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

1	Tan J, Jiang XZ, Yin GM, et al. Anacardic acid induces cell apoptosis of prostatic cancer through autophagy by ER stress/DAPK3/Akt signaling pathway[J]. Oncol Rep, 2017, 38(3):1373-1382.
2	Zhang H, Meng H, Huang X, et al. lncRNA MIR4435-2HG promotes cancer cell migration and invasion in prostate carcinoma by upregulating TGF-β1[J]. Oncol Lett, 2019, 18(4):4016-4021.
3	Gao RX, Zhang NW, Yang JY, et al. Long non-coding RNA ZEB1-AS1 regulates miR-200b/FSCN1 signaling and enhances migration and invasion induced by TGF-beta 1 in bladder cancer cells[J]. J Exp Clin Cancer Res, 2019, 38(1):111-125.
4	Chen YE, Tian T, Li ZY, et al. FSCN1 is an effective marker of poor prognosis and a potential therapeutic target in human tongue squamous cell carcinoma[J]. Cell Death Dis, 2019, 10(5):356-366.
5	Arlt MJ, Kuzmanov A, Snedeker JG, et al. Fascin-1 enhances experimental osteosarcoma tumor formation and metastasis and is related to poor patient outcome[J]. BMC Cancer, 2019, 19(1):83-91.
6	Chen JJ, Cai WY, Liu XW, et al. Reverse correlation between MicroRNA-145 and FSCN1 affecting gastric cancer migration and invasion[J]. PLoS One, 2015, 10(5):e0126890.
7	Yang XC, Lei PF, Huang Y, et al. MicroRNA-133b inhibits the migration and invasion of non small cell lung cancer cells via targeting FSCN1[J]. Oncol Lett, 2016, 12(5):3619-3625.
8	Fuse M, Nohata N, Kojima S, et al. Restoration of miR-145 expression suppresses cell proliferation, migration and invasion in prostate cancer by targeting FSCN1[J]. Int J Oncol, 2011, 38(4):1093-1101.
9	Gao AC, Isaacs JT. Expression of homeobox gene-GBX2 in human prostatic cancer cells[J]. Prostate, 1996, 29(6):395-398.
10	Choubey VK, Sankhwar SN, Carlus SJ, et al. SRD5A2 gene polymorphisms and the risk of benign prostatic hyperplasia but not prostate cancer[J]. Asian Pac J Cancer Prev, 2015, 16(3):1033-1036.
11	Xue M, Pang H, Li X, et al. Long noncoding RNA UCA1 promotes bladder cancer cell migration and invasion via hsa-miR-145/ZEB1/2/FSCN1 pathway[J]. Cancer Sci, 2016, 107(1):18-27.
12	Zheng K, Liu W, Liu Y, et al. MicroRNA-133a suppresses colorectal cancer cell invasion by targeting Fascin1[J]. Oncol Lett, 2015, 9(2):869-874.
13	皮文, 于长华, 刘志标. 慢病毒表达系统介导的RNAi技术靶向沉默Bi-1基因对鼻咽癌细胞生物学特性的影响[J]. 中国老年学杂志, 2017, 37(3):555-557.
14	Cui XP, Liu Y, Wang B, et al. Knockdown of GPR137 by RNAi inhibits pancreatic cancer cell growth and induces apoptosis[J]. Biotechnol Appl Biochem, 2015, 62(6):861-867.
15	Pan XH, Zhang XL, Sun HL, et al. Autophagy inhibition promotes 5-Fluorouraci-Induced apoptosis by stimulating ROS formation in human Non-Small cell lung cancer a549 cells[J]. PLoS One, 2013, 8(2):e56679.
16	Luo Y, Yang X, Zhao S, et al. Hydrogen sulfide prevents OGD/R-induced apoptosis via improving mitochondrial dysfunction and suppressing an ROS-mediated caspase-3 pathway in cortical neurons[J]. Neurochem Int, 2013, 63(8):826-831.
17	Gundala SR, Yang CH, Mukkayilli RA, et al. Hydroxychavicol, a betel leaf component, inhibits prostate cancer through ROS-driven DNA damage and apoptosis[J]. Toxicol Appl Pharmacol, 2014, 280(1):86-96.
18	张成娟. HOI-02通过ROS诱导食管癌细胞凋亡和G2-M期阻滞的机制研究[D]. 郑州: 郑州大学, 2015.
19	Yuan Y, Anbalagan D, Lee LH, et al. ANXA1 inhibits miRNA-196a in a negative feedback loop through NF-kB and C-Myc to reduce breast cancer proliferation[J]. Oncotarget, 2016, 7(19):27007-27020.
20	Qaiser F, Trembley JH, Sadiq S, et al. Examination of CK2 alpha and NF-kappa B p65 expression in human benign prostatic hyperplasia and prostate cancer tissues[J]. Mol Cell Biochem, 2016, 420(1/2):43-51.
21	Ho HH, Chang CS, Ho WC, et al. Anti-metastasis effects of Gallic acid on gastric cancer cells involves inhibition of NF-kappaB activity and downregulation of PI3K/AKT/small GTPase signals[J]. Food Chem Toxicol, 2010, 48(8/9):2508-2516.
22	彭昭, 田翠时, 高会斌, 等. PCNA、8-OHdG在胃癌组织中的表达及意义[J]. 实用医学杂志, 2016, 32(8):1274-1276.
23	Inserte J, Cardona M, Poncelas-Nozal M, et al. Studies on the role of apoptosis after transient myocardial ischemia:genetic deletion of the executioner caspases-3 and-7 does not limit infarct size and ventricular remodeling[J]. Basic Res Cardiol, 2016, 111(2):1-10.

