Krüppel样因子7对前列腺癌细胞生物学行为的影响及分子机制

Effect and molecular mechanism of Krüppel-like factor 7 on the biological behavior of castration-resistant prostate cancer cells

目的探讨Krüppel样因子7(KLF7)高表达对去势抵抗性前列腺癌(PCa)细胞增殖、侵袭与迁移的影响及分子机制。方法RNA-seq结合生物信息学方法预测KLF7下游靶基因。收集2017-09-01-2019-09-31在石河子市人民医院住院PCa患者肿瘤组织和前列腺增生(BPH)组织石蜡切片各10例,免疫组化法检测前列腺组织中KLF7及下游靶基因的表达水平。体外培养去势抵抗性PCa细胞系PC-3,分别设置上和下调KLF7组、上和下调下游靶基因组及上调KLF7同时下调下游靶基因组。qRT-PCR和蛋白质印迹法检测细胞中KLF7及下游靶基因的mRNA和蛋白表达水平,CCK-8法检测细胞增殖能力,Transwell法检测细胞的侵袭与迁移能力;双荧光素酶报告基因实验检测KLF7对下游靶基因是否具有转录激活作用。结果RNA-seq结合生物信息学预测结果显示,转化生长因子-α(TGF-α)可能是KLF7的下游靶基因。与前列腺组织相比,KLF7(0.89±0.26 vs 2.19±0.45,t=7.989,P<0.001)及TGF-α(2.06±0.54 vs 2.60±0.39,t=2.474,P=0.024)在PC-3组织中均高表达。上调(1.00±0.35 vs 2.09±0.08,t=5.345,P=0.006)、下调(1.00±0.17 vs 0.36±0.01,t=6.562,P=0.003)KLF7后TGF-α的mRNA和蛋白表达水平升高和降低;上调KLF7可显著促进PC-3细胞的增殖(72 h:1.73±0.07 vs 1.90±0.02,t=4.269,P=0.013)、侵袭(57.11±18.68 vs 95.78±30.84,t=3.217,P=0.005)与迁移(121.56±33.67 vs 240.11±36.56,t=7.156,P<0.001)能力;下调KLF7可显著抑制PC-3细胞的增殖(72 h:2.12±0.07 vs 1.77±0.02,t=8.671,P=0.001)、侵袭(210.00±56.73 vs 87.33±16.90,t=5.888,P<0.001)与迁移(208.33±43.44 vs 138.22±33.61,t=3.830,P=0.002)能力;上调TGF-α可显著促进PC-3细胞的增殖(72 h:1.90±0.06 vs 2.39±0.03,t=15.442,P<0.001)、侵袭(152.67±8.11 vs 201.22±7.30,t=13.356,P<0.001)与迁移(107.00±27.79 vs 163.00±15.80,t=5.255,P<0.001)能力;下调TGF-α可显著抑制PC-3细胞的增殖(72 h:2.31±0.07 vs 1.80±0.11,t=6.695,P=0.003)、侵袭(188.44±20.43 vs 108.56±27.96,t=6.922,P<0.001)与迁移(192.11±22.18 vs 110.67±30.67,t=6.456,P<0.001)能力;上调KLF7同时下调TGF-α后,可逆转KLF7对PC-3细胞增殖(72 h:2.40±0.11 vs 1.87±0.03,t=8.916,P<0.001)、侵袭(83.67±18.25 vs 61.56±12.10,t=3.029,P=0.008)和迁移(98.78±11.71 vs 60.67±6.00,t=8.688,P<0.001)的促进作用。双荧光素酶报告基因实验结果显示,KLF7对TGF-α无直接转录激活作用(1.00±0.17 vs 1.06±0.06),t=0.704,P=0.508。结论高表达的KLF7可通过上调TGF-α表达,促进去势抵抗性PCa细胞PC-3的增殖、侵袭与迁移能力。

