[信号转导与转录激活因子3反义寡核苷酸对非小细胞肺癌细胞系A549增殖和凋亡的影响]

[Effects of STAT3 antisense oligonucleotide on proliferation and apoptosis of non-small cell lung cancer cell line A549].

作者信息

Zhu Bao-Rang, Cai Jian-Ming, Tang Gu-Sheng, Li Bai-Long, Gao Fu, Cui Jian-Guo, Liu Han-Chen

机构信息

Department of Radiation and Nuclear Medicine, The Second Military Medical University,Shanghai, 200433, PR China.

出版信息

Ai Zheng. 2007 Aug;26(8):820-7.

PMID:17697540

Abstract

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 cell 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-x(L) 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-x(L) were down-regulated 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.

摘要

背景与目的

信号转导及转录激活因子3（STAT3）在多种人类肿瘤组织和肿瘤细胞系中高表达，可能参与肿瘤的发生发展。本研究旨在设计针对STAT3 mRNA的有效反义寡核苷酸，并探讨其对人非小细胞肺癌细胞系A549增殖和凋亡的影响。

方法

利用RNAstructure4.2软件和STAT3 mRNA全长序列设计10组针对STAT3的反义序列，分别转染A549细胞（反义组）。采用细胞计数试剂盒（CCK-8）检测细胞增殖抑制率。在倒置相差显微镜下观察细胞增殖和凋亡情况。通过Hoechst33258染色和Annexin V/PI双染，利用流式细胞术（FCM）检测细胞凋亡。采用蛋白质免疫印迹法检测STAT3、p-STAT3和Bcl-x(L)的表达。利用FCM检测细胞周期。

结果

设计的10组序列均能抑制A549细胞的增殖。转染AS10后A549细胞增殖抑制率达75.46%；反义寡核苷酸浓度越高，抑制作用越明显（P<0.01）。转染反义寡核苷酸后凋亡细胞增多。反义寡核苷酸诱导A549细胞早期凋亡：反义组早期凋亡率显著高于对照组（11.51%比5.18%，P<0.01）。转染反义寡核苷酸后STAT3、p-STAT3和Bcl-x(L)的表达下调。反义组A549细胞G1期比例显著高于对照组（63.96%比44.47%，P<0.01）。