Department of Pathology, the Affiliated Hospital of Zunyi Medical University, Zunyi 563003, Guizhou Province, China.
World J Gastroenterol. 2020 Feb 7;26(5):499-513. doi: 10.3748/wjg.v26.i5.499.
MicroRNA 34c (miR-34c) has been reported to be associated with malignant types of cancer, however, it remains unknown whether miR-34c is involved in chemoresistance in gastric cancer (GC).
To investigate the effect of miR-34c and its upstream transcription factor E2F1 on paclitaxel combined with cisplatin resistance in GC cells.
Paired GC tissues and adjacent normal tissues were randomly sampled from 74 GC patients. miR-34c and E2F1 were detected by real-time quantitative PCR (qPCR) and Western blot. In addition, the drug resistance of GC cells to paclitaxel and cisplatin was induced by concentration gradient increasing methods, and changes in miR-34c and E2F1 during this process were measured. Furthermore, E2F1 and miR-34c overexpression or underexpression vectors were constructed and transfected into drug-resistant GC cells. MTT was employed to test the sensitivity of cells to paclitaxel combined with cisplatin, qPCR was adopted to detect the expression of miR-34c, Western blot was applied to detect the expression levels of E2F1, drug resistance-related proteins and apoptosis-related proteins, and flow cytometry was used for the determination of cell apoptosis and cell cycle status.
E2F1 was overexpressed while miR-34c was underexpressed in GC. After inducing GC cells to be resistant to paclitaxel and cisplatin, E2F1 expression increased while miR-34c expression decreased. Both silencing E2F1 and over-expressing miR-34c could increase the sensitivity of drug-resistant GC cells to paclitaxel combined with cisplatin, promote cell apoptosis and inhibit cell proliferation. Among which, silencing E2F1 could reduce the expression of drug resistance-related proteins and apoptosis-related proteins, while over-expression of miR-34c could upregulate the expression of apoptosis-related proteins without affecting the expression of MDR-1, MRP and other drug resistance-related proteins. Rescue experiments demonstrated that inhibiting miR-34c could significantly weaken the sensitization of drug resistant cells, and Si E2F1 to paclitaxel combined with cisplatin.
E2F1 inhibits miR-34c to promote the proliferation of GC cells and enhance the resistance to paclitaxel combined with cisplatin, and silencing E2F1 is conducive to improving the efficacy of paclitaxel combined with cisplatin in GC cells.
MicroRNA 34c（miR-34c）已被报道与恶性癌症类型相关，但miR-34c 是否参与胃癌（GC）的化疗耐药性尚不清楚。
研究 miR-34c 及其上游转录因子 E2F1 对 GC 细胞中紫杉醇联合顺铂耐药性的影响。
从 74 例 GC 患者中随机抽取配对的 GC 组织和相邻正常组织。实时定量 PCR（qPCR）和 Western blot 检测 miR-34c 和 E2F1。此外，采用浓度梯度递增法诱导 GC 细胞对紫杉醇和顺铂的耐药性，并在此过程中测量 miR-34c 和 E2F1 的变化。进一步构建 E2F1 和 miR-34c 过表达或低表达载体，并转染至耐药 GC 细胞中。MTT 法检测细胞对紫杉醇联合顺铂的敏感性，qPCR 法检测 miR-34c 的表达，Western blot 法检测 E2F1、耐药相关蛋白和凋亡相关蛋白的表达水平，流式细胞术检测细胞凋亡和细胞周期状态。
GC 中 E2F1 过表达而 miR-34c 低表达。诱导 GC 细胞对紫杉醇和顺铂耐药后，E2F1 表达增加，miR-34c 表达减少。沉默 E2F1 和过表达 miR-34c 均可增加耐药 GC 细胞对紫杉醇联合顺铂的敏感性，促进细胞凋亡，抑制细胞增殖。其中，沉默 E2F1 可降低耐药相关蛋白和凋亡相关蛋白的表达，而过表达 miR-34c 可上调凋亡相关蛋白的表达，而不影响 MDR-1、MRP 等耐药相关蛋白的表达。挽救实验表明，抑制 miR-34c 可显著削弱耐药细胞对紫杉醇联合顺铂的敏感性，而 Si E2F1 则可增强其敏感性。
E2F1 抑制 miR-34c 促进 GC 细胞增殖，增强对紫杉醇联合顺铂的耐药性，沉默 E2F1 有利于提高 GC 细胞中紫杉醇联合顺铂的疗效。
