Oct4 is a transcription factor that is required for pluripotency during early embryogenesis and the maintenance of embryonic stem (ES) cell and pluripotent cell identity. miR-302, a cluster of eight microRNAs (miRNAs) that are expressed specifically in ES cells and pluripotent cells, is crucial for normal pluripotent cell self-renewal and pluripotency. But, the mechanism by which miR-302 participates in the core regulatory circuitry that controls self-renewal and pluripotency in P19 embryonic carcinoma cells has not been established. Here, we show that Oct4 is required for the expression and transcriptional activation of miR-302 and that Oct4 binds to the putative promoter of miR-302, suggesting that Oct4 activates the primary miR-302 transcript in P19 cells. This study proposes that the miR-302 cluster acts downstream of the Oct4 regulation network in P19 cells.
Resistance to chemotherapy is a primary problem for the effective treatment of ovarian cancer. Recently, increasing evidence has demonstrated that miRNAs modulate many important molecular pathways involved in chemotherapy. Previous studies demonstrated that miR-199a affected ovarian cancer cell resistance to cisplatin (DDP). However, the role of miR-199a and its target genes in determination of ovarian cancer sensitivity to DDP remains unclear. Quantitative reverse transcription polymerase chain reaction was used to detect the expression levels of miR-199a in ovarian cancer tissues and C13* and OV2008 cell lines. After transfection of miR-199a mimic or inhibitor, flow cytometry was used to detect cell apoptosis exposed to DDP. Enzyme-linked immunosorbent assay and Western blot assay were applied to detect tumor necrosis factor-α levels and protein expression levels of Bax, Fas, Fas-associated death domain, and caspase-8. The results indicated that the expression of miR-199a was downregulated and hypoxia-inducible factor 1α (Hif1α) upregulated in the ovarian tumors compared with those in the corresponding normal tissues. Besides, the expression levels of miR-199a were significantly higher in OV2008 cells compared with those in C13* cells. Moreover, suppression of Hif1α reversed the inhibiting function of miR-199a inhibitor on DDP-induced apoptosis in the OV2008 cells. However, overexpression of both miR-199a and Hif1α reduced DDP-induced apoptosis in C13* cells. In conclusion, miR-199a may change DDP resistance in ovarian cancer by regulating Hif1α.
To investigate the relationship between the expression of early growth response gene 1 (EGR-1) and p38MAPK pathway in the paclitaxel resistance of ovarian carcinoma cells, the effect of p38MAPK inhibitor SB203580 on cell apoptosis was examined by using Hoechst 33258 staining. The intracellular Rh123 (Rhodamine 123) accumulation was detected by the flow cytometry (FCM). The 50% inhibition concentration (IC50) of paclitaxel for A2780/Taxol cells was determined by MTT method. Electrophoretic motility shift assay (EMSA) was employed to examine the EGR-1DNA binding activity. MDR1 and EGR-1 mRNA were assessed by RT-PCR. The expressed of p-gp, phosphorylated p53 and p38 were detected by Western blotting. SB203580 could remarkably promote the apoptosis of A2780/Taxol cells, and the cell apoptosis was in a time-dependent manner. Cellular Rh123 accumulation was increased, and the IC50 of paclitaxel for A2780/Taxol cells was decreased significantly. A2780/Taxol cell line after SB203580 treatment was shown to have a significantly higher level of EGR-1 DNA binding activity. SB203580 down-regulated the activity of p38MAPK pathway, but up-regulated EGR-1 expression. SB203580 significantly increased the level of cellular phosphorylated p53 protein, but decreased the p-gp protein level and MDR1 mRNA level in A2780/Taxol cells. There existed a close relationship between p38MAPK pathway and the paclitaxel resistance of ovarian carcinoma cells. The expression of EGR-1 mediated by p38MAPK pathway plays a critical role in paclitaxel resistance of ovarian carcinoma cells.
Cross-reacting material 197 (CRM197), a specific HB-EGF inhibitor, has been proven to be a promising antitumor agent for ovarian cancer therapy. Our previous studies have shown that CRM197 has potent antitumor activity in human cisplatin-resistant ovarian cancer. However, the relationship between CRM197 and the resistance to cisplatin remains unclear. Here, we report that CRM197 significantly reverses the resistance to cisplatin in cisplatin-resistant ovarian carcinoma cell line (A2780/CDDP). We established xenograft nude mice models with A2780 and A2780/CDDP cells. Notably, we observed that CRM197 suppresses the expression of HB-EGF and epidermal growth factor receptor in A2780/CDDP cells and xenografts harboring the overexpression of HB-EGF and epidermal growth factor receptor. Experiments conducted in vitro and in vivo suggest that CRM197 markedly downregulates the expression of excision repair cross-complementing group 1 (P = 0.002) and DNA repair capacity in A2780/CDDP tumor (P < 0.001) by inactivation of extracellular signal-regulated kinase signaling, providing novel possible mechanisms for the ability of CRM197 to restore drug sensitivity. These results suggest that CRM197 as an HB-EGF inhibitor might be a cisplatin-chemosensitizing agent for the treatment of ovarian carcinoma with resistance to cisplatin.
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been proven to be a promising chemotherapeutic target for ovarian cancer. Our previous studies have demonstrated that inhibition of HB-EGF by the special inhibitor, cross-reacting material 197 (CRM197), potently inhibits the anti-tumor activity in paclitaxel-resistant ovarian cancer. Here, we found that inhibition of HB-EGF by CRM197 significantly reverses the resistance to paclitaxel in paclitaxel-resistant ovarian carcinoma cell line (A2780/Taxol). A2780/Taxol cells over-expressed HB-EGF and epidermal growth factor receptor (EGFR) and CRM197 notably suppressed the expression of HB-EGF and EGFR. Experiments performed in vitro and in vivo further suggested that CRM197 markedly down-regulated the ATP-binding cassette sub-family B member 1 (ABCB1/MDR1) messenger RNA (mRNA) expression (P = 0.01), plasma membrane glycoprotein (P-gp) protein (P = 0.009), and P-gp-mediated efflux (P = 0.007) through inhibition of nuclear factor-κB (NF-κB) expression, which were classical chemoresistance-related targets with respect to paclitaxel therapy. Meanwhile, inhibition of HB-EGF enhanced caspase-3 activity to induce apoptosis via MDR1 inhibition in A2780/Taxol cells (P = 0.038). Collectively, HB-EGF is a molecular target for the resistance of ovarian cancer to paclitaxel and CRM197 as a HB-EGF-targeted agent might be a chemosensitizing agent for paclitaxel-resistant ovarian carcinoma. Our findings provide novel possible mechanisms for HB-EGF to be a target to restore the chemosensitivity to paclitaxel.
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