shHOTAIR对上皮性卵巢癌细胞侵袭及裸鼠致瘤能力的影响

目的探讨短发夹RNA（shRNA）降低人上皮性卵巢癌SKOV3细胞中长链非编码RNA（lncRNA）HOTAIR基因表达后对SKOV3细胞的侵袭、裸鼠致瘤能力及锌指转录因子snail基因表达的影响。方法 RT-PCR检测SKOV3细胞lncRNA HOTAIR的表达；构建靶向人lncRNA HOTAIR基因表达的干扰质粒shHOTAIR，经脂质体转染SKOV3细胞后筛选稳定转染细胞株，实时荧光定量PCR（qRT-PCR）检测lncRNA HOTAIR在SKOV3转染细胞中的表达。利用Transwell小室基质胶侵袭试验及裸鼠致瘤实验，检测不同转染SKOV3细胞侵袭及裸鼠致瘤能力，获取荷瘤鼠肿瘤组织块，进行免疫组化染色及Western blot检测荷瘤鼠肿瘤组织中snail蛋白的表达，qRT-PCR检测E-cadherin、 snail mRNA的表达。结果 RT-PCR检测结果显示SKOV3细胞表达lncRNA HOTAIR；成功构建干扰质粒shHOTAIR，稳定转染SKOV3细胞（shHOTAIR-SKOV3）后，与对照组scramble-SKOV3细胞比较，lncRNA HOTAIR表达下降 (P<0.01)；shHOTAIR-SKOV3细胞与对照组scramble-SKOV3细胞相比，对transwell小室基质胶侵袭能力、裸鼠致瘤能力下降、荷瘤鼠肿瘤体积下降(P<0.01)，荷瘤鼠肿瘤组织免疫组化染色及Western blot检测结果显示，snail蛋白表达降低(P<0.05)，qRT-PCR检测显示，荷瘤鼠肿瘤组织snail表达降低(P<0.05)、E-cadherin表达增高(P<0.05)。结论 lncRNA HOTAIR表达下调可引起上皮性卵巢癌细胞snail表达降低、E-cadherin表达增高，并使卵巢癌细胞侵袭及裸鼠致瘤能力降低，提示lncRNA HOTAIR作为治疗靶点，可能具有靶向治疗上皮性卵巢癌细胞的潜在应用价值。

英文摘要:

Objective To explore the effects of shRNA-mediated downregulating lncRNA HOTAIR on the invasion, nude mouse tumorigenicity and snail expression of epithelial ovarian cancer SKOV3 cells. Methods The expression of lncRNA HOTAIR was detected by RT-PCR in SKOV3 cells. The shRNA targeting the lncRNA HOTAIR gene was cloned into RNA interference plasmid. The construction shHOTAIR vector was transfected into ovarian cancer SKOV3 cells by lipofectamine 2000, and the stably transfected cells were isolated by G418 and single clone selection. The downregulating expression of lncRNA HOTAIR was detected by quantitative real time PCR(qRT-PCR). The characteristics of shHOTAIR transfected SKOV3 cells were analyzed from the assays of invasion and nude mouse tumorigenicity, as well as the expression of snail and E-cadherin mRNA detected by qRT-PCR, and snail detected by immunohistochemistry and Western blot methods in xenograft tumor, respectively. Results The lncRNA HOTAIR expression was proved by RT-PCR in SKOV3 cells. The lncRNA HOTAIR expression in shHOTAIR transfected SKOV3 cells was significantly lower than the scramble control (P<0.01). The shHOTAIR transfected SKOV3 cells show that the invasion ability was significantly decreased compared with the scramble control (P<0.01). The nude mouse tumorigenicity, including tumorigenicity mouse number and tumor volume, was significantly decreased compared with the control group (P<0.01). The snail protein expression detected by IHC and Western blot in shHOTAIR-SKOV3 xenograft tumor was significantly decreased compared with the control scramble- SKOV3 group (P<0.05). The lncRNA HOTAIR low expression resulted in increasing E-cadherin and decreasing snail expression detected by qRT-PCR in the shHOTAIR transfected SKOV3 cells of xenograft tumor, compared with the scramble control (P<0.05). Conclusion Targeting lncRNA HOTAIR expression in SKOV3 cells with RNA interference can decrease snail, increase E-cadherin and significantly reduce the invasion and tumorigenicity of epithelial ovarian cancer SKOV3 cells. These results suggest that the lncRNA HOTAIR could be an effective therapeutic target for human epithelial ovarian caner treatment.

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