高迁移率族蛋白B1沉默对人端粒酶逆转录酶抑制作用及其与肺癌放疗敏感性关系研究

Relationship between inhibition of humantelomerasereversetranscriptase by high mobility group protein B1 silencing and radiosensitivity of lung cancer

目的探讨高迁移率族蛋白B1(HMGB1)沉默对人端粒酶逆转录酶(hTERT)的抑制作用,以及其与肺癌放疗敏感性的关系。方法建立稳定转染细胞系(H-1299)和HMGB1沉默(H-1299-shHMGB1)细胞系,采用实时荧光定量聚合酶链反应检测HMGB1 mRNA的表达。根据处理方式不同将细胞分为对照组(无X射线照射,转染空白质粒),HMGB1 KO组(转染HMGB1-shRNA,敲除HMGB1),KO+5 Gy组(转染HMGB1-shRNA,敲除HMGB1,照射剂量5 Gy),KO+10 Gy组(转染HMGB1-shRNA,敲除HMGB1,照射剂量10 Gy)。应用噻唑蓝(MTT)和克隆形成实验分析HMGB1对肺癌细胞H-1299增殖和放射敏感性的影响。采用划痕损伤实验、Transwell实验和流式细胞仪检测HMGB1对放射诱导的肿瘤细胞迁移、侵袭、细胞周期和凋亡的影响。通过蛋白免疫印迹法分析HMGB1沉默对hTERT表达的影响。结果与正常肺泡上皮细胞BEAS-2B比较,HMGB1在肺癌细胞H-1299中表达显著上调(P<0.05)。10 Gy放疗48、96 h后,HMGB1均较对照组显著降低(P<0.05)。MTT与细胞克隆形成实验结果显示,HMGB1沉默后,随着放疗剂量增加,H-1299细胞活力显著降低(P<0.05)。划痕实验结果显示,HMGB1沉默后,随着放疗剂量增加,H-1299细胞迁移所受影响越明显(P<0.05)。Transwell实验结果显示,HMGB1沉默后,随着放疗剂量增加,H-1299细胞侵袭能力显著降低(P<0.05)。HMGB1沉默后,H-1299细胞G1期减少,G2期增加;给予放疗后,细胞周期明显阻滞,G1期减少及G2期增加程度更明显。HMGB1沉默后,随着放疗剂量增加,H-1299细胞凋亡率明显增加(P<0.05)。HMGB1沉默导致hTERT、RFPL3、CBP、TRF1和TRF2蛋白表达均有不同程度的降低,而给予放疗后,这些蛋白的表达均显著降低(P<0.05)。结论HMGB1下调破坏人肺癌细胞的端粒稳态,下调hTERT蛋白表达,增强放射敏感性,HMGB1和hTERT可能是肺癌放射治疗疗效预测的潜在靶点。

Objective To investigate the relationship between the inhibition of humantelomerasereversetranscriptase(hTERT)by high mobility group protein B1(HMGB1)silencing and the radiosensitivity of lung cancer.Methods Stable transfected lung cancer cell line(H-1299)and HMGB1 silenced(H-1299-shHMGB1)cell line were established.The expression of HMGB1 mRNA was detected by Real-time fluorescence quantitative PCR,the transfection efficiency of shRNA was evaluated by Western blot analysis,and the effect of HMGB1 on proliferation and radiosensitivity of lung cancer cell line Hmur1299 was analyzed by methyl thiazolyl tetrazolium(MTT)and colony formation assay.Scratch injury test,Transwell test and flow cytometry were used to detect the effects of HMGB1 on radiation-induced tumor cell migration,invasion,cell cycle and apoptosis.Western blot was used to analyze the effect of HMGB1 silencing on hTERT expression.Results Compared with normal alveolar epithelial cell BEAS-2 B,HMGB1 expression was significantly up-regulated in lung cancer cell H-1299(P<0.05).After 48 and 96 hours of 10 Gy radiotherapy,HMGB1 was significantly reduced compared with that of the control group(P<0.05).MTT and cell colony formation experiments showed that the viability of H-1299 cells was significantly reduced with the increase of radiotherapy dose after HMGB1 silencing(P<0.05).The scratch test results showed that the effect of HMGB1 silencing on H-1299 cell migration was more obvious with the increase of radiotherapy dose(P<0.05).The results of Transwell experiment showed that the invasion ability of H-1299 cells was significantly reduced with the increase of radiotherapy dose after HMGB1 silencing(P<0.05).After HMGB1 silencing,the G1 phase of H-1299 cells reduced and the G2 phase increased;after radiotherapy,the cell cycle was arrested obviously,the G1 phase reduced and the G2 phase increased more obviously.After HMGB1 silencing,the apoptosis rate of H-1299 cells was significantly increased with the increase of radiotherapy dose(P<0.05).Silencing of HMGB1 resulted in reduced expression of hTERT,RFPL3,CBP,TRF1 and TRF2 proteins to varying degrees,and the expression of these proteins was significantly decreased after radiotherapy(P<0.05).Conclusion Down-regulation of HMGB1 destroys telomere homeostasis,down-regulates the expression of hTERT protein and enhanced radiosensitivity in human lung cancer cells.HMGB1 and hTERT may be potential targets for predicting the efficacy of radiotherapy for lung cancer.