Yang Xiao-Yun, Yu Hai, Lin Wei, Peng Zhi-Lan
Department of Obstetrics and Gynecology, West China Second Hospital, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2009 Jan;40(1):6-10.
To investigate the effect of ARLTS1 (ADP-ribosylation factor-like tumor suppressor gene 1) on the growth and apoptosis of ovarian cystadenocarcinoma cell line SKOV3.
The recombinant plasmid pCMV-Tag 3B-ARLTS1 was constructed and transfected into SKOV3 cells by liposome protocol. The expression of ARLTS1 mRNA and its protein were examined by RT-PCR and Western blotting. Effects of ARLTS1 gene on growth and apoptosis of the transfected cells were evaluated by MTT assay and flow cytometry (FCM). The alterations of caspase-3 and bcl-2 protein levels were examined by Western blotting.
The stable transfection of pCMV-Tag 3B-ARLTS1 in SKOV3 cells were obtained and verified by RT-PCR and Western blotting methods. After transfected with ARLTS1, more SKOV3 cells were observed in S phase by FCM compared with those in the other two groups (P is 0.035 and 0.011, respectively). The 36.7% apoptotic index of ARLTS1 transfected cell was significantly higher than that of the other two groups (P < 0.001). The growth of ARLTS1 transfected cells was dramatically inhibited compared with cells transfected with Vector (P < 0.05). Western blotting indicated a significantly decrease in caspase-3 and bcl-2 protein levels in cells transfected with ARLTS1 compared with SKOV3 cells (P is 0.021 and 0.013, respectively).
ARLTS1 transfection can inhibit proliferation and induce apoptosis of SKOV3 cells in vitro and down-regulate the expression of caspase-3 and bcl-2. ARLTS1 gene may be a candidate tumor suppressor gene in ovarian cancer.
研究ADP核糖基化因子样肿瘤抑制基因1（ARLTS1）对卵巢囊腺癌SKOV3细胞生长及凋亡的影响。
构建重组质粒pCMV-Tag 3B-ARLTS1，采用脂质体法转染至SKOV3细胞。通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测ARLTS1 mRNA及其蛋白的表达。采用噻唑蓝（MTT）比色法和流式细胞术（FCM）评估ARLTS1基因对转染细胞生长及凋亡的影响。通过蛋白质免疫印迹法检测半胱天冬酶-3（caspase-3）和bcl-2蛋白水平的变化。
采用RT-PCR和蛋白质免疫印迹法证实了pCMV-Tag 3B-ARLTS1在SKOV3细胞中的稳定转染。转染ARLTS1后，FCM检测显示，与其他两组相比，S期的SKOV3细胞增多（P值分别为0.035和0.011）。ARLTS1转染细胞的凋亡指数为36.7%，显著高于其他两组（P<0.001）。与载体转染细胞相比，ARLTS1转染细胞的生长受到显著抑制（P<0.05）。蛋白质免疫印迹法显示，与SKOV3细胞相比，ARLTS1转染细胞中caspase-3和bcl-2蛋白水平显著降低（P值分别为0.021和0.013）。
ARLTS1转染可在体外抑制SKOV3细胞增殖并诱导其凋亡，下调caspase-3和bcl-2的表达。ARLTS1基因可能是卵巢癌的候选抑癌基因。
