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自噬相关基因 BECLIN1 的敲低促进 A549 人肺癌细胞系的细胞生长并抑制细胞凋亡。

Knockdown of autophagy-related gene BECLIN1 promotes cell growth and inhibits apoptosis in the A549 human lung cancer cell line.

机构信息

Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430023, PR China.

出版信息

Mol Med Rep. 2013 May;7(5):1501-5. doi: 10.3892/mmr.2013.1379. Epub 2013 Mar 19.

Abstract

The expression of BECLIN1 is significantly reduced in non‑small cell lung cancer (NSCLC) compared with non‑cancerous tissue. However, the role of BECLIN1 in lung cancer is unclear. Using the RNA interference (RNAi) technique the present study investigated the effect of the knockdown of BECLIN1 on the cell growth and proliferation of the A549 human lung cancer cell line. The target site for the RNAi technique was designed and the lentivirus vector for the small interfering (si)RNA expression was constructed according to the encoding sequence of the mRNA for BECLIN1. The A549 cells were transfected with the siRNA virus against BECLIN1. BECLIN1 expression was detected by reverse transcription (RT)‑PCR and western blot analysis. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was applied to detect cell growth. Flow cytometry was used to determine cell apoptosis. The activity of caspase‑3 and caspase‑9 was also detected in the A549 cells with BECLIN1 knockdown. The results showed that siRNA virus transfection significantly decreased the mRNA and protein expression of BECLIN1 in the transfected A549 cells. The knockdown of BECLIN1 promoted cell growth and decreased apoptosis. Caspase‑3 and ‑9 activity in the A549 cells with BECLIN1 knockdown was significantly reduced. In conclusion, the siRNA expression vector effectively inhibited the expression of BECLIN1 in the A549 human lung cancer cell line and promote the growth and proliferation of the A549 cells.

摘要

BECLIN1 的表达在非小细胞肺癌 (NSCLC) 中明显低于非癌组织。然而,BECLIN1 在肺癌中的作用尚不清楚。本研究采用 RNA 干扰 (RNAi) 技术,研究了敲低 BECLIN1 对 A549 人肺癌细胞系细胞生长和增殖的影响。根据 BECLIN1 mRNA 的编码序列设计了 RNAi 技术的靶位,并构建了用于小干扰 (si)RNA 表达的慢病毒载体。用针对 BECLIN1 的 siRNA 病毒转染 A549 细胞。通过逆转录 (RT)-PCR 和 Western blot 分析检测 BECLIN1 表达。应用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐 (MTT) 法检测细胞生长。采用流式细胞术检测细胞凋亡。还检测了 BECLIN1 敲低的 A549 细胞中 caspase-3 和 caspase-9 的活性。结果显示,siRNA 病毒转染显著降低了转染的 A549 细胞中 BECLIN1 的 mRNA 和蛋白表达。BECLIN1 的敲低促进了细胞生长,降低了细胞凋亡。BECLIN1 敲低的 A549 细胞中 caspase-3 和 caspase-9 的活性显著降低。综上所述,siRNA 表达载体可有效抑制 A549 人肺癌细胞系中 BECLIN1 的表达,并促进 A549 细胞的生长和增殖。

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