KHSRP通过ANK3调节前列腺癌细胞对雄激素的反应性

doi:10.3969/j.issn.1674-8115.2024.04.001

摘要/Abstract

摘要：

目的·研究KH型剪接调节蛋白（KH-type splicing regulatory protein，KHSRP）对前列腺癌细胞增殖能力的影响及其下游基因的表达调控，考察KHSRP在前列腺癌由雄激素依赖型向非依赖型转变过程中的潜在作用及机制。方法·通过重组慢病毒感染雄激素依赖型前列腺癌LNCaP细胞和雄激素非依赖型前列腺癌DU145细胞，分别构建KHSRP功能性缺失/获得的稳定细胞株，并比较KHSRP在2种细胞之间的功能差异。采用蛋白质印迹法（Western blotting）检测稳定细胞株中KHSRP、雄激素受体（androgen receptor，AR）及锚定蛋白3（ankyrin 3，ANK3）的表达量。通过细胞增殖实验、平板克隆实验、小鼠成瘤实验等检测KHSRP对LNCaP细胞增殖能力的影响。通过转录组测序（RNA sequencing，RNA-seq）检测KHSRP影响的下游基因，并通过实时荧光定量PCR（quantitative real-time PCR，RT-qPCR）检测KHSRP下游基因的mRNA表达量。结合癌细胞系百科全书（Cancer Cell Line Encyclopedia，CCLE）、癌症基因组图谱（The Cancer Genome Atlas，TCGA）、基因表达综合（Gene Expression Omnibus，GEO）数据库，分析研究ANK3和KHSRP在前列腺组织样本中的表达情况以及ANK3在不同前列腺癌细胞株中的表达差异。结果·GEO数据分析结果显示KHSRP在前列腺癌组织中的表达高于良性前列腺组织，提示其与前列腺癌的发生相关。细胞增殖实验、平板克隆实验、小鼠成瘤实验结果显示KHSRP的表达与LNCaP细胞增殖能力呈负相关，提示KHSRP可以抑制前列腺癌细胞的增殖，且KHSRP对LNCaP细胞增殖能力的影响强于对DU145细胞的影响。对LNCaP稳定细胞株的Western blotting和RT-qPCR检测显示KSHRP过表达后LNCaP细胞中AR蛋白质水平下降，但mRNA水平未产生变化，提示KHSRP可以间接调节AR的蛋白质水平。RNA-seq和RT-qPCR结果显示KHSRP与影响AR蛋白稳定性的调节因子ANK3的表达呈正相关，随后的Western blotting证明KHSRP过表达后ANK3蛋白质的表达量明显升高，TCGA数据分析结果进一步说明KHSRP与ANK3的mRNA表达的相关性。根据CCLE和GEO数据，ANK3的表达与前列腺癌的恶性程度也密切相关。结论·KHSRP可能通过ANK3间接调控AR的蛋白稳定性，影响雄激素依赖型前列腺癌细胞的增殖能力，并介导前列腺癌细胞对雄激素反应性的改变。

关键词: KH型剪接调节蛋白（KHSRP）, 雄激素受体（AR）, 锚定蛋白3（ANK3）, 前列腺癌, 细胞增殖

Abstract:

Objective ·To investigate the impact of KH-type splicing regulatory protein (KHSRP) on the proliferation of prostate cancer cells and the regulation of downstream gene expression, and explore the potential role and mechanism of KHSRP in the transition of prostate cancer from androgen-dependent to androgen-independent. Methods ·Recombinant lentivirus was used to infect androgen-dependent prostate cancer LNCaP cells and androgen-independent prostate cancer DU145 cells to establish stable cell lines with functional deficiency/acquisition of KHSRP, and the functional differences of KHSRP between the two cell types were compared. Western blotting was used to detect the expression levels of KHSRP, androgen receptor (AR), and ankyrin 3 (ANK3) in the stable cell lines. Cell proliferation assay, colony formation assay, and mouse xenograft assay were performed to assess the impact of KHSRP on the proliferation ability of LNCaP cells. RNA sequencing (RNA-seq) was performed to identify downstream genes affected by KHSRP, and the mRNA expression levels of these genes were measured by quantitative real-time PCR (RT-qPCR). The expression of ANK3 and KHSRP in prostate tissue samples and the difference of ANK3 expression in different prostate cancer cell lines were analyzed by combining Cancer Cell Line Encyclopedia (CCLE), The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Results ·GEO data analysis showed that KHSRP was upregulated in prostate cancer tissues compared to benign prostate tissues, suggesting its association with prostate tumorigenesis. Cell proliferation assay, colony formation assay, and mouse xenograft assay demonstrated a negative correlation between KHSRP expression and the proliferation ability of LNCaP cells, indicating that KHSRP can inhibit the proliferation of prostate cancer cells, with a stronger effect on LNCaP cells than on DU145 cells. Western blotting and RT-qPCR analysis of the stable LNCaP cell lines showed that KHSRP overexpression led to a decrease in AR protein level without affecting its mRNA level, suggesting that KHSRP can indirectly regulate AR protein level. RNA-seq and RT-qPCR results showed KHSRP was positively correlated with the expression of ANK3, a regulatory factor affecting AR protein stability, and subsequent Western blotting confirmed an increase in ANK3 protein expression after KHSRP overexpression. TCGA data analysis further supported the correlation between KHSRP and ANK3 mRNA expression. Interestingly, according to CCLE and GEO data, the expression of ANK3 was closely related to prostate cancer malignancy. Conclusion ·KHSRP may indirectly regulate AR protein stability through ANK3, thereby influencing the proliferation of androgen-dependent prostate cancer cells and mediating the altered responsiveness to androgen in prostate cancer cells.

