RNA干扰HMGB1基因对食管鳞癌细胞X线照射后增殖与迁移能力影响

目的 RNAi技术干扰人食管鳞癌细胞中HMGB1基因表达,观察X线照射后细胞增殖活性、迁移和存活能力及细胞周期分布。方法 RT-PCR和Western blot法检测HMGB1 mRNA和蛋白表达;分别采用MTS和Transwell方法检测食管鳞癌细胞ECA109、KYSE30细胞增殖活性和迁移能力;克隆形成实验检测各组细胞的存活能力;流式细胞术检测照射后各组的细胞周期分布。结果 照射组ECA109、KYSE30细胞中HMGB1 mRNA和蛋白表达水平较未照射组明显增加(P<0.05),且存在剂量-效应和时间-效应关系;MTS结果显示食管鳞癌细胞的增殖水平在照射后不同时间点均明显降低(P<0.01);HMGB1沉默组细胞中HMGB1 mRNA和蛋白的表达均明显低于对照组和NC组(P<0.01);克隆形成实验结果显示HMGB1表达降低后,肿瘤细胞的放射敏感性明显增加(P<0.01);Tanswell侵袭小室模型检测显示照射后4 h沉默组穿膜细胞数明显降低(P<0.01),且沉默组G0/G1期比例明显高于对照组和NC组,S期比例显著低于其他组,照射后这种趋势更明显(P<0.01)。结论 RNAi干扰技术降低食管鳞癌细胞中HMGB1的表达,降低了细胞增殖和迁移能力,通过诱导照射后G0/G1期阻滞增加了放射敏感性。

Effect of HMGB1 knockdown by RNA interference on cell proliferation and migration of esophageal squamous cell carcinoma after X-ray radiation

Objective To evaluate the effect of X-ray radiation on cell proliferation, migration, survival ability and cell cycle of human esophageal squamous cell carcinoma after RNA interference-mediated down-regulation of HMGB1 gene expression. Methods The expression of HMGB1 at mRNA and protein levels in the human esophageal squamous cell carcinoma cell lines ECA109 and KYSE30 was determined using RT-PCR and Western blot assays. MTS and Transwell assays were employed to examine the proliferation and migration of ECA109 and KYSE30 cell lines. The cellular survival ability in vitro was assessed by clone formation assay. The cell cycle after X-ray radiation in different groups was detected by flow cytometry. Results The expression of HMGB1 at mRNA and protein levels in ECA109 and KYSE30 cells were markedly higher in a dose-dependent and time-dependent manner in the radiation group than that in the control group (all P<0.05). MTS results demonstrated that the proliferation of ECA109 and KYSE30 cells was obviously lower at each time point after radiation than that in the group without radiation (all P<0.01). The expression of HMGB1 at mRNA and protein levels was significantly inhibited in the HMGB1 siRNA group than those in the control and NC groups (both P<0.01). The data from the clone formation assay revealed that the radiosensitivity was significantly increased after down-regulation of HMGB1 expression (P<0.01). Transwell migration assay revealed that the number of migrating cells at the fourth hour after X-ray irradiation in the HMGB1 siRNA group was significantly lower than those in the control and negative groups (both P<0.01). In the HMGB1 siRNA group, the percentage of cells at G0/G1 phase was obviously higher, whereas the percentage of S phase was significantly lower than those in the control and NC groups, and the trend was even more significant after X-ray radiation (all P<0.01). Conclusion Inhibition of HMGB1 expression by siRNA can suppress the proliferation and migration of ECA109 and KYSE30 cells and enhance the radiosensitivity by increasing the cell cycle arrest at G0/G1 stage after X-ray irradiation in vitro.