Fig 1: E2F1 is predicted to regulate hMSH2. (a) Two predicted binding sites of E2F1 upstream of transcription start site (TSS) of hMSH2. (b) Correlation between expression of E2F1 and hMSH2 in samples of epithelial ovarian cancer (EOC) from the Cancer Genome Atlas (TCGA) database. TPM, transcript per million and (c) Correlation of E2F1 and hMSH2 mRNA levels in samples from 77 patients with EOC.
Fig 2: Overexpression of E2F1 increased susceptibility of ovarian cancer cells to cisplatin. (a) Percentage of viable cells (OVCAR-3 and SKOV-3) with or without E2F1 overexpression incubated with cisplatin (5–30 μM) after 24 h and (b and c) Percentage of apoptotic cells with or without E2F1 overexpression counted by flow cytometry after cisplatin treatment. Con, control; OE, over expressed. *P < 0.05 and **P < 0.01.
Fig 3: E2F1 levels in tissues from patients with ovarian cancer correlated with prognosis following platinum-based therapy. (a) mRNA levels of E2F1 and hMSH2 in platinum sensitive or resistant patients were statistically significantly different. (b) Immunohistochemistry confirmed that higher E2F2 and hMSH2 protein levels were associated with platinum sensitivity and (c) The progression-free survival (PFS) and overall survival (OS) curves of patients with high or low levels of E2F1. The median PFS of patients with E2F1high was 29 months, compared with 9.5 months for patients with E2F1low. Patients with E2F1high had a more favourable median survival (34 months) compared with patients with E2F1low (23 months). The differences between the two groups were statistically significant.
Fig 4: E2F1 regulates the expression of hMSH2 in ovarian cancer cells. (a) E2F1 knockdown resulted in hMSH2 under-expression. E2F1 was knocked down in OVCAR-3 and SKOV-3 cells and the mRNA (left, middle) and protein (right) levels of hMSH2 were also downregulated correspondingly. GADPH, glyceraldehyde 3-phosphate dehydrogenase; si-NC, random control; si-E2F1, E2F1 knockdown. *P < 0.05, **P < 0.01, and ***P < 0.001. (b) E2F1 overexpression resulted in hMSH2 upregulation. E2F1 was overexpressed in OVCAR-3 and SKOV-3 cells, and hMSH2 was also upregulated correspondingly at mRNA (left) and protein (right) levels. GFP, green fluorescent protein; GADPH, glyceraldehyde 3-phosphate dehydrogenase; NC: empty vector control; OE-E2F1, E2F1 overexpression. *P < 0.05 and **P < 0.01. (c) Luciferase showed the regulation of E2F1 on the hMSH2 promoter. The promoter region of hMSH2 was subcloned into a reporter vector and the luciferase signal intensity altered in coordination with the E2F1 level. *P < 0.05, **P < 0.01, and ***P < 0.001 and (d) Electrophoretic Mobility Shift Assay showing binding of E2F1 to the promoter region of hMSH2. Synthesized 146 bp promoter region of hMSH2 was incubated with 50, 100, and 150 ng recombinant E2F1 and separated in native gel. Upper bands show the shifted mobility of DNA/E2F1 complex.
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