摘要
目的探讨高迁移率族蛋白B1(HMGB1)介导癌症相关成纤维细胞(CAFs)活化在结直肠癌转移中的作用。方法从结直肠癌及癌旁组织中,分离提纯癌旁正常成纤维细胞(NFs)和CAFs,分别与人结直肠癌细胞SW480共培养,划痕实验和Transwell侵袭实验检测NFs和CAFs对SW480迁移、侵袭的作用。免疫组化法检测HMGB1在结直肠癌、癌旁正常结直肠组织中表达。蛋白质印迹法和实时荧光定量聚合酶链反应(qRT-PCR)法检测NFs、CAFs中HMGB1 mRNA和蛋白的表达水平。ELISA法检测上清液中HMGB1表达。应用小干扰RNA(siRNA)技术抑制CAFs细胞内HMGB1基因表达,qRT-PCR和蛋白质印迹法分别检测阴性对照转染组(CAFs-siNC组)与HMGB1-siRNA转染组(CAFs-siHMGB1组)中HMGB1、α平滑肌肌动蛋白(α-SMA)基因和蛋白的表达情况,2组细胞分别与SW480细胞共培养后,划痕实验和Transwell侵袭实验验证HMGB1表达对SW480迁移和侵袭的影响。结果NFs与SW480细胞共培养迁移率为(25.51±46.96)%,侵袭细胞数为(92.97±9.36)个,CAFs与SW480细胞共培养迁移率为(63.38±9.25)%,侵袭细胞数为(129.56±10.93)个,差异均有统计学意义,t值分别为5.665和4.405,P值分别为0.005和0.012。HMGB1在正常结直肠组织中表达较低或不表达,而在结直肠癌组织中高表达。CAFs细胞中HMGB1 mRNA和蛋白表达分别为1.918±0.082和0.834±0.051,细胞上清液HMGB1浓度值为253.538±22.873,均高于NFs细胞,均P<0.05。CAFs-siHMGB1组中HMGB1 mRNA、α-SMA mRNA、HMGB1蛋白、α-SMA蛋白表达分别为0.385±0.038、0.511±0.029、0.649±0.040、0.475±0.039,CAFs-siNC组中分别为1.002±0.051、0.998±0.047、0.964±0.031、0.746±0.047,差异均有统计学意义,均P<0.05。CAFs-siNC与SW480细胞共培养迁移率为(62.99±})%,侵袭细胞数为(125.38±11.54)个,CAFs-siHMGB1与SW480细胞共培养迁移率为(26.22±6.48)%,侵袭细胞数为(83.58±10.51)个,差异均有统计学意义,t值分别为5.522和4.638,P值分别为0.005和0.010。结论CAFs能促进SW480细胞迁移、侵袭。HMGB1在CAFs细胞和结直肠癌组织中均高表达,抑制CAFs细胞中HMGB1表达能够减弱CAFs对SW480细胞迁移、侵袭的促进作用。
Objective To investigate the role of high mobility group protein B1(HMGB1)-mediated activation of carcinoma-associated fibroblasts(CAFs)in colorectal cancer metastasis.Methods Para-carcinoma normal fibroblasts(NFs)and CAFs were isolated and purified from colorectal cancer and para-carcinoma tissues,and co-cultured with human colorectal cancer cells SW480,respectively.The effects of NFs and CAFs on the migration and invasion of SW480were detected by scratch and transwell invasion experiments.Immunohistochemistry was used to detect the expression of HMGB1in colorectal cancer and adjacent normal colorectal tissues.Western blotting and quantitative real-time polymerase chain reaction(qRT-PCR)were used to detect HMGB1mRNA and protein expression levels in NFs and CAFs.The expression of HMGB1in supernatant was detected by ELISA.Small interfering RNA(siRNA)technique was applied to inhibit HMGB1 gene expression in CAFs cells.qRT-PCR and western blot were used to detect the expression of HMGB1andα-smooth muscle actin(α-SMA)gene and protein in the negative control transfection group(CAFs-siNC group)and HMGB1-siRNA transfection group(CAFs-siHMGB1group),respectively.After the cells in the two groups were co-cultured with SW480,the effects of HMGB1expression on migration and invasion of SW480were verified by scratch and transwell invasion experiments.Results The co-culture migration rate of NFs and SW480cells was(25.51±46.96)%,and the number of invasive cells was 92.97±9.36.The co-culture migration rate of CAFs and SW480cells was(63.38±9.25)%,and the number of invasive cells was 129.56±10.93.The differences were statistically significant,witht values of 5.665and 4.405,and Pvalues of 0.005and 0.012,respectively.HMGB1was less expressed or not expressed in normal colorectal tissue,but highly expressed in colorectal cancer tissue.The expression of HMGB1mRNA and protein in CAFs cells were 1.918±0.082and 0.834±0.051,respectively.The concentration of HMGB1in cell supernatant was 253.538±22.873,both higher than that in NFs cells,both P<0.05.HMGB1mRNA,α-SMA mRNA,HMGB1protein,α-SMA protein was 0.385±0.038,0.511±0.029,0.649±0.040,0.475±0.039in the CAFs-si-HMGB1group,and 1.002±0.051,0.998±0.047,0.964±0.031,0.746±0.047in the CAFs-siNC group,respectively.The differences were statistically significant(all P<0.05).The co-culture migration rate of CAFs-siNC and SW480cells was(62.99±9.55)%,and the number of invasive cells was 125.38±11.54.The co culture migration rate of CAFs-siHMGB1and SW480 cells was(26.22±6.48)%,and the number of invasive cells was 83.58±10.51.The differences were statistically significant.The t values were 5.522and 4.638,respectively,with Pvalues of 0.005and 0.010.Conclusions CAFs can promote the migration and invasion of SW480cells.HMGB1is highly expressed in CAFs cells and colorectal cancer tissues.Inhibiting HMGB1expression in CAFs cells can attenuate the promotion of migration and invasion of SW480cells by CAFs.
作者
孟凯
鲍慧婧
亢健
赵云霞
王明玉
李哲
于冰
MENG Kai;BAO Huijing;KANG Jian;ZHAO Yunxia;WANG Mingyu;LI Zhe;YU Bing(Taian Central Hospital Affiliated to Qingdao University,Taian271000,China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2023年第11期654-661,共8页
Chinese Journal of Cancer Prevention and Treatment
基金
青岛大学附属泰安市中心医院苗圃项目(2021MPM17)。
关键词
结直肠癌
高迁移率族蛋白B1
癌症相关成纤维细胞
肿瘤侵袭
肿瘤转移
colorectal cancer
high mobility family protein B1
carcinoma-associated fibroblasts
neoplasm invasion
neoplasm metastasis
