慢病毒介导的microRNA-19b表达对鼻咽癌细胞凋亡及Wnt/β-catenin信号的影响

Effect of miR-19b gene expression mediated by lentivirus on apoptosis and Wnt/β-catenin signal in nasopharyngeal carcinoma cells

摘要:

目的 研究慢病毒介导的microRNA-19b（miR-19b）表达对鼻咽癌细胞凋亡及Wnt/β-catenin信号的影响。方法 实时荧光定量聚合酶链反应（qRT-PCR）检测鼻咽癌组织及人永生化鼻咽上皮细胞系NP69和鼻咽癌细胞系HONE1、5-8F和CNE1中miR-19b表达。构建miR-19b抑制表达（miR-19b inhibitor）、miR-19b过表达和无意义序列（Scrambled）的慢病毒载体（miR-19b mimics），并设定空白组，转染鼻咽癌细胞系HONE1，qRT-PCR检测慢病毒转染细胞后细胞中miR-19b的表达；MTT法检测慢病毒转染细胞24、48和72?h后的细胞活力；流式细胞仪检测慢病毒转染细胞48?h的细胞凋亡率；Western blotting检测 ki-67、p53、β-连环蛋白（β-catenin），Cyclin D1和C-myc的蛋白表达。结果 miR-19b在鼻咽癌组织及细胞中表达升高（P?<0.05）。Scrambled组与空白组比较，差异无统计学意义（P?>0.05），与空白组比较，miR-19b inhibitor组miR-19b的表达降低，miR-19b mimics组miR-19b的表达升高（P?<0.05）；与空白组比较，miR-19b inhibitor组在48和72 h的细胞活力降低，而miR-19b mimics组在48和72?h的细胞活力升高（P?<0.05）；与空白组比较，miR-19b inhibitor组细胞凋亡率及p53的表达升高，ki-67、β-catenin、Cyclin D1和C-myc的表达降低，miR-19b mimics组细胞凋亡率及p53的表达降低，ki-67、β-catenin、Cyclin D1和C-myc的表达升高（P?< 0.05）。结论 抑制miR-19b表达可降低鼻咽癌细胞活力，诱导细胞凋亡及下调Wnt/β-catenin信号通路，而过表达miR-19b则反之。

Abstract:

Objective To study the effect of miR-19b gene expression mediated by lentivirus on apoptosis and Wnt/β-catenin signal in nasopharyngeal carcinoma cells. Methods The expression of miR-19b in nasopharyngeal carcinoma tissues and human immortalized nasopharyngeal epithelial cell lines NP69 and nasopharyngeal carcinoma cell lines HONE1, 5-8F and CNE1 was detected by qRT-PCR. MiR-19b inhibition expression (miR-19b inhibitor), miR-19b overexpression and meaningless sequence (scrambled) lentivirus vector (miR-19b mimics) were constructed, and a blank group was set. Cells were transfected into the EBV negative nasopharyngeal carcinoma cell line HONE1, and RT-PCR was used to detect the expression of miR-19b in the transfected cells; cell viability was measured by MTT method at 24, 48 and 72 h after transfection; cell apoptosis was detected by flow cytometry at 48 h after transfection; the expression of Ki-67, p53, β-catenin, cyclin D1 and c-myc protein were detected by Western blotting. Results The expression of miR-19b in nasopharyngeal carcinoma tissues and cells increased significantly (P??0.05); compared with the control group, the expression of miR-19b was decreased significantly in miR-19b inhibitor group, miR-19b expression increased significantly in miR-19b mimics group (P?