Meng Da-wei, Bao Ji-min, Ma Yun-peng, Li Zhe, Li Su-jie, Li Dan
Department of Otorhinolaryngology, Jinqiu Hospital, Shenyang 110016, China.
Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2009 Jun;44(6):504-8.
To investigate whether down-regulation of Twist1 could change sensitivity of nasopharyngeal carcinoma cell line HNE1 to taxol.
HNE1 cells were transfected with the small interfering RNA (siRNA) expression vector pSuppressor-Retro-Si-Twist, containing the short hairpin RNA (shRNA) sequence targeting the Twist gene-coding region by Fugene 6. Positive clones were then selected in Neomycin (400 microg/ml) for 21 days. The low expressions of Twist1 were examined by real-time reverse-transcription polymerase chain reaction (RT-PCR) and Western blot. Drug sensitivity of si-Twist1 HNE1 to taxol was determined by Annexin V-fluorescein isothiocyanate( FITC)/propidium lodide (PI) double-labeled flow cytometry and detection of DNA ladder. The Effect of Twist1 inactivation on HNE1 cell proliferation was observed by MTT assay and flow cytometry.
Annexin V- FITC-PI assay showed that apoptosis ratio was 40.2% in si-Twist HNE1 after treated with 10 ng/ml taxol, significantly higher than that in the control siRNA group 24.3%. The deference had statistic meaning. After the re-expression of HNE1, apoptosis ratio was 44.80% +/- 4.80% (x +/- s) in low Twist1 protein expression group and that was 27.00% +/- 2.91% in high expression group. The deference had statistic meaning (t = 4.374, P = 0.049). Real time PCR test revealed apoptosis protein bcl-2 expression in si-Twist HNE1 was 0.28 +/- 0.05, significantly lower than that in the control siRNA HNE1 (0.57 +/- 0.08, t = 6.710, P = 0.021), nevertheless, significant bax and bcl-XL changes were not observed (t = 2.000, P = 0.184 and t = 1.502, P = 0.272). MTT and FCM showed that down-regulation of Twist1 did not alter cell proliferation rate (P>0.05).
Down-regulation of Twist1 could increase drug sensitivity of nasopharyngeal carcinoma cell line HNE1 to taxol by inducing apoptosis. These results suggested that Twist1 may be a promising treatment target for nasopharyngeal carcinoma therapy.
探讨Twist1基因表达下调是否能改变人鼻咽癌HNE1细胞株对紫杉醇的敏感性。
采用脂质体Fugene 6将携带有针对Twist基因编码区的短发夹RNA（shRNA）序列的小干扰RNA（siRNA）表达载体pSuppressor-Retro-Si-Twist转染至HNE1细胞中，用含400μg/ml新霉素的培养基筛选阳性克隆21天。采用实时逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测Twist1的低表达情况。采用膜联蛋白V-异硫氰酸荧光素（FITC）/碘化丙啶（PI）双染法和DNA梯状条带检测法，通过流式细胞仪检测si-Twist1 HNE1对紫杉醇的药物敏感性。采用MTT法和流式细胞仪观察Twist1基因沉默对HNE1细胞增殖的影响。
膜联蛋白V-FITC-PI双染法检测结果显示，用10ng/ml紫杉醇作用后，si-Twist HNE1细胞的凋亡率为40.2%，显著高于对照siRNA组（24.3%），差异具有统计学意义。HNE1细胞重新表达后，Twist1蛋白低表达组细胞凋亡率为44.80%±4.80%（x±s），高表达组为27.00%±2.91%，差异具有统计学意义（t=4.374，P=0.049）。实时PCR检测结果显示，si-Twist HNE1细胞中凋亡蛋白bcl-2的表达量为0.28±0.05，显著低于对照siRNA HNE1细胞（0.57±0.08，t=6.710，P=0.021），但bax和bcl-XL表达量无显著变化（t=2.000，P=0.184；t=1.502，P=0.272）。MTT法和流式细胞仪检测结果显示，Twist1基因表达下调未改变细胞增殖率（P>0.05）。
Twist1基因表达下调可通过诱导凋亡增加人鼻咽癌HNE1细胞株对紫杉醇的药物敏感性。上述结果提示，Twist1可能是鼻咽癌治疗中一个有前景的治疗靶点。