Key words: KH-type splicing regulatory protein (KHSRP), androgen receptor (AR), ankyrin 3 (ANK3), prostate cancer, cell proliferation

中图分类号:

R737.25

蔡人杰, 徐明. KHSRP通过ANK3调节前列腺癌细胞对雄激素的反应性[J]. 上海交通大学学报（医学版）, 2024, 44(4): 417-426.

CAI Renjie, XU Ming. KHSRP regulates the responsiveness of prostate cancer cells to androgens through ANK3[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(4): 417-426.

图/表 7

表 1 慢病毒质粒构建所用引物序列

Tab 1 Primer sequences used for lentiviral vector plasmid construction

Primer	Sequence (5′→3′)
sh-KHSRP-UTR-F	GATCCGCTGAGGATAAAGCAATTCATTCTCGAGAATGAATTGCTTTATCCTCAGCTTTTTG
sh-KHSRP-UTR-R	AATTCAAAAAGCTGAGGATAAAGCAATTCATTCTCGAGAATGAATTGCTTTATCCTCAGCG
sh-KHSRP-CDS-F	GATCCGCCCGAGAAGATTGCTCATATACTCGAGTATATGAGCAATCTTCTCGGGCTTTTTG
sh-KHSRP-CDS-R	AATTCAAAAAGCCCGAGAAGATTGCTCATATACTCGAGTATATGAGCAATCTTCTCGGGCG
HA-KHSRP-F	TTTTGACCTCCATAGAAGATTATGGGTACCTATCCTTACGA
HA-KHSRP-R	GCGTCTAGGAAGAGCTCGATCAGTTAGAGGAGAACTTGTCT

表 2 RT-qPCR 所用引物序列

Tab 2 Primer sequences used for RT-qPCR

Primer	Forward (5′→3′)	Reverse (5′→3′)
GAPDH	ATGAGGTCCACCACCCTGTT	CTCAAGGGCATCCTGGGCTA
ANK3	GAAGATGCAATGACCGGGGA	AAAGCCCATGTAACCCTCTG
AR	GACGACCAGATGGCTGTCATT	GGGCGAAGTAGAGCATCCT

图1 KHSRP 对LNCaP细胞增殖能力的影响Note： A. Verification of KHSRP expression in stable LNCaP cell lines through Western blotting. B. Results of the cell proliferation assay in stable LNCaP cell lines.C. Results of the colony formation assay in stable LNCaP cell lines. D. Results of the mouse xenograft assay in stable LNCaP cell lines. ①P=0.044, ②P=0.000, ③P=0.022, ④P=0.036, ⑤P=0.004, ⑥P=0.047, ⑦P=0.045.

Fig 1 Impact of KHSRP on the proliferation of LNCaP cells

图2 KSHRP对DU145细胞增殖能力的影响Note： A. Detection of KHSRP expression in 59 prostate cancers and 39 benign tissue samples. B. Verification of KHSRP expression in DU145 stable cell lines through Western blotting. C. Bar chart showing the results of the cell viability experiment in stable DU145 cell lines. D. Bar chart showing the tumor weight results of the mouse xenograft experiment in stable DU145 cell lines. ①P=0.012, ②P=0.001, ③P=0.011.

Fig 2 Effects of KHSRP on the proliferation of DU145 cells

图3 过表达 KHSRP 后LNCaP细胞中AR表达情况的检测Note： A. Detection of AR protein expression in stable LNCaP cell lines through Western blotting. B. Detection of AR mRNA expression in stable LNCaP cell lines through RT-qPCR. ns—not significant.

Fig 3 Detection of AR expression in LNCaP cells with KHSRP overexpression

图4 LNCaP细胞中 KHSRP 所调控基因的检测Note： A. Heatmap of differentially expressed genes between LNCaPsh-KHSRPand LNCaPsh-KHSRP-HA-KHSRP stable cell lines based on RNA-seq. B. Bubble plot of KEGG enrichment analysis of differential genes between LNCaPsh-KHSRP and LNCaPsh-KHSRP-HA-KHSRP stable cell lines from RNA-seq. C. Bar chart showing the expression level of ANK3 in LNCaPsh-KHSRP and LNCaPsh-KHSRP-HA-KHSRP stable cell lines based on RNA-seq. FPKM—fragments per kilobase of transcript per million mapped reads. D. Detection of ANK3 mRNA expression in stable LNCaP cell lines through RT-qPCR. E. Detection of ANK3 protein expression in stable LNCaP cell lines through Western blotting. F. Analysis of the correlation between ANK3 and KHSRP expression levels in prostate cancer using the TCGA database.①P=0.010, ②P=0.001.

Fig 4 Detection of genes regulated by KHSRP in LNCaP cells

图5 ANK3 与前列腺癌组织和细胞的恶性程度的关联Note：A. Relative expression levels of ANK3 mRNA in different prostate cancer cell lines. B. Relative expression levels of AR mRNA in different prostate cancer cell lines. C. Comparison of ANK3 mRNA levels in 25 low-grade (Gleason score=6) and 27 high-grade (Gleason score=8) prostate cancer tissues from the GEO database. ①P=0.004.

Fig 5 Correlation of ANK3 expression and malignancy of prostate cancer tissues and cells

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