Moergel Maximilian, Goldschmitt Jutta, Stockinger Marcus, Kunkel Martin
Department of Oral and Maxillofacial Surgery, University of Mainz, Medical Center, Augustusplatz 2, 55131, Mainz, Germany.
Department of Oral and Maxillofacial Surgery, Johannes Gutenberg-University, Medical Center, Augustusplatz 2, 55131, Mainz, Rheinland-Pfalz, Germany.
Clin Oral Investig. 2014 May;18(4):1259-1268. doi: 10.1007/s00784-013-1078-0. Epub 2013 Aug 15.
This study investigated the expression of ΔNp63α in carcinoma cell lines of the upper aerodigestive tract and their potential influence on radioresistance using a small interfering RNA (siRNA) knockdown approach.
Four carcinoma cell lines were investigated for the expression of the ΔNp63 isoform by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (0, 24, 48 h) with and without single dose irradiation of 6 Gy. Furthermore, all cell lines were transfected with siRNA against the ΔNp63α isoform over 24 h. Knockdown effectiveness was controlled by qRT-PCR and Western blot. Apoptotic events were evaluated by terminal transferase dUTP nick end labeling (TUNEL) assay and cross-checked by a test for cell viability (WST-1, Roche) over 48 h.
All cell lines presented varying expression of the ΔNp63α isoform with and without irradiation. A sufficient knockdown rate was established by siRNA transfection. Knockdown of the ΔNp63 isoform showed an effect on radiation sensitivity proven by an increase of apoptotic events detectable by immunofluorescence (TUNEL assay) and likewise a significant reduction of formazan production (WST-1 test) in three cell lines.
We found overexpression of ΔNp63α with and without irradiation in three cell lines, and the knockdown of ΔNp63α led to increased apoptotic events and fewer viable cells. Thus, the overexpression of ΔNp63α might protect carcinoma cells against irradiation effects.
The present work supports the hypothesis that protein 63 might serve as a negative predictor for irradiation response and survival in a clinical setting and may be a target for future therapeutic strategies.
本研究采用小干扰RNA（siRNA）敲低方法，调查了上消化道癌细胞系中ΔNp63α的表达及其对放射抗性的潜在影响。
通过定量逆转录聚合酶链反应（qRT-PCR）（0、24、48小时），在有或无单次6 Gy照射的情况下，研究了四种癌细胞系中ΔNp63亚型的表达。此外，在24小时内用针对ΔNp63α亚型的siRNA转染所有细胞系。通过qRT-PCR和蛋白质印迹法控制敲低效果。通过末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）测定评估凋亡事件，并在48小时内通过细胞活力检测（WST-1，罗氏公司）进行交叉检查。
在有或无照射的情况下，所有细胞系中ΔNp63α亚型均呈现不同表达。通过siRNA转染建立了足够的敲低率。敲低ΔNp63亚型对放射敏感性有影响，这通过免疫荧光（TUNEL测定）可检测到的凋亡事件增加以及同样在三种细胞系中噻唑蓝产生的显著减少（WST-1试验）得到证实。
我们发现在三种细胞系中，无论有无照射，ΔNp63α均有过表达，敲低ΔNp63α导致凋亡事件增加且存活细胞减少。因此，ΔNp63α的过表达可能保护癌细胞免受辐射影响。
本研究支持以下假设，即蛋白63可能作为临床环境中辐射反应和生存的阴性预测指标，并且可能是未来治疗策略的靶点。