[1]	钟雅雯, 王煜, 王海臻, 黄莉萍. 肌苷通过抑制线粒体通透性转换孔开放缓解缺氧/复氧诱导的人绒毛膜滋养层细胞凋亡[J/OL]. 中华妇幼临床医学杂志(电子版), 2024, 20(05): 525-533.
[2]	唐亦骁, 陈峻, 连正星, 胡海涛, 鲁迪, 徐骁, 卫强. 白果内酯对小鼠肝缺血再灌注损伤保护作用研究[J/OL]. 中华移植杂志(电子版), 2024, 18(05): 278-282.
[3]	李伟, 宋子健, 赖衍成, 周睿, 吴涵, 邓龙昕, 陈锐. 人工智能应用于前列腺癌患者预后预测的研究现状及展望[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(06): 541-546.
[4]	祝炜安, 林华慧, 吴建杰, 黄炯煅, 吴婷婷, 赖文杰. RDM1通过CDK4促进前列腺癌细胞进展的研究[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(06): 618-625.
[5]	王功炜, 李书豪, 魏松, 吕博然, 胡成. 溶瘤病毒M1对不同前列腺癌细胞杀伤效果的研究[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(06): 626-632.
[6]	施一辉, 张平新, 朱勇, 杨德林. 机器人辅助前列腺根治术后切缘阳性的研究进展[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(06): 633-637.
[7]	胡思平, 熊性宇, 徐航, 杨璐. 衰老相关分泌表型因子在前列腺癌发生发展中的作用机制[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(05): 425-434.
[8]	杨勇军, 曾一鸣, 贺显雅, 卢强, 李远伟. ASA分级≥Ⅲ级患者局麻经会阴前列腺多模态影像融合穿刺的安全性和有效性[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(05): 441-447.
[9]	李鑫钊, 张廷涛, 朱峰, 刘金山, 刘大闯. 血纤维蛋白原、D-二聚体及碱性磷酸酶诊断前列腺癌骨转移的价值分析[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(05): 459-463.
[10]	郑俊, 吴杰英, 谭海波, 郑安全, 李腾成. EGFR-MEK-TZ三联合分子的构建及其对去势抵抗性前列腺癌细胞增殖与凋亡的影响[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(05): 503-508.
[11]	李勇, 彭天明, 王倩倩, 陈育纯, 蒲小勇, 刘久敏. 基于失巢凋亡相关基因的膀胱癌预后模型构建及分析[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(04): 331-339.
[12]	黄程鑫, 陈莉, 刘伊楚, 王水良, 赖晓凤. OPA1 在乳腺癌组织的表达特征及在ER阳性乳腺癌细胞中的生物学功能研究[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(05): 275-284.
[13]	杜霞, 马梦青, 曹长春. 造影剂诱导的急性肾损伤的发病机制及干预靶点研究进展[J/OL]. 中华肾病研究电子杂志, 2024, 13(05): 279-282.
[14]	王国强, 张纲, 唐建坡, 张玉国, 杨永江. LINC00839 调节miR-17-5p/WEE1 轴对结直肠癌细胞增殖、凋亡和迁移的影响[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(06): 491-499.
[15]	靳英, 付小霞, 陈美茹, 袁璐, 郝力瑶. CD147调控MAPK信号通路对结肠癌细胞增殖和凋亡的影响及机制研究[J/OL]. 中华临床医师杂志(电子版), 2024, 18(05): 474-480.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

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

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

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