FBXO6介导的ERO1L泛素化降解对膀胱癌细胞增殖、迁移和侵袭的作用研究

Role of FBXO6-mediated ubiquitination and degradation of ERO1L in the proliferation,migration and invasion of bladder cancer cells

目的探究F盒蛋白6(FBXO6)对膀胱癌细胞的作用及其作用机制。方法体外培养人正常膀胱上皮细胞株(SV-HUC-1)和人膀胱癌细胞株(T24)。用过表达载体阴性对照(oe-NC)、过表达FBXO6(oe-FBXO6)、过表达内质网氧化还原蛋白-1样蛋白(oe-ERO1L)及oe-FBXO6和oe-ERO1L慢病毒液(MOI=20)感染T24细胞。RT-qPCR检测细胞FBXO6和ERO1L mRNA表达;放线菌酮(CHX)蛋白合成抑制实验检测T24细胞ERO1L蛋白稳定性;免疫共沉淀(Co-IP)实验检测FBXO6对ERO1L泛素化调控;Western blot检测细胞FBXO6和ERO1L蛋白表达;CCK-8检测细胞活力;克隆形成实验检测细胞增殖;Transwell实验检测细胞迁移和侵袭。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果与SV-HUC-1相比,T24细胞中FBXO6 mRNA(1.00±0.05比0.33±0.02)和蛋白表达(1.00±0.11比0.31±0.03)均降低(P均<0.05),而ERO1L mRNA(1.00±0.05比2.70±0.12)和蛋白表达(1.00±0.16比3.27±0.09)均升高(P均<0.05)。FBXO6可降低ERO1L蛋白稳定性并促进ERO1L泛素化。与空白对照和oe-NC相比,oe-FBXO6细胞中FBXO6 mRNA(1.00±0.06比3.74±0.18)和蛋白表达(1.00±0.10比2.25±0.06)均升高,ERO1L蛋白表达(0.99±0.08比0.21±0.03),细胞活力、克隆形成数[(78.00±3.00)比(41.67±2.52)个]、迁移[(150.67±5.03)比(91.67±5.51)个]和侵袭细胞数[(122.00±7.00)比(74.67±5.51)个]均降低(P均<0.05);与oe-NC相比,oe-ERO1L细胞中ERO1L蛋白表达(1.01±0.06比2.58±0.02)、细胞活力、克隆形成数[(78.00±3.00)比(121.67±7.64)个]、迁移[(150.67±5.03)比(230.33±12.01)个]和侵袭细胞数[(122.00±7.00)比(203.00±11.53)个]均升高(P均<0.05);与oe-FBXO6相比,oe-FBXO6+oe-ERO1L细胞中ERO1L蛋白表达(0.54±0.02比1.02±0.06),细胞活力、克隆形成数[(41.67±2.52)比(62.00±3.61)个]、迁移[(91.67±5.51)比(131.67±6.03)个]和侵袭细胞数[(74.67±5.51)比(102.67±7.51)个]均升高(P均<0.05)。结论FBXO6通过介导ERO1L泛素化降解抑制膀胱癌细胞增殖、迁移和侵袭。

Objective To explore the effect of F-box protein 6(FBXO6)on bladder cancer cells and its possible mechanism.Methods Human normal bladder epithelial cell line(SV-HUC-1)and human bladder cancer cell line(T24)were cultured in vitro.T24 cells were infected with negative control for overexpression vector(oe-NC),overexpression FBXO6(oe-FBXO6),overexpression ERO1L(oe-ERO1L)and oe-FBXO6 and oe-ERO1L lentivirus(MOI=20).RT-qPCR was used to detect the expression of FBXO6 and endoplasmic reticulum oxidoreductin-1-like protein(ERO1L)mRNA in T24 cells;the cycloheximide protein synthesis inhibition test was used to detect the stability of ERO1L protein;co-immunoprecipitation experiment was used to detect the effect of FBXO6 on the ubiquitination of ERO1L;Western blot was used to detect the expression of FBXO6 and ERO1L protein in cells;CCK-8 was used to detect cell viability;clone formation experiment was used to detect the proliferation of cells;Transwell experiment was used to detect the migration and invasion of cells.Independent sample t test was used for comparison between two groups,one-way ANOVA was used for comparison between multiple groups,and LSD-t test was used for comparison between two groups.Results Compared with the SV-HUC-1 cells,the expression of FBXO6 mRNA and protein cells(1.00±0.05 vs 0.33±0.02)(1.00±0.11 vs 0.31±0.03)was significantly lower in T24 cells(both P<0.05),while the expression of ERO1L mRNA and protein(1.00±0.05 vs 2.70±0.12)(1.00±0.16 vs 3.27±0.09)was significantly increased(both P<0.05).FBXO6 could inhibit the stability of ERO1L protein and promote ERO1L ubiquitination.Compared with the blank control and the OE-NC cells,the expression of FBXO6 mRNA and protein(1.00±0.06 vs 3.74±0.18)(1.00±0.10 vs 2.25±0.06)was significantly increased in the oe-FBXO6 cells(both P<0.05),while the expression of ERO1L protein(0.99±0.08 vs 0.21±0.03),cell viability,number of clones(78.00±3.00 vs 41.67±2.52),the number of migration(150.67±5.03 vs 91.67±5.51)and invasion cells(122.00±7.00 vs 74.67±5.51)were significantly reduced(all P<0.05);Compared with the oe-NC cells,the expression of ERO1L protein(1.01±0.06 vs 2.58±0.02),cell viability,number of clones(78.00±3.00 vs 121.67±7.64),number of migrating(150.67±5.03 vs 230.33±12.01)and invading cells(122.00±7.00 vs 203.00±11.53)were significantly increased in the ox-ERO1L cells(all P<0.05);Compared with the oe-FBXO6 cells,the expression of ERO1L protein(0.54±0.02 vs 1.02±0.06),cell viability,number of clones(41.67±2.52 vs 62.00±3.61),number of migrating(91.67±5.51 vs 131.67±6.03)and invasive cells(74.67±5.51 vs 102.67±7.51)were significantly increased(all P<0.05).Conclusion FBXO6 inhibits the proliferation,migration and invasion of bladder cancer cells by mediating the degradation of ERO1L ubiquitination.