MicroRNA-26b下调MALAT-1抑制乳腺癌MCF-7细胞恶性生物学行为的机制研究

MicroRNA-26b inhibits the malignant biological behavior of MCF-7 breast cancer cells by down-regulating MALAT-1

摘要:

目的 探索microRNA-26b（miR-26b）通过MALAT-1抑制乳腺癌MCF-7细胞恶性生物学行为的分子机制。方法 以乳腺癌细胞系MCF-7作为研究对象，采用慢病毒LV-miR-26b-ctrl和LV-miR-26b转染肿瘤细胞，逆转录聚合酶链反应检测MALAT-1、miR-26a和miR-26b的mRNA表达，平板克隆形成实验、CCK-8细胞增殖实验、软琼脂成瘤实验、细胞划痕实验、Transwell细胞侵袭实验分别检测肿瘤细胞的增殖、体外成瘤、迁移和侵袭能力。结果 E2组与Mock组MCF-7细胞24、48、72和96 h时吸光度值比较，经重复测量设计的方差分析，结果 ①不同时间点吸光度值比较，差异有统计学意义（P <0.05）；②两组吸光度值比较，差异有统计学意义（P <0.05），与Mock组比较，E2组72和96 h时细胞增殖能力较Mock组提高（P <0.05）；③两组吸光度值变化趋势比较，差异有统计学意义（P <0.05）。与Mock组相比，E2组MALAT-1相对表达量升高（P <0.05），miR-26a、miR-26b mRNA相对表达量下降（P <0.05）。高表达miR-26b的乳腺癌患者的生存情况优于低表达miR-26b组，死亡风险更低（P <0.05），低表达miR-26a组患者的生存情况优于高表达miR-26a组（P <0.05），用Cox比例风险回归模型，将miR-26a作为一个独立的预后因素来比较，两组患者的死亡风险比较无差异（P >0.05）。与LV-miR-26b-ctrl组比较，LV-miR-26b-ctrl/E2组乳腺癌细胞的MALAT-1相对表达量升高（P <0.05），与LV-miR-26b-ctrl/E2组比较，LV-miR-26b/E2组乳腺癌细胞的MALAT-1相对表达量降低（P <0.05）。LV-miR-26b-ctrl组、LV-miR-26b-ctrl/E2组、LV-miR-26b/E2组细胞在24、48、72和96 h时吸光度值比较，经重复测量设计的方差分析，结果 ①不同时间点吸光度值比较，差异有统计学意义（P <0.05）；②各组吸光度值比较，差异有统计学意义（P <0.05），LV-miR-26b-ctrl/E2组72和96 h时细胞增殖能力明显较LV-miR-26b-ctrl组增强（P <0.05），LV-miR-26b/E2组72和96 h时细胞增殖能力较LV-miR-26b-ctrl/E2组降低（P <0.05）；③各组吸光度值变化趋势比较，差异有统计学意义（P <0.05）。E2处理后的LV-miR-26b-ctrl肿瘤细胞增殖和体外成瘤能力增强。与LV-miR-26b-ctrl/E2组比较，LV-miR-26b/E2组肿瘤细胞的增殖和体外成瘤能力降低。在24 h时，LV-miR-26b-ctrl/E2组细胞划痕间隙较LV-miR-26b-ctrl组变窄，LV-miR-26b/E2组24 h时细胞的划痕间隙较LV-miR-26b-ctrl/E2组明显增宽。LV-miR-26b-ctrl/E2组Transwell小室下方的乳腺癌细胞数量较LV-miR-26b-ctrl组增多，LV-miR-26b/E2组的肿瘤细胞数量较LV-miR-26b-ctrl/E2组减少。结论 下调MALAT-1可能是miR-26b抑制乳腺癌恶性生物学行为的分子机制之一，miR-26b有望成为乳腺癌治疗的调控靶点。

Abstract:

Objective To explore the molecular mechanism of microRNA-26b (miR-26b) inhibiting the malignant biological behavior of MCF-7 breast cancer cells through MALAT-1.Methods MCF-7 breast cancer cell line was transduced with lentivirus-based vectors LV-miR-26b-ctrl and LV-miR-26b, and expressions of MALAT-1, miR-26a and miR-26b were measured by RT-PCR. The colony formation assay, CCK-8 assay, soft agar colony formation assay, scratch assay, and Transwell invasion assay were performed to detect the proliferation ability, in vitro tumorigenic capability, and migration and invasion abilities, respectively.Results The optical density values of MCF-7 cells at 24 h, 48 h, 72 h and 96 h in the E2 group and the Mock group were compared via the repeated measures analysis of variance, which demonstrated that they were different among the time points (P < 0.05) and between the two groups (P < 0.05), and that the proliferation ability of cells at 72 h and 96 h in the E2 group was higher than that in the Mock group (P < 0.05). There were differences in the change trends of the optical density values between the two groups (P < 0.05). Compared with the Mock group, the expression of MALAT-1 in the E2 group was higher (P < 0.05), and the expressions of miR-26a and miR-26b were lower (P < 0.05). Compared with the miR-26b low-expression group, the position of the survival curve of patients with breast cancer was higher in the miR-26b high-expression group, indicating a lower risk of death (P < 0.05). Compared with the miR-26a low-expression group, the position of the survival curve of patients with breast cancer was lower in the miR-26a high-expression group (P < 0.05). However, the risk of death was not significantly different between the two groups when including miR-26a as an independent prognostic factor in the Cox regression model (P > 0.05). The expression of MALAT-1 in breast cancer cells of the LV-miR-26b-ctrl/E2 was higher than that in the LV-miR-26b-ctrl group (P < 0.05), while the expression of MALAT-1 in breast cancer cells of the LV-miR-26b/E2 group was lower than that in the LV-miR-26b-ctrl/E2 (P < 0.05). The optical density values of cells at 24 h, 48 h, 72 h and 96 h in the LV-miR-26b-ctrl group, LV-miR-26b-ctrl/E2 group, and LV-miR-26b/E2 group were compared via the repeated measures analysis of variance, and the results showed that they were different among the time points (P < 0.05) and among the groups (P < 0.05). The proliferation ability of cells at 72 h and 96 h in the LV-miR-26b-ctrl/E2 group was higher than that in the LV-miR-26b-ctrl group (P < 0.05), and the proliferation ability of cells at 72 h and 96 h in the LV-miR-26b/E2 group was lower than that in the LV-miR-26b-ctrl/E2 group (P < 0.05). There were differences in the change trends of the optical density values of cells among the groups (P < 0.05). After E2 treatment, the proliferation ability and in vitro tumorigenic capability of cancer cells in the LV-miR-26b-ctrl group were enhanced. Compared with the LV-miR-26b-ctrl/E2 group, the proliferation ability and in vitro tumorigenic capability of cancer cells in the LV-miR-26b/E2 group were weakened. At 24 h, the gap in the scratch assay in the LV-miR-26b-ctrl/E2 group was narrower than that in the LV-miR-26b-ctrl group, while the gap in the scratch assay in the LV-miR-26b/E2 group was wider than that in the LV-miR-26b-ctrl/E2. The number of breast cancer cells in the bottom chamber of the Transwell in the LV-miR-26b-ctrl/E2 group was higher than that in the LV-miR-26b-ctrl group, whereas that in the LV-miR-26b/E2 group was lower compared with the LV-miR-26b-ctrl/E2 group.Conclusions Down-regulation of MALAT-1 may be one of the mechanisms underlying the role of miR-26b in inhibiting the malignant biological behavior of MCF-7 breast cancer cells, and miR-26b holds promise as a target for breast cancer therapy.