IL-17A对肝癌细胞系干性的影响

Effect of interleukin-17A on stemness of hepatoma cell lines

目的探讨IL-17A对人肝癌细胞系Hep 3B、MHCC97H及MHCC97L干性的影响,以期分析IL-17A与肝癌发展之间的关系。方法选用人肝癌细胞系Hep 3B、MHCC97H及MHCC97L通过体外3D成球实验分析IL-17A对其成球能力的影响,分为只含有普通培养液的对照组和加50 ng/ml IL-17A的实验组。采用实时无标记动态细胞分析技术检测IL-17A对成球能力增强的肝癌细胞增殖及迁移的影响;采用实时定量PCR检测分析IL-17A对成球能力增强的肝癌细胞所表达的IL-17A受体IL-17RA、IL-17RC以及干性相关基因SOX2、NANOG、OCT4、BMI1的mRNA表达的影响;通过Western Blot检测上皮-间质转换(EMT)相关蛋白E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(vimentin)的表达。计量资料组间比较采用t检验。结果在存在50 ng/ml IL-17A及接种细胞数均为500个的条件下,Hep 3B肝癌细胞的成球数目显著增加[由(113.0±10.3)个增加至(180.0±7.2)个,t=5.533,P<0.001],MHCC97H(t=1.087,P=0.325)及MHCC97L(t=0.279,P=0.785)的成球数目无明显变化。对于成球数目增加的Hep 3B细胞,进一步分析发现IL-17A促进了其增殖和迁移。并且,加入50 ng/ml IL-17A后,IL-17A受体IL-17RA及IL-17RC mRNA的表达随着作用时间的增加而升高;加入50 ng/ml IL-17A作用后,Hep 3B细胞的干性相关基因SOX2(t=4.749,P=0.042)、NANOG(t=19.600,P=0.003)、OCT4(t=37.310,P<0.001)和BMI1(t=16.810,P=0.004)的mRNA表达均显著上调;通过Western blot检测发现EMT相关上皮源性标志物E-Cadherin表达无明显的降低趋势,vimentin表达未出现稳定升高,间质性标志物N-Cadherin在50 ng/ml IL-17A作用48 h时后其mRNA表达增加(t=17.620,P=0.036)。结论 IL-17A在一定程度上增强了Hep 3B肝癌细胞的干性。

Objective To investigate the effect of interleukin - 17A （ IL - 17A） on stemness of human hepatoma cell lines Hep 3B, MH- CC97H, and MHCC97L and the association between IL - 17A and the progression of liver cancer. Methods Human hepatoma cell lines Hep 3B, MHCC97H, and MHCC97L were selected, and in vitro 3D sphere formation assay was used to analyze the effect of IL - 17A on sphere formation ability. The control group with common culture solution and the experimental group with 50 ng/ml IL - 17A were estab- lished. Real - time cellular analysis was used to determine the effect of IL - 17A on the proliferation and migration of hepatoma cells with en- hanced sphere formation ability; quantitative real - time PCR was used to measure the changes in the mRNA expression of IL - 17A receptors IL- 17RA and IL- 17RC and stemness- related genes SOX2, NANOG, OCT4, and BMI1 in hepatoma cells with enhanced sphere forma- tion ability ; Western blot was used to measure the expression of epithelial - mesenchymal transition - related proteins E - cadherin, N - cad- herin, and vimentin. The t - test was used for comparison of continuous doota betwwen groups. Results With the presence of 50 ng/ml IL - 17A and 500 inoculated cells, Hep 3B cells had a significant increase in the number of spheres formed （ 113.0 ± 10.3 vs 180.0 ± 7.2, t = 5. 533, P 〈0.001 ） , while MHCC97H and MHCC97L ceils showed no significant changes （t = 1. 087 and 0. 279, P =0.325 and 0. 785）. The analysis showed that IL - 17A promoted the proliferation and migration of Hep 3B cells with an increased number of spheres formed. Af- ter the addition of 50 ng/ml IL - 17A, there was an increase in the mRNA expression of IL - 17A receptors IL - 17RA and IL - 17RC over the time of treatment ; Hep 3B cells showed significant increases in the mRNA expression of sternness - related genes SOX2 （ t = 4. 749, P = 0. 042）, NANOG （ t = 19. 600, P = 0. 003 ）, OCT4 （ t = 37.310, P 〈 0. 001 ）, and BMI1 （ t = 16. 810, P = 0. 004）. Western blot showed no significant change in the expression of the epithelium - derived marker E - Cadherin ; there was an increase in the expression of the interstitial marker N - cadherin after the treatment with 50 ng/ml IL - 17A for 48 hours （t = 17. 620,P = 0. 036） ; there was no significant change in the expression of vimentin. Conclusion To a certain degree, IL - 17A enhances the sternness of Hep 3B hepatoma cells.