Here we explored the role of Bach in epithelial ovarian
Here, we explored the role of Bach1 in epithelial ovarian cancer (EOC). We found that Bach1 was highly expressed and positively cor-related with Slug and HMGA2 expression in EOC and that higher levels of Bach1 were associated with invasive progression and poorer prog-nosis in human ovarian cancer. Bach1 promoted EMT and EOC me-tastasis in a mouse model. Mechanistically, Bach1 interacted with HMGA2 and promoted cell migration by recruiting HMGA2 in the human EOC cell line A2780. In addition, Bach1 increased the expres-sion of p-AKT, p-p70S6K and cyclin D1 and promoted the growth of ovarian cancer FUB-NPB22 and tumor xenografts. Thus, our findings identify Bach1 as a key transcriptional regulator of ovarian cancer growth and metastasis.
2. Materials and methods
The epithelial ovarian cancer cell line A2780 and human embryonic kidney (HEK) 293T cells were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS, Life Technologies, Inc., Burlington, ON, Canada) and glutamine or in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS and antibiotics. Normoxic incubation (CO2 water-jacketed incubator; Thermo Electron, Waltham, MA) was performed at 37 °C in 5% CO2 with 95% humidity.
2.2. Transfection and establishment of a stable cell line
For the in vitro study, Bach1 expression in A2780 cells was tran-siently downregulated by small interfering RNA transfection, desig-nated Bach1-siRNA (with Con-siRNA as the corresponding control), or upregulated by Bach1-adenovirus infection, designated Ad-Bach1 (with Ad-GFP as the corresponding control). A2780 cells were transfected with Bach1 siRNA or negative control siRNA (100 nmol/L) using Lipofectamine 2000 transfection reagent according to the manufac-turer's instructions (Invitrogen, Carlsbad, CA, USA). Forty-eight hours after transfection, the cells were used for diﬀerent subsequent experi-ments. Recombinant adenoviruses encoding the human Bach1 gene (Ad-GFP-Bach1) or the GFP control (Ad-GFP) were purchased from GenePharma (Shanghai, China) and used to infect A2780 cells. For transduction, a multiplicity of infection (MOI) of 25 was used. To elu-cidate the molecular mechanisms underlying Bach1-mediated tumor cell migration and growth, a mixture of 3 pairs of HMGA2-siRNA or negative control siRNA was transfected into Ad-Bach1 A2780 cells. The siRNA sequences targeting Bach1 or HMGA2 in this study are listed in Supplementary Table 1.
For the in vivo study, Bach1 expression in A2780 cells was stably knocked out using the CRISPR/Cas9 genome editing technology, de-signated KO (with WT as the corresponding control), or overexpressed by Bach1-lentivirus, designated Lenti-Bach1 (with Lenti-Con as the corresponding control). The sgRNAs targeting Bach1 were designed Cancer Letters 445 (2019) 45–56
using CRISPR DESIGN (http://crispr.mit.edu/). Then, sgRNA expres-sion vectors (lenti crispr V2) were constructed and transfected into A2780 cells (5 × 105/well in 24-well plates) with Lipofectamine 2000. Twenty-four hours after transfection, puromycin (1 μg/ml) was added to select stably transfected cells. Positive clones were identified by se-quencing the gene of interest and confirmed by analyzing protein le-vels. Similarly, Lenti-Bach1-transfected cells were isolated with pur-omycin selection, and clones were chosen for use in subsequent experiments.
Cell proliferation was evaluated by cell counting. A2780 cells were transfected with Bach1 siRNA (100 nmol/L) or control siRNA (100 nmol/L) using Lipofectamine 2000 transfection reagent and cul-tured for 48 h. Ad-GFP or Ad-Bach1 was used to infect the cells, and the cells were then transfected with HMGA2 siRNA (100 nmol/L) or control siRNA (100 nmol/L) for 24 h. Cells were seeded in 12-well plates (7 × 104 cells/well) and cultured for the indicated time periods; the cells were then counted with a cell counting chamber as described previously . Each experiment was performed independently three times.
2.4. Soft agar colony formation
The anchorage-independent growth ability of cells was evaluated by a soft agar colony formation assay. In this assay, a bottom layer of 1.2% agar in complete media (1:1) was poured into 30-mm-diameter culture dishes and solidified, followed by an upper layer containing 1 × 103 cells suspended in a mixture of medium with 0.6% agar (1:1). After two weeks of incubation, the colonies were stained with a 0.5% crystal violet solution, photographed and counted using image analysis software .
2.5. Wound healing assay
In brief, 1.0 × 106 A2780 cells were seeded into each well of 6-well plates and incubated to form a confluent monolayer. Forty-eight hours after transient transfection with Con-siRNA, Bach1-siRNA, Ad-GFP or Ad-Bach1, the cell monolayer was scraped in a straight line with a p200 pipette tip to create a “scratch;” the debris was removed, and the edge of the scratch was smoothed by washing the cells twice with 1 ml of PBS. Then, the cells were cultured with serum-free medium and imaged at 0, 24, and 48 h after wounding. The wound area was calculated by manually tracing the cell-free area in the captured images using ImageJ public domain software (NIH, Bethesda, MD, USA) .