小干扰 RNA 靶向脱嘌呤/脱嘧啶内切酶增强人胰腺癌细胞对吉西他滨的体外敏感性。

Small interfering RNA against the apurinic or apyrimidinic endonuclease enhances the sensitivity of human pancreatic cancer cells to gemcitabine in vitro.

机构信息

Department of Gastroenterology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Institute of Digestive Disease, Shanghai, China.

出版信息

J Dig Dis. 2010 Aug;11(4):224-30. doi: 10.1111/j.1751-2980.2010.00442.x.

DOI:10.1111/j.1751-2980.2010.00442.x

PMID:20649735

Abstract

OBJECTIVE

To investigate whether the downregulation of human apurinic or apyrimidinic endonuclease/redox factor-1 gene (APE1/Ref-1) expression by ribonucleic acid interference (RNAi) would increase the sensitivity of SW1990 cells to gemcitabine.

METHODS

Chemically synthesized small interfering RNA (siRNA) directed against human APE1/Ref-1 (si-APE1) was transfected into SW1990 cells through transfection reagents. The mRNA expression of APE1/Ref-1 was detected by semi-quantitative RT-PCR and the protein expression of APE1/Ref-1 was detected by Western blot; cell proliferation and apoptosis were studied by a Cell Counting Kit 8 (CCK-8) and flow cytometry (FCM) and fluorescence microscopy.

RESULTS

After transfecting the SW1990 cells with siRNA directed against human APE1/Ref-1, the mRNA expression of APE1/Ref-1 of these cells was reduced, and its protein expression was reduced by 55.41 +/- 3.58%. The CCK-8 assay showed that the absorbance and the inhibition of cell growth transfected with si-APE1 were significantly different from the blank (cultured with Dulbecco's modified Eagle's medium) and negative control (given 50 nmol/L scrambled control siRNA). The inhibition rates of cell growth of the si-APE1 group at 24, 48, 72 h were 41.69 +/- 2.78%, 24.83 +/- 3.70% and 21.27 +/- 9.82%, respectively. A FCM analysis and cell morphology study showed that the apoptotic rate of SW1990 cells transfected with si-APE1 combined with gemcitabine treatment was significantly different from the blank control and others.

CONCLUSION

To knock down APE1/Ref-1 gene expression may significantly sensitize the SW1990 cells to gemcitabine and enhance cell apoptosis.

摘要

目的

通过 RNA 干扰（RNAi）下调人类脱嘌呤/脱嘧啶内切核酸酶/氧化还原因子-1 基因（APE1/Ref-1）的表达，观察其对 SW1990 细胞吉西他滨敏感性的影响。

方法

化学合成针对人 APE1/Ref-1 的小干扰 RNA（siRNA）（si-APE1），通过转染试剂转染至 SW1990 细胞，半定量 RT-PCR 检测 APE1/Ref-1mRNA 的表达，Western blot 检测 APE1/Ref-1 蛋白的表达，CCK-8 法和流式细胞术（FCM）及荧光显微镜观察细胞增殖和凋亡。

结果

转染靶向人 APE1/Ref-1 的 siRNA 后，SW1990 细胞 APE1/Ref-1mRNA 的表达降低，其蛋白表达降低 55.41%±3.58%。CCK-8 法显示，转染 si-APE1 的细胞吸光度和生长抑制与空白组（DMEM 培养）和阴性对照组（转染 50 nmol/L 错配对照 siRNA）差异有统计学意义。si-APE1 组 24、48、72 h 的细胞生长抑制率分别为 41.69%±2.78%、24.83%±3.70%、21.27%±9.82%。FCM 分析和细胞形态学研究显示，转染 si-APE1 联合吉西他滨处理的 SW1990 细胞的凋亡率与空白对照组和其他组差异有统计学意义。