Objective To explore the effects of proteasome 20S subunit beta 8 (PSMB8) on the proliferation,migration,and invasion of clear cell renal cell carcinoma (ccRCC) cells and whether PSMB8 promotes tumor progression by activating the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway. Methods The Cancer Genome Atlas was employed to analyze the mRNA levels of PSMB8 in ccRCC and normal tissue,and the expression levels of PSMB8 in ccRCC tissue and cells were determined by real-time quantitative PCR,Western blotting,and immunohistochemistry.Furthermore,the cell lines with stable overexpression and knockdown of PSMB8 were constructed.The CCK-8 assay and colony formation assay were employed to examine the cell proliferation,and the wound healing assay and Transwell assay were employed to examine the invasion and migration of cells.Kyoto Encyclopedia of Genes and Genomes pathway enrichment was performed to analyze the co-expressed genes of PSMB8.Western blotting was used to measure the phosphorylation levels of the proteins in the MEK/ERK signaling pathway.Finally,the rescue experiment was carried out with the ERK agonist C16-PAF. Results Compared with the normal tissue,the ccRCC tissue showed up-regulated mRNA and protein levels of PSMB8 (both P<0.001),which were associated with the TNM stage of patients with ccRCC (P<0.001).Compared with the negative control group,overexpression of PSMB8 promoted the proliferation (P=0.021,P=0.039),migration and invasion (all P<0.001) of 786-O and ACHN cells,and the knockdown of PSMB8 inhibited the proliferation (P=0.022,P=0.005),migration and invasion (all P<0.001) of 786-O and ACHN cells.The pathway enrichment analysis of co-expressed genes of PSMB8 predicted the mitogen-activated protein kinase signaling pathway (P<0.001).After the knockdown of PSMB8,786-O and ACHN cells showed lowered phosphorylation levels of MEK1/2 (P=0.017,P=0.016) and ERK1/2 (P=0.010,P=0.040) and down-regulated transcription levels of ERK downstream factors c-Myc (P=0.043,P=0.038),c-Fos (P=0.025,P=0.008),and CyclinD1 (P=0.006,P=0.047).Compared with the ERK agonist C16-PAF group,the PSMB8 knockdown + C16-PAF group showed inhibited proliferation (P=0.003,P=0.002),migration and invasion (all P<0.001) of 786-O and ACHN cells. Conclusion PSMB8 may promote the proliferation,migration,and invasion of ccRCC cells by activating the MEK/ERK signaling pathway.
目的 探究蛋白酶体20S亚基β8(PSMB8)对肾透明细胞癌(ccRCC)细胞增殖、迁移和侵袭的影响,以及是否通过调控丝裂原活化蛋白激酶激酶(MEK)/细胞外信号调节激酶(ERK)信号通路发挥其作用。方法 采用癌症基因组图谱数据库分析ccRCC与正常组织PSMB8 mRNA的表达水平,并通过实时荧光定量PCR、Western blot和免疫组织化学染色等方法进一步检测PSMB8在ccRCC组织和细胞中的表达情况。构建稳定过表达和敲减PSMB8的细胞株,分别采用CCK-8法和平板克隆实验检测细胞的增殖能力,划痕愈合实验和Transwell实验检测细胞迁移和侵袭的能力。对PSMB8共表达基因进行京都基因与基因组百科全书通路富集分析,Western blot检测MEK/ERK通路相关蛋白的磷酸化水平,并加用ERK激动剂C16-PAF处理进行细胞功能学挽救实验。结果 与正常组织比较,PSMB8 mRNA和蛋白在ccRCC组织中呈高表达(P均<0.001),且与临床患者的TNM分期显著相关(P<0.001);与阴性对照组比较,过表达PSMB8可以促进786-O、ACHN细胞的增殖(P=0.021,P=0.039)、迁移和侵袭(P均<0.001),敲减PSMB8可以抑制786-O、ACHN细胞的增殖(P=0.022,P=0.005)、迁移和侵袭(P均<0.001);PSMB8共表达基因通路富集分析提示其可能与丝裂原活化蛋白激酶通路相关(P<0.001);敲减PSMB8后786-O和ACHN细胞MEK1/2(P=0.017,P=0.016)、ERK1/2(P=0.010,P=0.040)蛋白磷酸化水平及ERK下游因子c-Myc(P=0.043,P=0.038)、c-Fos(P=0.025,P=0.008)和CyclinD1(P=0.006,P=0.047)转录水平均下调;与ERK激动剂C16-PAF处理组比较,敲减PSMB8 + C16-PAF组明显抑制786-O、ACHN细胞的增殖(P=0.003,P=0.002)、迁移和侵袭能力(P均<0.001)。结论 PSMB8通过激活MEK/ERK信号通路从而促进ccRCC细胞的增殖、迁移及侵袭。.
Keywords: clear cell renal cell carcinoma; mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathway; proteasome 20S subunit beta 8.