外源性HMGB1/mtDNA促进放疗残存胰腺癌细胞侵袭与迁移的作用及其机制

Radiotherapy-induced exogenous HMGB1/mtDNA promotes residual pancreatic cancer cells invasion and migration

目的探讨放疗诱导胰腺癌细胞释放的高迁移率族蛋白B1/线粒体DNA(HMGB1/mtDNA)对残存胰腺癌细胞侵袭与迁移的作用及其机制。方法不同剂量(0、8 Gy)X射线处理胰腺癌PaTu8988细胞后,酶联免疫吸附测定(ELISA)试剂盒检测放疗后上清HMGB1浓度、实时荧光定量PCR(qRT-PCR)检测放疗后上清mtDNA相对拷贝数。将放疗处理的胰腺癌细胞、外源性重组人高迁移率族蛋白B1(rhHMGB1)/mtDNA分别与PaTu8988细胞共培养,Transwell检测侵袭与迁移细胞数。外源性rhHMGB1/mtDNA培养PaTu8988细胞,蛋白质印迹法检测环鸟苷酸-腺苷酸合成酶(cGAS)、干扰素基因刺激蛋白(STING)、TANK结合激酶1(TBK1)和干扰素调节因子3(IRF3)蛋白水平变化;qRT-PCR检测干扰素-β(IFN-β)转录水平变化。同时构建HMGB1低表达(sh-HMGB1)、mtDNA部分缺失(ρ0)胰腺癌PaTu8988细胞株,放疗后与PaTu8988细胞共培养,蛋白质印迹法检测TBK1蛋白磷酸化水平变化。结果放疗诱导HMGB1(t=-18.429,P<0.001)和mtDNA相关基因COX1(t=-12.370,P=0.006)、COX2(t=-8.503,P=0.014)和COX3(t=-10.321,P=0.009)释放至胰腺癌细胞外。放疗的胰腺癌细胞与rhHMGB1/mtDNA均能够促进PaTu8988细胞的侵袭与迁移(t=-4.016,P=0.015;t=-0.563,P=0.006;t=-25.489,P=0.001;t=-19.157,P=0.003)。外源性rhHMGB1/mtDNA抑制PaTu8988细胞中cGAS(t=2.895,P=0.048)、STING(t=-13.354,P=0.005)、p-TBK1(Ser172)/TBK1(t=-7.347,P=0.012)和p-IRF3(Ser396)/IRF3(t=-3.698,P=0.021)蛋白的表达;同时降低了PaTu8988细胞中IFN-β的转录水平,t=3.186,P=0.033。结论放疗促进胰腺癌细胞释放HMGB1和mtDNA至胞外;放疗诱导的HMGB1/mtDNA通过抑制cGAS-STING通路的激活和IFN-β的生成促进残存胰腺癌细胞的侵袭与迁移。

Objective To investigate the effects of radiotherapy-induced HMGB1/mtDNA on the invasion and migration of residual pancreatic cancer cells and its mechanism.Methods After the treatment of pancreatic cancer PaTu8988cells with different doses of X-ray(0Gy and 8Gy),the concentration of HMGB1and the relative copy number of mtDNA in the supernatant after radiotherapy were detected by the enzyme linked immunosorbent assay(ELISA)kit and quantitative realtime fluorescence PCR(qRT-PCR)respectively.The pancreatic cancer cells treated with radiation or rhHMGB1/mtDNA were further co-cultured with PaTu8988cells respectively,and the cell numbers of invasion and migration were detected by Transwell.With the treatment of exogenous rhHMGB1/mtDNA in PaTu8988cells,the protein expression levels of cGAS,STING,TBK1and IRF3were detected by Western blotting and the interferon-beta(IFN-β)transcription level were also detected by qRT-PCR.Meanwhile,the pancreatic cancer cell line with the low expression of HMGB1(sh-HMGB1)or the partial deletion of mtDNA(ρ0)were constructed,which were co-cultured with PaTu8988cells after radiation.Western blotting were utilized to detect the protein phosphorylation level of TBK1.Results Radiotherapy promoted the release of HMGB1(t=-18.429,P<0.001)and mtDNA related genes like COX1(t=-12.370,P=0.006),COX2(t=-8.503,P=0.014)and COX3(t=-10.321,P=0.009)to the extracellular of PaTu8988cells.Both pancreatic cells treated with radiotherapy and rhHMGB1/mtDNA could promote the invasion and migration of PaTu8988cells(t=-4.016,P=0.015;t=-0.563,P=0.006;t=-25.489,P=0.001;t=-19.157,P=0.003).Exogenous rhHMGB1/mtDNA down-regulated the protein expression level of cGAS(t=2.895,P=0.048),STING(t=-13.354,P=0.005),p-TBK1(Ser172)/TBK1(t=-7.347,P=0.012)and p-IRF3(Ser 396)/IRF3(t=-3.698,P=0.021)and also reduced the transcription level of IFN-β(t=3.186,P=0.033).Conclusions Radiotherapy can promote the release of HMGB1and mtDNA to the extracellular of PaTu8988cells.Radiotherapy-induced HMGB1/mtDNA can cause the invasion and migration of residual pancreatic cancer cells by inhibiting the activation of the cGAS-STING pathway and the production of IFN-β.