HMGB1 siRNA干扰对食管鳞癌细胞放射敏感性的影响

目的： 采用siRNA技术干扰人食管鳞癌细胞中高迁移率族蛋白B1（HMGB1）基因表达，观察经X射线照射后细胞增殖活性、存活能力及细胞凋亡率的变化。方法： 针对HMGB1 mRNA序列，设计合成有效的干扰序列HMGB1 siRNA，并转染食管鳞癌KYSE30细胞，同时设置转染阴性对照序列的阴性对照组以及未进行转染的空白对照组。采用实时荧光定量PCR（qPCR）和Western blot法检测各组细胞HMGB1 mRNA和蛋白的表达；分别用MTS比色、克隆形成实验、流式细胞术和Western blot法检测HMGB1 siRNA干扰对食管鳞癌细胞KYSE30细胞的增殖活力、存活能力、细胞凋亡率及凋亡相关蛋白表达的影响。结果： HMGB1 siRNA干扰后，KYSE30细胞HMGB1 mRNA和蛋白的表达水平明显降低（P均 < 0.01）；MTS数据显示HMGB1 siRNA干扰后经X射线照射显著抑制了食管鳞癌细胞的增殖水平（与空白对照组和阴性对照组比较，P < 0.05）；克隆形成实验结果显示空白对照组、阴性对照组、HMGB1 siRNA组的D₀值分别为2.57、2.54、1.55 Gy；D_q值分别为1.69、1.65、1.30 Gy；SF₂值分别为0.27、0.27、0.13；外推数N值分别为1.93、1.91、2.31；X射线照射后HMGB1 siRNA组肿瘤细胞的凋亡率明显增加，同时bcl-2蛋白表达显著降低，而bax、caspase-3蛋白的表达明显增加（与空白对照组和阴性对照组比较，P < 0.01）。结论： siRNA干扰技术可用来抑制食管鳞癌细胞中HMGB1基因的表达，联合X射线照射降低了肿瘤细胞的增殖水平和存活能力，诱导了细胞凋亡，增加了放射敏感性，该作用可能与调控bcl-2、bax和caspase-3基因表达有关。

Effect of HMGB1 silencing by siRNA on radiosensitivity of esophageal carcinoma KYSE30 cells

OBJECTIVE: To examine the effect of HMGB1 silencing by siRNA on cell proliferation, survival ability, and cell apoptosis of human esophageal squamous cell carcinoma, KYSE30, after X-ray radiation. METHODS: There were three study groups. The HMGB1 siRNA group:siRNA based on the sequences of the HMGB1 mRNA were synthesized and were transfected into the cultured KYSE30 cells. The negative control group:a negative siRNA was synthesized and transfected. The blank control group:no transfection was conducted. Expression of HMGB1 at the mRNA and protein levels was determined using quantitative real-time PCR (qPCR) and Western blot, respectively. The effect of HMGB1 knockdown on the proliferation,survival ability,cell apoptosis rate and the expression of cell apoptosis-related proteins of tumor cells were examined by MTS, clonal formation, flow cytometry and Western blot assays, respectively. RESULTS: Both mRNA and protein expression of HMGB1 were significantly reduced after silencing the HMGB1 gene (P < 0.01). MTS data demonstrated that HMGB1 knockdown,followed by irradiation,significantly inhibited the proliferation of KYSE30 cells compared with the negative control and the blank control groups (P < 0.05). Data from the clonal formation assay revealed that the values of D₀ were 2.57 Gy,2.54 Gy,1.55 Gy; the values of D_q were 1.69 Gy,1.65 Gy,1.30 Gy;the values of SF₂ were 0.27,0.27,0.13;the values of N were 1.93,1.91,2.31 in the blank control,negative control and HMGB1 siRNA groups,respectively (all P < 0.01). The apoptosis rate of tumor cells in the HMGB1 siRNA group after irradiation was markedly increased, followed by the downregulation of bcl-2 expression and the upregulation of bax, caspase-3 expression compared with negative control and blank control groups (P < 0.01). CONCLUSION: SiRNA interference technology inhibited the expression of HMGB1 gene in KYSE30 cells,reduced the proliferation and viability ability of cells, induced cell apoptosis, and increased radiosensitivity after X-ray exposure, which may be associated with regulating the expression of bcl-2,bax and caspase-3.