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目的观察Hsa-miR-15a/16-1靶向抑制B细胞淋巴瘤/白血病-2基因(B-cell lymphoma gene 2,Bcl-2gene)表达对鼻咽癌(nasopharyngeal carcinoma,NPC)CNE-2细胞的生长抑制作用。方法将构建成功的重组质粒pENTR-CMV-EGFP-hsa-mir-15a/16-1经Lipofectamine 2000转染CNE-2细胞,RT-qPCR检测miR-15a/16-1、Bcl-2 mRNA表达水平,流式细胞术和WesternBlot检测Bcl-2、caspase-3蛋白表达水平,CCK-8法分析细胞生长抑制情况,4,6-二脒基-2-苯基吲哚(4,6-diamidino-2-phenylindole,DAPI)染色观察细胞凋亡状况。结果转染后miR-15a表达增高(F=547.525,P均<0.001);miR-16-1表达增高(F=99.979,P均<0.001);Bcl-2 mRNA表达无明显差别(F=0.220,P>0.05);Bcl-2蛋白表达下调,caspase-3蛋白表达增高;细胞在体外凋亡明显;细胞增殖受到抑制,作用呈明显的时效关系。结论 Hsa-miR-15a/16-1对Bcl-2基因的靶向抑制,可体外对CNE-2细胞产生增殖抑制和促进凋亡作用。
Objective To investigate the effect of Hsa-miR-15a / 16-1 on inhibiting the expression of Bcl-2 gene in CNE-2 cells of nasopharyngeal carcinoma (NPC) Growth inhibitory effect. Methods The recombinant plasmid pENTR-CMV-EGFP-hsa-mir-15a / 16-1 was transfected into CNE-2 cells by Lipofectamine 2000. The expression of miR-15a / 16-1 and Bcl- , The expression of Bcl-2 and caspase-3 protein were detected by flow cytometry and Western Blot. The cell growth inhibition was analyzed by CCK-8 method. The 4,6-diamidino- 2-phenylindole, DAPI) staining to observe the apoptosis status. Results The expression of miR-15a was significantly increased after transfection (F = 547.525, P <0.001), and the expression of miR-16-1 was higher (F = 99.979, P <0.001) , P> 0.05). The expression of Bcl-2 protein was down-regulated and the expression of caspase-3 protein was increased. The apoptosis of cells was obvious in vitro. The cell proliferation was inhibited. Conclusion The targeted inhibition of Bcl-2 gene by Hsa-miR-15a / 16-1 can inhibit proliferation and promote apoptosis of CNE-2 cells in vitro.