Department of Gastrointestinal Surgery, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, P.R. China.
The First Department of Endoscopy, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, P.R. China.
Int J Oncol. 2017 May;50(5):1785-1791. doi: 10.3892/ijo.2017.3922. Epub 2017 Mar 21.
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.
结直肠癌（CRC）在全球的发病率呈上升趋势，其对化疗的耐药性在临床治疗中已经观察到。肿瘤抑制因子 p53 在 CRC 中研究得较多，但对其在 CRC 细胞 DNA 损伤时的作用知之甚少。本研究旨在揭示 p53 在 CRC 细胞 DNA 损伤过程中与 microRNA-374b 和 v-akt 鼠胸腺瘤病毒癌基因同源物 1（AKT1）相关的潜在机制。将 p53 特异性小干扰 RNA（siRNA）、p53 过表达载体或 miR-374b 抑制剂转染 CRC 细胞 HCT116 和 HT29，然后用 10μM 博来霉素（BLM）处理 24 小时诱导 DNA 损伤。用 qRT-PCR 定量测定原（pri）、前体（pre）和成熟 miR-374b 水平。用 Western blot 检测 AKT1 和 p53 蛋白水平。用流式细胞术评估细胞凋亡变化。BLM 处理诱导 AKT1 mRNA 表达（P<0.05），但其蛋白水平受到强烈抑制。p53 敲低逆转了 BLM 对 AKT1 蛋白的抑制。p53 敲除的 HCT116 和 HT29 细胞中 p53 的过表达上调了 AKT1 调节因子 miR-374b（P<0.05），p53 敲低逆转了 BLM 诱导的 miR-374b 表达。qRT-PCR 表明，p53 除了能促进成熟 miR-374b 外（P<0.05），还能促进 pre-miR-374b 水平（P<0.05），而不能促进 pri-miR-374b。此外，miR-374b 抑制缓解了 BLM 诱导的 AKT1 蛋白抑制，并减少了 BLM 诱导的细胞凋亡。这些数据描绘了 p53/miR-374b/AKT1 信号通路，该通路可能调节 CRC 细胞中 BLM 诱导的细胞凋亡，从而有助于改善 CRC 化疗的效果。
