miR-221促进神经母细胞瘤SH-SY5Y细胞N-MYC表达和细胞增殖

miR-221 promotes N-MYC expression and cell proliferation in neuroblastoma SH-SY5Y cells

目的探讨过表达miR-221对神经母细胞瘤细胞SH-SY5Y中N-MYC表达及其细胞增殖能力的影响。方法构建miR-221过表达慢病毒载体并建立稳定转染细胞系,实验设未处理组、空载组和miR-221组,以实时定量荧光PCR(RT-q PCR)检测miR-221表达;以RT-q PCR和Western blot检测N-MYC mRNA及蛋白表达;流式细胞术检测细胞周期;CCK8法检测细胞增殖。结果经测序证实慢病毒载体构建成功,与未处理的SH-SY5Y细胞相比,稳定表达miR-221的SH-SY5Y细胞(miR-221组)miR-221表达升高约7倍(P<0.01);miR-221组N-MYC mRNA和蛋白表达水平较未处理组显著增多(P<0.01);miR-221组G0/G1期细胞比例明显减少(P<0.05),S期细胞比例显著增多(P<0.05);miR-221组细胞增殖能力明显增强(P<0.01)。结论过表达miR-221能够上调神经母细胞瘤SH-SY5Y细胞中N-MYC的表达,并可能通过促发细胞从G0/G1期向S期转化促进细胞增殖。

Objective To explore the impact of miR-221 overexpression on the expression of N-MYC level and the cell proliferation in neuroblastoma SH-SY5 Y cells. Methods miR-221 overexpression lentiviral vector was constructed and its stable transfection cells were established. The expression of miR-221 was detected by RT-q PCR.The N-MYC mRNA and protein expression were analyzed by RT-q PCR and Western blot respectively. The distribution of cell cycle was tested by flow cytometry. The cell proliferation was measured by CCK8 assay. Results The recombinant vectors were verified by DNA Sanger sequencing. The results of RT-q PCR indicated that the miR-221 expression in cells transfected with miR-221 lentiviral vector as seven times as SH-SY5 Y cells（ P 〈0. 01）,with no difference between control and SH-SY5 Y cells. Compared with SH-SY5 Y cells,the mRNA and protein in N-MYC dramatically increased in cells transfected with miR-221（ P 〈0. 01）. The results of Flow cytometrydemonstrated that miR-221 significantly decreased G1-S checkpoint arrest（ P〈 0. 05）. The results from CCK8 assay showed that overexpression of miR-221 promoted cell proliferation（ P 〈0. 01）. Conclusions miR-221 may contribute to the enhancement of N-MYC and cell proliferation by rescue of G1-S checkpoint arrest in neuroblastoma SH-SY5 Y cells.