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    2020-08-28

    r> HMGA2, a chromatin-binding protein, contains three AT-hook do-mains that enable its binding to the minor groove of DNA, which allows it to organize protein complexes on enhancers of various genes reg-ulating EMT gene expression (e.g., Snail and Twist) [16,34] and cell differentiation [19,35]. HMGA2 overexpression is associated with ag-gressive tumor growth, early metastasis, and poor prognosis [36,37], whereas knockdown of HMGA2 suppresses tumor metastatic invasion, homing and osteolysis [38]; thus, the HMGA2 gene is a promising target
    Fig. 4. Bach1 promotes the metastasis of the epithelial ovarian cancer cell A2780 in vivo. A. The exact loci of the CRISPR/Cas-9 genomic target within the Bach1 gene nucleotide sequences. B. Western blot validation of the construction of stable Bach1 knockout and Bach1 overexpression cell lines. C-D. Metastatic tumors in the liver (C) and diaphragm (D). To establish the mouse model of dissemination, 5 × 106 A2780 cells were injected into nude mice by the i.p. route, resulting in tumor growth. Left: Representative images of metastatic tumors in the liver and diaphragm. Right: Number and volume of metastatic tumors in the liver and diaphragm. n = 8. t-test. E. IHC staining for Bach1 and Slug in mouse liver. Higher expression of Bach1 in metastatic tumors of the liver was associated with increased levels of Slug. Scale bar, 50 μm. t-test.
    for ovarian cancer-silencing therapy [37]. Both HMGA2 and Bach1 are targets of let-7 [31], which is regulated by RKIP, but the relationship between HMGA2 and Bach1 is unknown. We found that HMGA2 is a binding partner of Bach1 in the EOC cell line and that Bach1 recruits HMGA2 to occupy the Lipo2000 of the promoter regions of EMT-related genes. The effect of Bach1 overexpression on EMT-related genes and cell migration in the EOC cell line can be partially abolished by HMGA2 silencing, indicating that Bach1 promotes EMT and cell 
    motility at least in part via HMGA2. Interestingly, the enhancement of CXCR4 expression by Bach1 does not seem to be mediated by HMGA2. Moreover, we observed that Bach1 expression was positively correlated with HMGA2 expression in human ovarian cancer tissues. Taken to-gether, these results suggest that Bach1 plays an important role in ovarian cancer metastasis via HMGA2-mediated EMT. Both HMGA2 and Bach1 are targets of let-7 in breast cancer cells [39]. We found that HMGA2 knockdown repressed the protein expression of Bach1 in the
    Fig. 5. Correlation of Bach1 with Slug and HMGA2 in human ovarian cancers. A. Comparison of the protein expression of Bach1 and EMT-related genes in normal ovarian tissues and EOC samples by western blotting. n = 5. **P < 0.01 vs normal. t-test. BeC. The protein expression of Slug and HMGA2 was determined by immunostaining in 5 normal ovary samples, 21 samples of early-stage EOC (stage I or II), and 9 samples of advanced-stage EOC (stage III or IV). The stage of EOC was defined using the International Federation of Gynecology and Obstetrics (FIGO) staging systems. B: Representative images of immunostaining for Slug (upper) and HMGA2 (lower). Scale bar, 200 μm. C: The IHC scores for Slug and HMGA2 in EOC and normal ovarian tissues. ANOVA with post hoc test. D. Correlation analysis. Bach1 expression was positively correlated with Slug and HMGA2 expression in human ovarian cancers. Pearson's coefficient tests were performed to assess statistical significance.
    EOC cell line (Fig. 3F). It is possible that HMGA2 positively regulates Bach1 transcription. Future studies are expected to elucidate the re-lationship between HMGA2 and Bach1. Bach1 serves as an oncogene to promote tumor progression. Bach1 overexpression in human colorectal cancer accelerated the growth of tumor xenografts in vivo [10], and deficiencies in Bach1 expression have been associated with decreases in proliferation and migration of a human breast adenocarcinoma cell line [40]. Both of these observations 
    are consistent with the results from our studies: higher levels of Bach1 expression were associated with increases in the proliferation of the EOC cell line and tumor xenograft growth in nude mice. Our results show that Bach1-mediated cell growth was not affected by HMGA2 silencing, indicating that HMGA2 is not required for the tumor cell growth induced by Bach1. We observed that Bach1 increased the ex-pression of p-AKT, p-p70S6K and cyclin D1 in both the EOC cell line and xenograft tumors in mice; these increases may be responsible for the
    **P < 0.01 vs Con-siRNA. t-test. C: t-test. D. Comparison by western blotting of protein levels of AKT pathway proteins and Cyclin D1 between AdBach1-cells and AdGFP-cells as well as between Bach1-siRNA cells and Con-siRNA-cells. The data are representative of three independent experiments. NS, not significant. t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)