下调HOXB7基因对卵巢癌SKOV3细胞增殖的影响及机制探讨

Silencing of HOXB7 by RNAi influences proliferation and relative mechanism of ovarian serous adenocarcinoma cell line SKOV3

背景与目的:HOXB7基因在卵巢癌中过表达,可能与细胞恶性转化相关。本研究采用RNA干扰技术下调HOXB7基因表达,并探讨其对卵巢浆液性囊腺癌SKOV3细胞增殖的影响及可能的机制。方法:将SKOV3细胞分为3组:空白组(未作转染处理)、阴性对照组(转染阴性质粒pRNAT-neg)和HOXB7siRNA干扰组(转染干扰质粒pRNAT-hoxb7)。荧光显微镜观察转染效率,RT-PCR、蛋白质印迹法(Western blot)检测干扰效应。BrdU实验检测细胞增殖;流式细胞仪测定细胞周期、凋亡的变化;Western blot方法检测与细胞周期相关蛋白CyclinD1、Cyclin E的表达变化。结果:转染干扰质粒pRNAT-hoxb7到SKOV3细胞后,HOXB7基因的表达受到高效且特异的抑制(P<0.01);细胞增殖量减少;G1期阻滞,Cyclin D1、Cyclin E表达下降;细胞凋亡率增加。结论:HOXB7基因的表达下调可抑制SKOV3细胞增殖,其机制可能与转录下调细胞周期相关蛋白Cyclin D1、CyclinE,使细胞周期阻滞在G1期有关;同时可促进其凋亡。

Background and purpose： The expression ofHOXB7 gene was higher expressed in ovarian cancer and may be involved in the cellular malignant transformation.This study used the sequence-specific siRNA knocking down the expression of HOXB7 gene and aimed to investigate its effect on cell proliferation and relative mechanism of ovarian serous adenocarcinoma cell line SKOV3. Methods： SKOV3 cells were divided into 3 groups： untreated control group, non-specific siRNA group transfected with unrelated siRNA （pRNAT-neg） and HOXB7siRNA groups transfected with HOXB7s-iRNA （pRNAT-hoxb7）. RT-PCR, Western blot were used to examine the HOXB7mRNA and protein expression. Cell cycle and apoptosis were analyzed by flow cytometry. We further investigated the expression of Cyclin D1, Cyclin E by Western blot, which were critical in regulating cell proliferation. Results： The pRNAT-hoxb7 siRNA had high inhibition for HOXB7 gene （P〈0.01）. HOXB7 RNAi could also arrest of G1 cell cycle, induce inhibition of cell proliferation and increase apoptosis rate. Western blot showed the expression of Cyclin D1, Cyclin E decreased after HOXB7 expression downregulated. Conclusion： Down-regulated HOXB7 expression could inhibit the expression of Cyclin D1, Cyclin E and thus is involved in the G1 arrest and may increase the apoptosis rate of SKOV3 Cells.