p53/microRNA-374b/AKT1 regulates colorectal cancer cell apoptosis in response to DNA damage.

Colorectal cancer (CRC) has a rising morbidity worldwide and its resistance to chemotherapy has been observed in clinical treatment. Tumor suppressor p53 is well-studied in CRC, but little is known about its effects during DNA damage of CRC cells. This study was aimed at uncovering potential mechanisms of p53 regarding microRNA-374b and v-akt murine thymoma viral oncogene homolog 1 (AKT1) during DNA damage of CRC cells. CRC cells HCT116 and HT29 were transfected with p53-specific small interfering RNA (siRNA), p53 overexpression vector or miR-374b inhibitor, and then treated with 10 µM bleomycin (BLM) for 24 h to induce DNA damage. Primary (pri), precursor (pre) and mature miR-374b levels were quantified by qRT-PCR. AKT1 and p53 protein levels were detected by western blotting. Cell apoptosis changes were assessed by flow cytometry. AKT1 mRNA was detected to be induced by BLM treatment (P<0.05), but its protein level was strongly inhibited. Knockdown of p53 reversed the inhibition of AKT1 protein by BLM. Overexpression of p53 in p53-knockout HCT116 and HT29 cells upregulated the AKT1 regulator miR-374b (P<0.05), and knockdown of p53 reversed the induction of miR-374b by BLM. qRT-PCR suggested that besides mature miR-374b, p53 could also promote pre-miR-374b level (P<0.05), rather than pri-miR-374b. Moreover, inhibition on miR-374b relieved the suppressed AKT1 protein, and reduced cell apoptosis induced by BLM. These data depict the p53/miR-374b/AKT1 signaling that may regulate BLM-induced apoptosis in CRC cells, thus facilitating to improve the outcome of chemotherapy in CRC.