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

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mi R-19b表达。构建mi R-19b抑制表达(mi R-19b inhibitor)、mi R-19b过表达和无意义序列(Scrambled)的慢病毒载体(mi R-19b mimics),并设定空白组,转染鼻咽癌细胞系HONE1,q RT-PCR检测慢病毒转染细胞后细胞中mi R-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,mi R-19b inhibitor组mi R-19b的表达降低,miR-19b mimics组mi R-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mi R-19b mimics组在48和72 h的细胞活力升高(P <0.05);与空白组比较,mi R-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,mi R-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mi R-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。

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, 5-8 F 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);there was no significant difference between the Scrambled group and the control group(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 < 0.05);compared with the control group, cell viability were significantly reduced in miR-19b inhibitor group in 48 h and 72 h, while cells activity increased significantly in miR-19b mimics group(P < 0.05);compared with the control group, the apoptosis rate of cells and p53 expression were significantly increased in miR-19b inhibitor group, the expression of Ki-67, β-cateninc were decreased significantly, the apoptosis rate of cells and p53 expression were decreased significantly in miR-19b mimics group, and the expression of Ki-67, β-catenin, cyclin D1 and c-myc were increased significantly(P < 0.05). Conclusions Inhibition of miR-19b expression can reduce the viability of nasopharyngeal carcinoma cells, induce apoptosis and down regulate the signaling pathway of Wnt/β-catenin, and the overexpression of miR-19b is opposite.