Hsa-miR-15a/16-1靶向抑制Bcl-2基因对鼻咽癌CNE-2细胞的抑制作用

Inhibitory effect of nasopharyngeal carcinoma CNE-2 cells through targeted inhibitory effect on the Bcl-2 gene expression by Hsa-mir-15a/16-1

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 observe the inhibitory effect of Targeted inhibition of Bcl-2 gene expression by Hsa-miR-15a/16-1 on the growth of nasopharyngeal carcinoma CNE-2 cells.METHODS The recombinant plasmid pENTR-CMV-EGFP-hsa-mir-15a/16-1 which had been successfully constructed was transfected into CNE-2 cells with the help of Lipofectamine 2000.The expression of miR-15a/16-1,Bcl-2 mRNA was detected by RT-qPCR.The levels of Bcl-2 and Caspase-3 Protein were measured by Western blot and FCM.The inhibitory effect of Bcl-2 gene on growth of CNE-2 cells was detected by CCK-8.Apoptosis of the CNE-2 cells was observed by analyzing of nucleus morphology with DAPI pigmentation.RESULTS After transfection of the recombinant plasmid,the expression of both miR15a and miR-16-1 increased significantly（F=547.525,P 0.001;F =99.979,P 0.001）,the expression of Bcl-2 mRNA had no significant difference（F=0.220,P 0.05）,the expression of Bcl-2 protein decreased,the expression of activated Caspase-3 protein increased.Apoptosis of CNE-2 cell in vitro was obvious.Meanwhile the cell proliferation was inhibited after transfection of the recombinant plasmid,and the effects showed an obvious time-effect relationship.CONCLUSION Targeted inhibition of Bcl-2 gene expression by Hsa-miR-15a/16-1 could inhibit cells proliferation and promote apoptosis on human nasopharyngeal carcinoma CNE-2 cells in vitro.