微小RNA-449a靶向B细胞淋巴瘤／白血病-2抑制神经母细胞瘤细胞增殖

MicroRNA- 449a inhibits neuroblastoma cell proliferation by down- regulating B cell lymphoma/leukemia - 2 expression

目的检测微小RNA（miRNA，miR）449a对神经母细胞瘤细胞株SK—N—SH增殖的影响并探讨其作用机制。方法人工设计合成miR449a模拟物，构建上调miR449a表达的细胞株SK-N-SH；实时定量反转录聚合酶链反应（1iT-qPCR）检测转染后miR-449a的表达水平；转染48h后采用细胞计数试剂盒（CCK-8）法检测细胞24、48、72h的增殖，膜联蛋白V（Annexin-V）／碘化丙锭（PI）法检测细胞凋亡；利用生物信息学方法预测miR-449a的候选靶基因。结果转染miR-449amimic的细胞中miR449a的表达水平升高3．5倍；过表达miR-49a后细胞生长速度较对照组细胞明显减慢；过表达miR449a后细胞凋亡明显增加，细胞总凋亡比例为（21．94±3．37）％，而对照组细胞的总凋亡比例为（11．01±3．51）％，两者差异有统计学意义（P〈0．05）；通过靶基因预测软件分析，抗凋亡基因B细胞淋巴瘤／自血病-2（bcl-2）是miR-449a的靶基因，过表达miR-449amimic后，内源性bcl-2蛋白的表达水平显著下降。结论miR449a可能通过调控bcl-2的转录水平抑制神经母细胞瘤细胞增殖，促进凋亡。

Objective To investigate the effect of microRNA （ miRNA, miR） - 449a on prolifera- tion and apoptosis of neuroblastoma SK - N - SH ceils and the mechanism. Methods SK - N - SH cells were transfected with mimic control and miR -449a mimic. The expression level of miR -449a was deter- mined by real -time quantitative reverse transcriptase -polymerase chain reaction （RT -qPCR）. Cell pro- liferation at 24, 48 and 72 h was examined by cell counting kit - 8 （ CCK - 8 ） kit after 48 h of transfec- tion. Cell apoptosis was examined by annexin- V/ropidium iodide （PI）. The putative targets for miR - 449a were predicted using bioinformatics. Results RT - qPCR analysis confirmed that miR - 449a was upregulated 3.5 times in miR -449a mimic cells than in mimic control cells, miR -449a over - ex- pression inhibited cells growth at 24, 48, and 72 h, respectively as compared with mimic control cells. miR - 449a over - expression could promote cell apoptosis with apoptosis rate being （ 21.94 ± 3.37 ） % , which was twofold higher than mimic control cells [ （ 11.01±3.51）% ,P 〈0. 05]. B cell lymphoma/leu- kemia-2 （bcl -2）, an antiapoptotic molecule, was identified to be a directly target of miR -449a. En- dogenous protein expression of bcl - 2 was inhibited by miR - 449a overexpression in SK - N - SH cells. Conclusion miR- 449a inhibited tumor growth, and promoted tumor apoptosis through targeting bcl -2 in human neuroblastoma.