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

doi:10.3877/cma.j.issn.2095-1221.2022.03.004

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。

关键词: 膀胱癌, F盒蛋白6, 内质网氧化还原蛋白-1样蛋白, 迁移, 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.

Key words: Bladder cancer, F-box protein 6, Endoplasmic reticulum oxidoreductin-1-like protein, Migration, Invasion

表1 引物序列信息

基因	序列（5'→3'）	预期扩增产物长度（bp）
FBXO6	上游ACAGCTTCAGGACACGCAG	320
	下游GAGGTTCCTATGCAGGCTCC
ERO1L	上游GACATCAGCCAGTGTGGAAGA	271
	下游TCTTCTTAGATGACCACCAGCA
GAPDH （内参）	上游AATGGGCAGCCGTTAGGAAA	168
	下游GCGCCCAATACGACCAAATC

表1 引物序列信息

基因	序列（5'→3'）	预期扩增产物长度（bp）
FBXO6	上游ACAGCTTCAGGACACGCAG	320
	下游GAGGTTCCTATGCAGGCTCC
ERO1L	上游GACATCAGCCAGTGTGGAAGA	271
	下游TCTTCTTAGATGACCACCAGCA
GAPDH （内参）	上游AATGGGCAGCCGTTAGGAAA	168
	下游GCGCCCAATACGACCAAATC

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（ ± s，n = 3）

分组	FBXO6		ERO1L
分组	mRNA	蛋白表达	mRNA	蛋白表达
SV-HUC-1细胞	1.00±0.05	1.00±0.11	1.00±0.05	1.00±0.16
T24细胞	0.33±0.02	0.31±0.03	2.70±0.12	3.27±0.09
t值	21.552	29.715	33.094	54.251
P值	0.001	0.001	0.001	0.001

表2 SV-HUC-1和T24细胞中FBXO6和ERO1L的表达（ ± s，n = 3）

分组	FBXO6		ERO1L
分组	mRNA	蛋白表达	mRNA	蛋白表达
SV-HUC-1细胞	1.00±0.05	1.00±0.11	1.00±0.05	1.00±0.16
T24细胞	0.33±0.02	0.31±0.03	2.70±0.12	3.27±0.09
t值	21.552	29.715	33.094	54.251
P值	0.001	0.001	0.001	0.001

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（ ± s，n = 3）

分组	FBXO6		ERO1L
分组	mRNA	蛋白表达	mRNA	蛋白表达
空白对照	1.00±0.04	1.00±0.10	1.00±0.05	0.99±0.08
oe-NC	1.00±0.06	0.98±0.12	0.99±0.04	1.02±0.06
oe-FBXO6	3.74±0.18^a	2.25±0.06^a	0.98±0.05	0.21±0.03^a
F值	93.534	74.385	0.147	69.356
P值	0.001	0.001	0.001	0.001

表3 FBXO6对T24细胞中FBXO6和ERO1L的影响（ ± s，n = 3）

分组	FBXO6		ERO1L
分组	mRNA	蛋白表达	mRNA	蛋白表达
空白对照	1.00±0.04	1.00±0.10	1.00±0.05	0.99±0.08
oe-NC	1.00±0.06	0.98±0.12	0.99±0.04	1.02±0.06
oe-FBXO6	3.74±0.18^a	2.25±0.06^a	0.98±0.05	0.21±0.03^a
F值	93.534	74.385	0.147	69.356
P值	0.001	0.001	0.001	0.001

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（ ± s，n = 3）

分组	FBXO6蛋白表达	ERO1L蛋白表达
空白对照	1.00±0.07	1.00±0.06
oe-NC	0.99±0.06	1.01±0.06
oe-FBXO6	2.27±0.06^a	0.54±0.02^a
oe-ERO1L	1.10±0.05	2.58±0.02^a
oe-FBXO6+oe-ERO1L	2.23±0.06^a	1.02±0.06^b
F值	68.253	74.384
P值	0.001	0.001

表4 Western blot检测细胞中FBXO6和ERO1L蛋白表达（ ± s，n = 3）

分组	FBXO6蛋白表达	ERO1L蛋白表达
空白对照	1.00±0.07	1.00±0.06
oe-NC	0.99±0.06	1.01±0.06
oe-FBXO6	2.27±0.06^a	0.54±0.02^a
oe-ERO1L	1.10±0.05	2.58±0.02^a
oe-FBXO6+oe-ERO1L	2.23±0.06^a	1.02±0.06^b
F值	68.253	74.384
P值	0.001	0.001

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（ ± s，n = 3）

分组	OD值（450 nm）
分组	0 h	24 h	48 h	72 h
空白对照	0.26±0.02	0.46±0.02	0.79±0.03	1.24±0.09
oe-NC	0.25±0.01	0.46±0.03	0.78±0.03	1.23±0.08
oe-FBXO6	0.25±0.01	0.38±0.01^a	0.53±0.04^a	0.77±0.02^a
oe-ERO1L	0.26±0.01	0.54±0.03^a	0.92±0.03^a	1.53±0.09^a
oe-FBXO6+oe-ERO1L	0.26±0.01	0.46±0.02^b	0.65±0.03^b	1.03±0.07^b
F值	2.402	12.483	21.579	33.583
P值	0.294	0.005	0.001	0.001

表5 FBXO6/ERO1L轴对T24细胞活力的影响（ ± s，n = 3）

分组	OD值（450 nm）
分组	0 h	24 h	48 h	72 h
空白对照	0.26±0.02	0.46±0.02	0.79±0.03	1.24±0.09
oe-NC	0.25±0.01	0.46±0.03	0.78±0.03	1.23±0.08
oe-FBXO6	0.25±0.01	0.38±0.01^a	0.53±0.04^a	0.77±0.02^a
oe-ERO1L	0.26±0.01	0.54±0.03^a	0.92±0.03^a	1.53±0.09^a
oe-FBXO6+oe-ERO1L	0.26±0.01	0.46±0.02^b	0.65±0.03^b	1.03±0.07^b
F值	2.402	12.483	21.579	33.583
P值	0.294	0.005	0.001	0.001

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（ ± s，n = 3）

分组	迁移细胞数（个/每个视野）	侵袭细胞数（个/每个视野）
空白对照	149.33±7.51	124.00±8.00
oe-NC	150.67±5.03	122.00±7.00
oe-FBXO6	91.67±5.51^a	74.67±5.51^a
oe-ERO1L	230.33±12.01^a	203.00±11.53^a
oe-FBXO6+oe-ERO1L	131.67±6.03^b	102.67±7.51^b
F值	48.384	39.293
P值	0.001	0.001

表6 FBXO6/ERO1L轴对T24细胞迁移和侵袭的影响（ ± s，n = 3）

分组	迁移细胞数（个/每个视野）	侵袭细胞数（个/每个视野）
空白对照	149.33±7.51	124.00±8.00
oe-NC	150.67±5.03	122.00±7.00
oe-FBXO6	91.67±5.51^a	74.67±5.51^a
oe-ERO1L	230.33±12.01^a	203.00±11.53^a
oe-FBXO6+oe-ERO1L	131.67±6.03^b	102.67±7.51^b
F值	48.384	39.293
P值	0.001	0.001

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

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