中文摘要：

背景与目的：研究表明,STAT3（signal transducer and activator of transcription 3）蛋白在多种肿瘤组织或细胞中高表达,STAT3蛋白可能参与了肿瘤的发生与发展。本研究拟设计筛选出针对STAT3的高效反义核酸,研究STAT3反义核酸对人肺腺癌细胞A549增殖及凋亡的影响。方法：用RNAStructure4.2软件和STAT3 mRNA全长序列设计出10组反义STAT3序列,并分别转染A549细胞;Cell Count Kit（CCK-8）检测细胞增殖变化,倒置相差显微镜观察细胞增殖和凋亡的变化;Hoechst33258染色观察细胞凋亡形态变化,AnnexinⅤ/PI复染检测细胞早期凋亡,Western blot检测STAT3蛋白表达和磷酸化,及其下游Bcl-xL蛋白表达的变化,用碘化丙啶（propidium iodide,PI）单染结合流式细胞仪检测细胞周期变化。结果：设计出的10条反义序列对A549细胞增殖有明显抑制作用,AS10对细胞的增殖抑制率达到75.46%;反义核酸浓度越高,对A549细胞的增殖抑制效应越强（P〈0.01）。反义转染后,胞膜皱缩和核固缩形态的典型细胞凋亡明显增多,AnnexinⅤ/PI双标流式细胞仪检测发现,反义转染后能够促进细胞的早期凋亡（P〈0.01）,早期凋亡率达11.51%;而正常对照组细胞的早期凋亡率为5.18%。STAT3蛋白及其下游Bcl-xL蛋白表达变化明显下降,STAT3蛋白磷酸化水平也降低。PI单染结合流式细胞仪检测发现,正常对照组的S期细胞为44.47%,G1期的细胞则为48.49%;反义转染后的S期细胞明显减少（23.70%）,G1期细胞明显增多（63.96%）。结论：通过计算机辅助设计筛选得到的高效STAT3反义序列对A549细胞有强烈的增殖抑制作用,能有效促进A549细胞的凋亡,其机理与反义STAT3抑制STAT3蛋白表达、降低磷酸化水平、下调Bcl-xL基因以及使细胞发生G1期阻滞有关。

英文摘要：

BACKGROUND ＆ OBJECTIVE： Signal transducer and activator of transcription 3 （STAT3） is highly expressed in various human tumor tissues and tumor cell lines, and may be involved in tumor genesis and development. This study was to design effective antisense oligonucleotide targeting STAT3 mRNA, and explore its effect on the proliferation and apoptosis of human non-small ceil lung cancer cell line A549. METHODS： Ten sets of antisense sequences targeting STAT3 were designed with RNAstructure4.2 software and STAT3 mRNA total sequences, and transfected respectively into A549 cells （AS group）. Cell proliferation inhibition was measured by Cell Count Kit （CCK-8） assay. Cell proliferation and apoptosis were observed under inverted phase contrast microscope. Cell apoptosis was determined by flow cytometry （FCM） with Hoechst33258 staining and Annexin Ⅴ/PI double staining. The expression of STAT3, p-STAT3, and Bcl-xL were detected by Western blot. Cell cycle was detected by FCM. RESULTS： The 10 sets of designed sequences inhibited the proliferation of A549 cells. The inhibition rate of A549 cell proliferation reached 75.46% after transfection of AS10; the higher the concentration of the antisense oligonucleotide was, the heavier the inhibitory effect was displayed （P〈0.01）. Apoptotic cells were increased after transfection of antisense oligonucleotide. Antisense oligonucleotide induced early apoptosis in A549 cells：the early apoptosis rate was significantly higher in AS group than in control group （11.51% vs. 5.18%, P〈0.01）. The expression of STAT3, p-STAT3, and Bcl-xL were downregulated after transfection of antisense oligonucleotide. The G1 phase proportion of A549 cells was significantly higher in AS group than in control group （63.96% vs. 44.47%, P〈0.01）. CONCLUSION： The antisense oligonucleotide sequences targeting STAT3 designed with computer could inhibit the proliferation and induce the apoptosis of A549 cells.