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中华细胞与干细胞杂志(电子版) ›› 2022, Vol. 12 ›› Issue (03) : 153 -160. doi: 10.3877/cma.j.issn.2095-1221.2022.03.004

论著

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
邓凌钢1, 孙建明1,(), 陈晓峰1   
  1. 1. 423000 郴州,湖南省郴州市第一人民医院泌尿外科
  • 收稿日期:2021-06-21 出版日期:2022-06-01
  • 通信作者: 孙建明

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

Linggang Deng1, Jianming Sun1,(), Xiaofeng Chen1   

  1. 1. Chenzhou First People's Hospital Urology, Hunan Province, chenzhou 423000, China
  • Received:2021-06-21 Published:2022-06-01
  • Corresponding author: Jianming Sun
引用本文:

邓凌钢, 孙建明, 陈晓峰. 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[J/OL]. 中华细胞与干细胞杂志(电子版), 2022, 12(03): 153-160.

Linggang Deng, Jianming Sun, Xiaofeng Chen. Role of FBXO6-mediated ubiquitination and degradation of ERO1L in the proliferation, migration and invasion of bladder cancer cells[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(03): 153-160.

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.

表1 引物序列信息
图1 Western blot检测细胞FBXO6和ERO1L蛋白表达
表2 SV-HUC-1和T24细胞中FBXO6和ERO1L的表达( ± sn = 3)
图2 Western blot检测细胞FBXO6和ERO1L蛋白表达
图3 FBXO6对T24细胞中ERO1L蛋白稳定性的影响注:Western blot检测细胞中FBXO6和ERO1L蛋白表达;放线菌酮CHX蛋白合成抑制实验检测细胞ERO1L蛋白稳定性,aP < 0.05,n = 3
表3 FBXO6对T24细胞中FBXO6和ERO1L的影响( ± sn = 3)
图4 免疫共沉淀实验检测FBXO6对ERO1L泛素化调控
图5 Western blot检测细胞中FBXO6和ERO1L蛋白表达
表4 Western blot检测细胞中FBXO6和ERO1L蛋白表达( ± sn = 3)
图6 倒置显微镜下观察FBXO6/ERO1L轴对T24细胞克隆形成数的影响(结晶紫染色,×1)注:与oe-NC比较,aP < 0.05;与oe-FBXO6比较,bP < 0.05;n = 3;ns为差异无统计学意义
表5 FBXO6/ERO1L轴对T24细胞活力的影响( ± sn = 3)
图7 倒置显微镜下观察FBXO6/ERO1L轴对T24细胞迁移和侵袭的影响(结晶紫染色,×200)注:迁移和侵袭结果显示,空白对照被染色的细胞数较多;oe-NC细胞数与空白对照相比差异无统计学意义;oe-FBXO6细胞被染色的细胞数比oe-NC减少;oe-ERO1L细胞被染色的细胞数比oe-NC增多;oe-FBXO6+oe-ERO1L细胞被染色的细胞数比oe-FBXO6减少
表6 FBXO6/ERO1L轴对T24细胞迁移和侵袭的影响( ± sn = 3)
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