干扰FSCN1基因表达对前列腺癌细胞凋亡、活性氧水平影响的研究

doi:10.3877/cmj.j.issn.2095-1221.2020.01.001

目的

探讨干扰FSCN1基因表达对前列腺癌细胞凋亡、活性氧（ROS）水平影响及机制。

方法

以正常前列腺上皮细胞RWPE-1为对照细胞，通过RT-PCR及Western blot检测前列腺癌LNCaP、DU145和PC-3细胞中FSCN1 mRNA及蛋白表达；以Lipofectamine^TM 2000为载体，DU145细胞分为si-FSCN1组（靶向抑制FSCN1的小干扰RNAs转染DU145细胞）、阴性对照组（随机序列转染DU145细胞）及空白对照组（未转染的细胞），siRNA转染48 h，Western blot检测FSCN1、PCNA、NF-κB p65、p-NF-κB p65、IKKα和p-IKKα的蛋白表达。CCK8检测细胞活力，流式细胞术检测细胞凋亡率及ROS水平。多组间比较采用单因素方差分析，组间两两比较采用SNK-q检验。

结果

与RWPE-1细胞比较，LNCaP、DU145和PC-3细胞中FSCN1 mRNA （1比2.561±0.189、7.183±0.882、4.796±0.567、4.796±0.567）及蛋白表达（0.053±0.007比0.217±0.013、0.654±0.058、0.316±0.035）均升高，差异具有统计学意义（P均< 0.05）。与阴性对照组比较，si-FSCN1组FSCN1表达（0.473±0.052比0.086±0.010）降低，差异具有统计学意义（P均< 0.05）。与si-FSCN1组比较，空白对照组、阴性对照组细胞活力（0.302±0.033比0.787±0.069、0.764±0.063）均升高，凋亡率（24.54﹪±1.47﹪比3.04﹪ ± 0.36﹪、3.28﹪±0.40﹪）和ROS相对荧光强度（90.04±5.73比47.88±3.62、49.62±4.11）均降低，差异具有统计学意义（P均< 0.05）。与si-FSCN1组比较，空白对照组、阴性对照组PCNA （0.255±0.032比0.654±0.062、0.631±0.058）、NF-κB p65 （0.092±0.011比0.296±0.032、0.318±0.037）、p-NF-κB p65 （0.042± 0.008比0.155±0.018、0.151±0.016）、IKKα （0.112±0.01比0.172±0.020、0.192±0.023）和p-IKKα的蛋白表达（0.051±0.005比0.102±0.011、0.091± 0.009）均升高，Cleaved caspase3蛋白表达（0.206±0.018比0.074±0.009、0.085±0.010）均降低，差异具有统计学意义（P均< 0.05）。阴性对照组与空白对照组细胞活力、凋亡率、ROS水平及FSCN1、PCNA、Cleaved caspase3、NF-κB p65、p-NF-κB p65、IKKα和p-IKKα的蛋白表达差异均无统计学意义（P > 0.05）。

结论

干扰FSCN1基因表达可降低前列腺癌细胞活力及诱导凋亡，机制可能与ROS水平升高及NF-κB信号下调有关。

关键词: 前列腺癌, FSCN1基因, ROS水平, 凋亡, NF-κB信号通路

Objective

To investigate the effect and mechanism of interfering FSCN1 gene expression on apoptosis and reactive oxygen species (ROS) level in prostate cancer cells.

Methods

The normal prostate epithelial cell RWPE-1 was used as the control, and the expression of FSCN1 mRNA and protein in the LNCaP, DU145 and PC-3 cells of prostate cancer were detected by RT-PCR and Western blot, respectively. Lipofectamine^TM 2000 was used as the carrier, DU145 cells were divided into si-FSCN1 group (small interfering RNAs targeting FSCN1 transfected DU145 cells) , negative control group (negative control siRNA transfected DU145 cells) and blank control group (untransfected cells) , siRNA was transfected for 48 h, and Western blot was used to detect the protein expression of FSCN1, PCNA, Cleaved caspase3, NF-κB p65, p-NF-κB p65, IKK and p-IKKα. Cell viability was detected by CCK8, and apoptosis rate and ROS level were detected by flow cytometry. One-way analysis of variance was used for comparison between groups, and SNK-q test was used for pairwise comparisons between groups.

Results

Compared with RWPE-1 cells, the expressions of FSCN1 mRNA in LNCaP, DU145 and PC-3cells (1 vs 2.561±0.189, 7.183±0.882, 4.796±0.567, 4.796±0.567) and protein (0.053±0.007 vs 0.217±0.013, 0.654±0.058, 0.316±0.035) were increased (all P< 0.05) . Compared with the negative control group, the expression of FSCN1 in the si-FSCN1 group were decreased (0.473±0.052 vs 0.086±0.010, P< 0.05) . Compared with the si-FSCN1 group, the cell viability of the blank control group and the negative control group (0.302±0.033 vs 0.787±0.069, 0.764±0.063) were increased, while the apoptotic rate (24.54±1.47﹪vs 3.04﹪±0.36﹪, 3.28﹪±0.40﹪) and ROS relative fluorescence intensity (90.04±5.73 vs 47.88±3.62, 49.62±4.11) were decreased (all P< 0.05) . Compared with si-FSCN1 group, the expression of PCNA (0.255±0.032 vs 0.654±0.062, 0.631± 0.058) , NF-κB p65 (0.092±0.011 vs 0.296±0.032, 0.318±0.037) , p-NF-κB p65 (0.042±0.008 vs 0.155±0.018, 0.151±0.016) , IKKα (0.112±0.01 vs 0.172±0.020, 0.192±0.023) and p-IKKα protein (0.051±0.005 vs 0.102±0.011, 0.091±0.009) were increased in blank control group and negative control group, while the expression of Cleaved caspase3 protein (0.206±0.018 vs 0.074±0.009, 0.085±0.010) decreased (all P< 0.05) . There was no significant difference in cell viability, apoptosis rate, ROS levels, and protein expression of FSCN1, PCNA, Cleaved caspase3, NF-κB p65, p-NF-κB p65, IKKα, and p-IKKα in the negative control group and the blank control group (P> 0.05) .

