Zhai J J, Du X R, Li C X
Emergency Department, Shanxi People's Hospital, Taiyuan 030000, China.
Oncology Department, Shanxi People's Hospital, Taiyuan 030000, China.
Zhonghua Yi Xue Za Zhi. 2020 May 12;100(18):1419-1425. doi: 10.3760/cma.j.cn112137-20190928-02130.
To investigate the effect of down-regulating long non-coding RNA (lncRNA) and HOX transcript antisense RNA (HOTAIR) targeting miR-761 on the radiosensitivity of HCCLM3. The expression of HOTAIR in liver cancer cells HuH-7, SNU-449, HCCLM3 and normal liver cells L-02 were measured by real-time quantitative PCR. HCCLM3 cells were divided into control, Sh-NC (transfected shRNA negative control), Sh-HOTAIR (transfected HOTAIR shRNA), RAD+Sh-NC (transfected shRNA negative control and irradiated with 8Gy dose), and RAD+Sh-HOTAIR (HOTAIR shRNA was transfected and irradiated with 8Gy dose) group. Apoptosis was detected by flow cytometry, Bcl-2 Associated X Protein (Bx-2), cleaved cysteine-containing Cleaved cysteinyl aspartate specific proteinase 3 (C-Caspase-3) protein expression. Sh-NC, Sh-HOTAIR cells were irradiated with 0, 2, 4, 6, 8 Gy, and plate-clone experiments were used to determine radiosensitivity. Bioinformatics software predicted that miR-761 might be a target gene of HOTAIR, and the luciferase reporter system identified the targeting relationship. The miR-761 inhibitor, HOTAIR shRNA and inhibitor negative control, and HOTAIR shRNA were co-transfected into HCCLM3 cells, respectively. Cell apoptosis and Bax and C-cysteine-containing aspartate proteins were also measured using the above method, as well as the hydrolase-3 protein expression and cell survival fraction. The expression levels of HOTAIR in liver cancer cells HuH-7, SNU-449, and HCCLM3 were higher than those in normal liver cells L-02 (1.85±0.12, 2.27±0.23, 2.68±0.15 vs 1.00±0.09, 0.05). Compared with Sh-NC, the apoptosis rate of Sh-HOTAIR, RAD+Sh-NC cells and Bax, C-Caspase-3 protein levels are higher [Apoptotic rate: (13.47±1.32)%, (12.84±1.19)% vs (2.98±0.27)%; Bax protein: 0.74±0.08, 0.72±0.06 vs 0.42±0.06; C-Caspase-3 protein: 0.56±0.06, 0.54±0.08 vs 0.25±0.04, all 0.05]. Compared with Sh-HOTAIR and RAD+Sh-NC, RAD+Sh-HOTAIR cell apoptosis rate and Bax, C-Caspase-3 protein levels are higher [apoptosis rate:(22.57±2.36)% vs (13.47±1.32)%, (12.84±1.19)%, Bax protein: 0.99±0.11 vs 0.74±0.08, 0.72±0.06, C-Caspase-3 protein: 1.03±0.12 vs 0.56±0.06, 0.54±0.08,all 0.05]. Compared with Sh-NC, Sh-HOTAIR cells had lower survival scores and higher radiosensitivity (0.05). HOTAIR targets negative regulation of miR-761 expression. Compared with cells co-transfected with inhibitor negative control and HOTAIR shRNA, cells co-transfected with miR-761 inhibitor and HOTAIR shRNA had lower apoptosis rate after radiation treatment [(10.24±1.32)% vs (21.84±2.01))%], Bax (0.50±0.06 vs 1.01±0.10) and C-Caspase-3 protein (0.56±0.07 vs 1.05±0.14) had lower expression and higher cell survival scores (all 0.05). Down-regulating lncRNA HOTAIR targets miR-761 to increase the radiosensitivity of HCCLM3.
探讨下调靶向miR-761的长链非编码RNA（lncRNA）和HOX转录本反义RNA（HOTAIR）对肝癌细胞系HCCLM3放射敏感性的影响。采用实时定量PCR检测肝癌细胞HuH-7、SNU-449、HCCLM3及正常肝细胞L-02中HOTAIR的表达。将HCCLM3细胞分为对照组、Sh-NC（转染shRNA阴性对照）、Sh-HOTAIR（转染HOTAIR shRNA）、RAD+Sh-NC（转染shRNA阴性对照并接受8Gy剂量照射）和RAD+Sh-HOTAIR（转染HOTAIR shRNA并接受8Gy剂量照射）组。采用流式细胞术检测细胞凋亡，检测Bcl-2相关X蛋白（Bax-2）、裂解的含半胱氨酸的天冬氨酸特异性蛋白酶3（C-Caspase-3）蛋白表达。对Sh-NC、Sh-HOTAIR细胞分别给予0、2、4、6、8Gy照射，采用平板克隆实验检测放射敏感性。生物信息学软件预测miR-761可能是HOTAIR的靶基因，荧光素酶报告系统鉴定二者靶向关系。分别将miR-761抑制剂、HOTAIR shRNA及抑制剂阴性对照与HOTAIR shRNA共转染入HCCLM3细胞。采用上述方法检测细胞凋亡及Bax和含半胱氨酸的天冬氨酸蛋白水解酶-3蛋白表达以及细胞存活分数。肝癌细胞HuH-7、SNU-449及HCCLM3中HOTAIR表达水平高于正常肝细胞L-02（1.85±0.12、2.27±0.23、2.68±0.15比1.00±0.09，P<0.05）。与Sh-NC组比较，Sh-HOTAIR组、RAD+Sh-NC组细胞凋亡率及Bax、C-Caspase-3蛋白水平升高[凋亡率：（13.47±1.32）%、（12.84±1.19）%比（2.98±0.27）%；Bax蛋白：0.74±0.08、0.72±0.06比0.42±0.06；C-Caspase-3蛋白：0.56±0.06、0.54±0.08比0.25±0.04，P均<0.05]。与Sh-HOTAIR组和RAD+Sh-NC组比较，RAD+Sh-HOTAIR组细胞凋亡率及Bax、C-Caspase-3蛋白水平升高[凋亡率：（22.57±2.36）%比（13.47±
