He Dongmei, Fang Baoying
Institute of Hematology, Medical College of Jinan University, Guangzhou, China.
Acta Biochim Biophys Sin (Shanghai). 2009 Feb;41(2):131-6. doi: 10.1093/abbs/gmn015.
Bcl-2, a prominent member of the Bcl-2 family proteins, is responsible for the dysregulation of apoptosis and resistance to chemotherapy. In this study, we investigated whether small hairpin RNA (shRNA) targeting at Bcl-2 mRNA could enhance cytarabine (Ara-C)-induced apoptosis in Raji cells. Bcl-2 shRNA was transfected into Raji cells and the expression levels of Bcl-2 mRNA and protein were assayed by RT-PCR and immunofluorescence. Cell proliferation was determined by MTT assay. Apoptosis was determined by morphological observation and flow cytometric analysis. Our results show that expression levels of Bcl-2 mRNA and protein from Raji cells transfected with Bcl-2 shRNA decreased, compared with either negative control shRNA group or untransfected cells group (P < 0.05). Viability of cells transfected with Bcl-2 shRNA was less than the cells transfected with control shRNA and untransfected Raji cells, respectively (P < 0.05). Bcl-2 shRNA combined with Ara-C significantly inhibited the growth of cells (P < 0.05). There was no difference in cell survival between control shRNA/Ara-C combination and cells treated with Ara-C alone. Using Giemsa staining, cells treated with Bcl-2 shRNA plus Ara-C at 48 h displayed changes of apoptosis. Apoptotic rates of Raji cells treated with Bcl-2 shRNA combined with Ara-C significantly increased (P < 0.05), compared with either control shRNA/Ara-C combination or Ara-C-treated cells alone. Our results suggest that the shRNA against Bcl-2 mRNA could increase Ara-C-induced apoptosis in Raji cells.
Bcl-2是Bcl-2家族蛋白中的重要成员，它负责细胞凋亡的失调以及对化疗的抗性。在本研究中，我们调查了靶向Bcl-2 mRNA的小发夹RNA（shRNA）是否能增强阿糖胞苷（Ara-C）诱导的Raji细胞凋亡。将Bcl-2 shRNA转染到Raji细胞中，通过RT-PCR和免疫荧光检测Bcl-2 mRNA和蛋白的表达水平。通过MTT法测定细胞增殖。通过形态学观察和流式细胞术分析确定细胞凋亡。我们的结果表明，与阴性对照shRNA组或未转染细胞组相比，转染了Bcl-2 shRNA的Raji细胞中Bcl-2 mRNA和蛋白的表达水平降低（P < 0.05）。转染Bcl-2 shRNA的细胞活力分别低于转染对照shRNA的细胞和未转染的Raji细胞（P < 0.05）。Bcl-2 shRNA与Ara-C联合使用显著抑制细胞生长（P < 0.05）。对照shRNA/Ara-C组合与单独用Ara-C处理的细胞之间的细胞存活率没有差异。使用吉姆萨染色，在48小时用Bcl-2 shRNA加Ara-C处理的细胞显示出凋亡变化。与对照shRNA/Ara-C组合或单独用Ara-C处理的细胞相比，用Bcl-2 shRNA联合Ara-C处理的Raji细胞的凋亡率显著增加（P < 0.05）。我们的结果表明，针对Bcl-2 mRNA的shRNA可以增加Ara-C诱导的Raji细胞凋亡。
