【摘要】 目的研究人类同源异型盒基因HOXB7小分子干扰RNA(siRNA)对卵巢透明细胞癌ES-2细胞侵袭的影响。方法采用半定量RT-PCR,Western blot印迹方法分别检测卵巢透明细胞癌细胞株ES-2,卵巢浆液性囊腺癌细胞株SKOV3的HOXB7基因mRNA及蛋白质表达水平。将ES-2细胞分为3组:空白组、阴性对照组(转染阴性质粒pRNAT-neg)、HOXB7 siRNA组(转染干扰质粒pRNAT-hoxb7)。荧光显微镜观察转染效率、RT-PCR、Western blot检测干扰效应。Transwell小室检测3组细胞侵袭、运动能力的变化;明胶酶谱实验检测3组细胞上清中基质金属蛋白酶活性;Western blot检测细胞内基质金属蛋白酶-2(MMP-2)变化。结果RT- PCR、Western blot结果显示:HOXB7在卵巢癌SKOV3,ES-2细胞中均阳性表达。转染质粒pRNAT-hoxb7到ES-2细胞后,HOXB7基因的表达受到高效且特异的抑制(P<0．01);细胞侵袭力及趋向运动能力均下降。明胶酶谱、Western blot结果显示HOXB7干扰后,ES-2细胞MMP-2表达下降。结论HOXB7 siRNA可有效抑制卵巢透明细胞癌ES-2细胞HOXB7基因的表达与MMP-2表达,同时可抑制其迁移、侵袭能力,提示HOXB7可能在卵巢癌转移中发挥作用。更多还原

【Abstract】 Purpose To use the sequence-specific siRNA to knock down the expression of HOXB7 gene and to investigate its effect on cell invasiveness of ovarian c-lear cell adenocarcinoma cell line ES-2. Methods Two human ovarian carcinoma cell lines ES-2,SKOV3 were analyzed for the expression of HOXB7 mRNA by RT-PCR and HOXB7protein by Western blot. ES-2 cells were divided into 3 groups: untreated control group,no-specific siRNA group transfected with unrelated siRNA (pRNAT-neg) and HOXB7 siRNA group transfected with HOXB7 siRNA (pRNAT-hoxb7). ES-2 cells were stably transfected with pRNAT-hoxb7-neg, pRNAT-hoxb7. RT-PCR and Western blot were used to examine the H0XB7 mRNA and protein expression. The invasion of cells transfected with pRNAT-hoxb7-neg,pRNAT-hoxb7 or non-transfected cells was observed through ma-trigel invasion assay, gelatin zymography and Western blotting. Results Both ES-2 and SKOV3 cells expressed HOXB7. The pRNAT-hoxb7 siRNA had high inhibitory action on the expression of HOXB7 gene. The motility and invasion of ES-2 cells stably expressing pRNAT-hoxb7 were decreased compared with cells stably expressing non-specific siRNA or non-transfected cells (P<0. 01 ). MMP-2 protein level and activity were lower in the cells stably expressing pRNAT-hoxb7 than that in negative control groups (P< 0. 05). Conclusions HOXB7 siRNA can effectively inhibit HOXB7 expression and res-train the in- vasion of the ES-2 cell, suggesting that HOXB7 plays an important role in invasion of ovarian carcinoma.更多还原