Objective To investigate the effect of high expression of Krüppel-like factor 7(KLF7)on the biological behavior of castration-resistant prostate cancer(PCa)cells and the molecular mechanism.Methods RNA-seq combined with bioinformatics was used to predict the downstream target genes of KLF7;From September 1,2017to September 31,2019,10paraffin sections of tumor tissues from patients with PCa and paraffin sections of individual prostate tissues from patients with benign prostatic hyperplasia(BPH)were collected from Shihezi People's Hospital.The expression levels of KLF7and downstream target genes were detected in prostate tissues by immunohistochemistry;After the castration-resistant PCa cell(PC-3)was cultured in vitro,the up/down-regulated KLF7group,up/down-regulated downstream target genes group,and the group which up-regulated KLF7while down-regulated downstream target genes were established.qRT-PCR and Western Blot assay were performed to detect the mRNA and protein expression levels of KLF7and downstream target genes in cells;CCK8assay was used to detect the proliferation ability of cells;Transwell assay was used to detect the invasion and migration ability of cells;Dual luciferase reporter gene assay was performed to detect whether KLF7has transcriptional activation effect on downstream target genes.Results RNA-seq combined with bioinformatics prediction showed that transforming growth factor-α(TGF-α)might be a downstream target gene of KLF7;both KLF7and TGF-αwere significantly overexpressed in tumor tissues of PCa patients compared with BPH individuals(KLF7:0.89±0.26 vs 2.19±0.45,t=7.989,P<0.001;TGF-α:2.06±0.54 vs 2.60±0.39,t=2.474,P=0.024);After up/down-regulated KLF7,the mRNA(up-regulated:1.00±0.35 vs 2.09±0.08,t=5.345,P=0.006;downregulated:1.00±0.17 vs 0.36±0.01,t=6.562,P=0.003)and protein expression levels of TGF-αwere significantly increased/decreased.After up-regulated KLF7,the proliferation,invasion and migration ability of PCa cells were significantly enhanced(proliferation of 72h:1.73±0.07 vs 1.90±0.02,t=4.269,P=0.013;invasion:57.11±18.68 vs95.78±30.84,t=3.217,P=0.005;migration:121.56±33.67 vs 240.11±36.56,t=7.156,P<0.001).After down-regulated KLF7,the proliferation,invasion and migration ability of PCa cells were significantly weakened(proliferation of 72h:2.12±0.07 vs 1.77±0.02,t=8.671,P=0.001;invasion:210.00±56.73 vs 87.33±16.90,t=5.888,P<0.001;migration:208.33±43.44 vs 138.22±33.61,t=3.830,P=0.002).Next,after up-regulated TGF-α,the proliferation,invasion and migration ability of PCa cells were significantly enhanced(proliferation of 72h:1.90±0.06 vs2.39±0.03,t=15.442,P<0.001;invasion:152.67±8.11 vs 201.22±7.30,t=13.356,P<0.001;migration:107.00±27.79 vs 163.00±15.80,t=5.255,P<0.001).After down-regulated TGF-α,the proliferation,invasion and migration ability of PCa cells were significantly weakened(proliferation of 72h:2.31±0.07 vs 1.80±0.11,t=6.695,P=0.003;invasion:188.44±20.43 vs 108.56±27.96,t=6.922,P<0.001;migration:192.11±22.18 vs 110.67±30.67,t=6.456,P<0.001).Up-regulated KLF7and down-regulated TGF-αcould significantly reversed the promoting effect of KLF7on the biological behavior of PCa cells(proliferation of 72h:2.40±0.11 vs 1.87±0.03,t=8.916,P<0.001;invasion:83.67±18.25 vs 61.56±12.10,t=3.029,P=0.008;migration:98.78±11.71 vs 60.67±6.00,t=8.688,P<0.001).The results of dual luciferase reporter gene assay showed that KLF7had no direct transcriptional activation effect on TGF-α(1.00±0.17 vs 1.06±0.06,t=0.704,P=0.508).Conclusion Highly expressed KLF7can promote the proliferation,invasion and migration ability of PCa cells(PC-3)by upregulating TGF-αexpression.