression using qPCR. ChIP (Chromatin Immunoprecipitation) and luciferase system assay were utilized to identify the direct binding of YY1 to MIR372. MSP (Methylation specific PCR) and methylated Med-DNA IP (Methylated DNA Immunoprecipitation) were conducted to determine the effects of YY1 on the methylation level of MIR372 promoter. To further determine the long-term effect of MIR372 and YY1 on BC cells, we constructed stable YY1 knockdown cell line. We also established nude mice model to further verify the role of YY1 in cancer cell growth in vivo.ResultsIn this study we firstly demonstrated that YY1 could function as an oncogene through regulating autophagy in BC cells. Compared to breast epithelial cells and normal breast tissues, YY1 expression was upregulated in BC cells as well as in primary carcinoma tissues. In vivo and in vitro experiments suggested that YY1 and SQSTM1 contributed to the survival of BC cells. Besides, YY1 positively correlated with SQSTM1 in protein level (p<0.05). However, neither protein stability nor mRNA level of SQSTM1 was affected by YY1. Indeed, we identified that MIR372 could be the only YY1-regulating candidate miRNA that targeted SQSTM1 meanwhile. YY1 deletion brought about the decreased cell viability, blocked autophagy and SQSTM1-downregulation. The phenotype of MIR372 overexpression was consistent with the one of YY1 knockdown while YY1 and miRNA 372 double-knockdown could rescue the phenotype caused by YY1 deletion only. Finally, time course experiment was done to elucidate the YY1-MIR372-SQSTM1 regulatory axis in EBSS induced autophagy and the carcinoma promoting role of YY1 in BC.ConclusionsYY1 could act as an important epigenetic modulator of autophagy to promote BC carcinogenesis and development via YY1-MIR372-SQSTM1 axis.