摘要
目的探讨高迁移率蛋白-1(high mobility protein-1, HMGB1)对信号转导与转录激活子3(signal transducer and activator of transcription 3, STAT3)信号通路的影响及其对喉癌Hep-2细胞顺铂耐药的作用机制。方法对数生长期喉癌Hep-2细胞分为siRNA组、si-NC组和对照组,siRNA组和si-NC组细胞分别转染siRNA-HMGB1和si-NC,对照组正常培养,不做任何处理。转染48 h后,采用荧光显微镜观察siRNA组和si-NC组的转染效率,采用荧光定量PCR法检测细胞HMGB1 mRNA、蛋白酪氨酸激酶2(janus kinase signal transducer 2,JAK2)mRNA、STAT3 mRNA相对表达量,采用Western blot法检测HMGB1、JAK2、p-JAK2、STAT3、p-STAT3蛋白相对表达量。3组细胞分别加入含0.05、0.20、0.40、0.80、1.00、2.00 ng/L顺铂的DMEM培养基,采用MTT法检测各组细胞在不同顺铂浓度下的细胞增殖抑制率,计算顺铂对Hep-2细胞的半数抑制浓度。应用流式细胞仪检测细胞凋亡率,采用血管生成拟态实验检测血管生成拟态情况。结果转染48 h后,si-NC组和siRNA组转染效率均>80%。siRNA组细胞HMGB1 mRNA和HMGB1蛋白相对表达量(0.46±0.05、0.31±0.04)低于对照组(0.82±0.09、0.74±0.08)和si-NC组(0.85±0.10、0.75±0.09)(P<0.05),不同顺铂浓度下细胞增殖抑制率均高于对照组和si-NC组(P<0.05),顺铂对Hep-2细胞的半数抑制浓度[(0.49±0.07)ng/L]低于对照组[(0.88±0.12)ng/L]和si-NC组[(0.89±0.13)ng/L](P<0.05),细胞凋亡率[(50.73±7.05)%]高于对照组[(2.54±0.39)%]和si-NC组[(2.60±0.41)%](P<0.05),血管通道数[(2.00±0.36)个/视野]少于对照组[(10.40±1.57)个/视野]和si-NC组[(10.20±1.45)个/视野](P<0.05),p-JAK2和p-STAT3蛋白相对表达量(0.22±0.03、0.21±0.03)及p-JAK2/JAK2(0.24±0.03)、p-STAT3/STAT3(0.23±0.04)低于对照组(0.67±0.07、0.80±0.07、0.74±0.08、0.91±0.08)和si-NC组(0.65±0.08、0.78±0.08、0.72±0.09、0.88±0.09)(P<0.05),对照组以上指标与si-NC组比较差异均无统计学意义(P>0.05)。3组细胞JAK2、ST
Objective To investigate the influence of high mobility protein-1(HMGB1) on signal transducer and activator of transcription 3(STAT3) signaling pathway and its mechanism on resistance of laryngeal carcinoma Hep-2 cells to cisplatin. Methods The laryngeal carcinoma Hep-2 cells in logarithmic growth phase were divided into siRNA group, si-NC group and control group. The cells in siRNA group and si-NC group were transfected with siRNA-HMGB1 and si-NC, respectively, and the cells in control group were cultured normally without any treatment. Fluorescence microscope was used to observe the transfection efficiency in siRNA group and si-NC group after 48 h of transfection. The relative expressions of HMGB1 mRNA, janus kinase signal transducer 2(JAK2) mRNA, and STAT3 mRNA were detected by fluorescence quantitative PCR. The relative expressions of HMGB1, JAK2, p-JAK2, STAT3 and p-STAT3 proteins were detected by Western blot. The cells in three groups were added with DMEM medium containing 0.05, 0.20, 0.40, 0.80, 1.00 and 2.00 ng/L cisplatin, MTT method was used to detect the cell proliferation inhibition rate of each group at different cisplatin concentrations, and the half inhibitory concentration(IC50) of Hep-2 cells was calculated. Flow cytometer was used to detect the apoptosis rate, and the angiogenic mimicry experiment was used to detect the angiogenic mimicry. Results The transfection efficiency was above 80% in si-NC group and siRNA group after 48 h of transfection. The relative expressions of HMGB1 mRNA and HMGB1 protein were lower in siRNA group(0.46±0.05, 0.31±0.04) than those in control group(0.82±0.09, 0.74±0.08) and si-NC group(0.85±0.10, 0.75±0.09)(P<0.05). The cell proliferation inhibition rates under different cisplatin concentrations were higher in siRNA group than those in control group and si-NC group(P<0.05). The IC50 value of cisplatin on Hep-2 cells was lower in siRNA group((0.49±0.07)ng/L)than that in control group((0.88±0.12)ng/L)and si-NC group((0.89±0.13)ng/L)(P<0.05).The cell apoptosis
作者
张炜
魏珍星
张杨
路武豪
ZHANG Wei;WEI Zhen-xing;ZHANG Yang;LU Wu-hao(Department of Otorhinolaryngology-Head and Neck Surgery,Luoyang Central Hospital Affiliated to Zhengzhou University,Luoyang,Henan 471000,China;Department of Otorhinolaryngology-Head and Neck Surgery,the First Affiliated Hospital of Zhengzhou University,Zhengzhou,Henan 450000,China)
出处
《中华实用诊断与治疗杂志》
2021年第2期116-121,共6页
Journal of Chinese Practical Diagnosis and Therapy
基金
河南省高等学校重点科研项目资助计划(18B310029)。
关键词
喉癌
高迁移率蛋白-1
顺铂
耐药
信号转导与转录激活子3
laryngeal carcinoma
high mobility protein-1
cisplatin
resistance
signal transducer and activator of transcription 3