Conclusion

Interfering with FSCN1 gene expression could reduce the activity and induce apoptosis of prostate cancer cells. The mechanism may be related to the increase of ROS level and the decrease of NF- κB signal.

Key words: Prostate cancer, FSCN1 gene, ROS level, Apoptosis, NF-κB signaling pathway

表1 PCR引物序列信息

基因	序列（5'-3'）
FSCN1	上游CACAGGCAAATACTGGACGGT
FSCN1	下游CCACCTTGTTATAGTCGCAGAAC
GAPDH	上游ACAACTTTGGTATCGTGGAAGG
GAPDH	下游GCCATCACGCCACAGTTTC

表1 PCR引物序列信息

基因	序列（5'-3'）
FSCN1	上游CACAGGCAAATACTGGACGGT
FSCN1	下游CCACCTTGTTATAGTCGCAGAAC
GAPDH	上游ACAACTTTGGTATCGTGGAAGG
GAPDH	下游GCCATCACGCCACAGTTTC

图1 前列腺癌细胞FSCN1蛋白表达

表2 前列腺癌细胞FSCN1 mRNA及蛋白表达（±s，n = 3）

细胞	FSCN1 mRNA表达	FSCN1蛋白表达
RWPE-1	1	0.053±0.007
LNCaP	2.561±0.189^a	0.217±0.013^a
DU145	7.183±0.882^a	0.654±0.058^a
PC-3	4.796±0.567^a	0.316±0.035^a
F值	76.759	160.657
P值	< 0.001	< 0.001

表2 前列腺癌细胞FSCN1 mRNA及蛋白表达（±s，n = 3）

细胞	FSCN1 mRNA表达	FSCN1蛋白表达
RWPE-1	1	0.053±0.007
LNCaP	2.561±0.189^a	0.217±0.013^a
DU145	7.183±0.882^a	0.654±0.058^a
PC-3	4.796±0.567^a	0.316±0.035^a
F值	76.759	160.657
P值	< 0.001	< 0.001

图2 FSCN1 siRNA转染DU145细胞FSCN1蛋白表达

图3 FSCN1 siRNA对DU145细胞凋亡率的影响

表3 FSCN1 siRNA对DU145细胞活力及凋亡率、ROS水平的影响（±s，n = 3）

分组	细胞活力	凋亡率（﹪）	相对荧光强度
空白对照组	0.787±0.069	3.04±0.36	47.88±3.62
阴性对照组	0.764±0.063	3.28±0.40	49.62±4.11
si-FSCN1组	0.302±0.033^ab	24.54±1.47^ab	90.04±5.73^ab
F值	68.622	559.658	81.513
P值	< 0.001	< 0.001	< 0.001

表3 FSCN1 siRNA对DU145细胞活力及凋亡率、ROS水平的影响（±s，n = 3）

分组	细胞活力	凋亡率（﹪）	相对荧光强度
空白对照组	0.787±0.069	3.04±0.36	47.88±3.62
阴性对照组	0.764±0.063	3.28±0.40	49.62±4.11
si-FSCN1组	0.302±0.033^ab	24.54±1.47^ab	90.04±5.73^ab
F值	68.622	559.658	81.513
P值	< 0.001	< 0.001	< 0.001

图4 FSCN1 siRNA对DU145细胞NF-κB信号的影响

表4 三组PCNA、Cleaved caspase3、NF-κB p65、p-NF-κB p65、IKKα和p-IKKα的蛋白相对表达量（±s，n = 3）

分组	PCNA	Cleaved caspase3	NF-κB p65	P-NF-κB p65	IKKα	P-IKKα
空白对照组	0.654±0.062	0.074±0.009	0.296±0.032	0.155±0.018	0.172±0.020	0.102±0.011
阴性对照组	0.631±0.058	0.085±0.010	0.318±0.037	0.151±0.016	0.192±0.023	0.091±0.009
si-FSCN1组	0.255±0.032^ab	0.206±0.018^ab	0.092±0.011^ab	0.042±0.008^ab	0.112±0.015^ab	0.051±0.005^ab
F值	54.866	95.602	55.594	57.452	13.518	28.560
P值	< 0.001	< 0.001	< 0.001	< 0.001	0.006	0.001

表4 三组PCNA、Cleaved caspase3、NF-κB p65、p-NF-κB p65、IKKα和p-IKKα的蛋白相对表达量（±s，n = 3）

分组	PCNA	Cleaved caspase3	NF-κB p65	P-NF-κB p65	IKKα	P-IKKα
空白对照组	0.654±0.062	0.074±0.009	0.296±0.032	0.155±0.018	0.172±0.020	0.102±0.011
阴性对照组	0.631±0.058	0.085±0.010	0.318±0.037	0.151±0.016	0.192±0.023	0.091±0.009
si-FSCN1组	0.255±0.032^ab	0.206±0.018^ab	0.092±0.011^ab	0.042±0.008^ab	0.112±0.015^ab	0.051±0.005^ab
F值	54.866	95.602	55.594	57.452	13.518	28.560
P值	< 0.001	< 0.001	< 0.001	< 0.001	0.006	0.001

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Effect of interfering FSCN1 gene expression on apoptosis and ROS content in prostate cancer cells

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Effect of interfering FSCN1 gene expression on apoptosis and ROS content in prostate cancer cells