PSME3-NF-?资B途径介导三阴性乳腺癌的侵袭与转移

Invasion and metastasis of triple-negative breast cancer mediated by the PSME3-NF-kappaB pathway

摘要:

目的：研究蛋白酶体活化复合物3（proteasome activator complex subunit 3，PSME3）对三阴性乳腺癌侵袭及转移的影响及其机制。方法：收集临床三阴性乳腺癌患者标本，免疫组织化学检测PSME3蛋白的表达；以三阴性乳腺癌细胞株MDA-MB-231为对象，利用慢病毒包装系统构建PSME3过表达及干扰稳定株。Real-time PCR法检测PSME3的mRNA表达，Western blot检测PSME3的蛋白表达。实验分为PSME3过表达（expression of PSME3，exPSME3）组、PSME3干扰（short hairpin RNA targeting PSME3，shPSME3）组和野生型对照组。在以Western blot法进行稳定株鉴定时，为排除载体本身对PSME3表达的影响，特增加PSME3过表达阴性对照（expression negative control，exNC）组、PSME3干扰阴性对照（short hairpin RNA targeting negative con－trol，shNC）组。划痕实验检测细胞迁移能力，Transwell实验检测细胞侵袭能力，Western blot进一步检测迁移相关蛋白血管内皮生长因子-A（vascular endothelial growth factor-A，VEGF-A）、基质金属蛋白酶-9（matrix metalloproteinase-9，MMP-9）在各组的表达情况。细胞爬片免疫荧光与Western blot分别检测核因子-?资B（nuclear factor-?资B，NF-?资B）及其活性基团，磷酸化的P65蛋白（phosphorylated-P65，P-P65）的表达。结果：免疫组织化学结果显示PSME3在三阴性乳腺癌患者肿瘤组织表达明显高于癌旁组织（t=36.700，P=0.000）。划痕实验结果显示exPSME3组的迁移能力高于对照组（P=0.000），shPSME3组的迁移能力低于对照组（P=0.009）。迁移相关蛋白检测示VEGF-A、MMP9均在exPSME3组高表达，在shPSME3组低表达，与对照组相比有统计学差异。Transwell实验结果显示exPSME3组的侵袭能力高于对照组（P=0.000），shPSME3组的侵袭能力低于对照组（P=0.000）。细胞爬片免疫荧光结果提示各组间NF-?资B蛋白表达无明显差异，而exPSME3组P-P65表达明显高于对照组（P=0.001），shPSME3组P-P65表达低于对照组（P=0.001），Western blot检测结果与之一致。结论：PSME3- NF-?资B途径可介导三阴性乳腺癌的侵袭及转移。

Abstract:

Objective：To investigate the effect of proteasome activator complex subunit 3（PSME3） on the invasion and metastasis of triple-negative breast cancer（TNBC） and its mechanism. Methods：The samples collected from TNBC patients were used to detect the expression of PSME3 by immunohistochemistry. The TNBC cell line MDA-MB-231 was used to construct PSME3 overexpression and interference stable strains using the lentiviral packaging system. The mRNA expression of PSME3 was determined by real-time PCR and the protein expression of PSME3 was measured by Western blot. The cell lines were divided into PSME3 overexpression（exPSME3） group，short hairpin RNA targeting PSME3（shPSME3） group，and wild-type control group. When Western blot was used to identify stable strains，PSME3 expression negative control（exNC） group and short hairpin RNA targeting negative control（shNC） group were included in order to exclude the effect of the vector itself on PSME3 expression. The cell migration ability and in－vasion ability were evaluated by wound healing assay and Tran－swell assay，respectively. The expression of vascular endothelial growth factor-A（VEGF-A） and matrix metalloproteinase-9（MMP-9）（migration-related proteins） in each group was mea－sured by Western blot. The protein expression of nuclear factor-kappaB（NF-kappaB） and its active group — phosphorylated-P65（P-P65） was determined by cell slide immunofluorescence and Western blot，respectively. Results：Immunohistochemical results showed that PSME3 expression was significantly higher in the tumor tissues of TNBC patients than in the adjacent tissues（t=36.700，P=0.000）. The wound healing assay indicated that compared with the control group，the migration ability was significantly higher in the exPSME3 group（P=0.000） and significantly lower in the shPSME3 group（P=0.009）. Western blot results showed that compared with the control group，the expression of VEGF-A and MMP-9 was signif－icantly higher in the exPSME3 group and significantly lower in the shPSME3 group. Transwell assay revealed that compared with the control group，the invasion ability was significantly higher in the exPSME3 group（P=0.000） and significantly lower in the shPSME3 group（P=0.000）. Cell slide immunofluorescence results showed that there was no significant difference in the expression of NF-kappaB between the three groups；compared with the control group，the expression of P-P65 was significantly higher in the exPSME3 group（P=0.001） and significantly lower in the shPSME3 group（P=0.001），which was in accordance with Western blot results. Conclusion：The PSME3-NF-kappaB pathway can mediate the invasion and metastasis of TNBC.