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中华腔镜泌尿外科杂志(电子版)

中华腔镜泌尿外科杂志(电子版) ›› 2022, Vol. 16 ›› Issue (03) : 262 -269. doi: 10.3877/cma.j.issn.1674-3253.2022.03.016

实验研究

ASF1B通过调控P53相关信号通路促进前列腺癌迁移和增殖的研究
雷震1, 郭正辉1, 唐晨1, 彭圣萌1, 任艳婷2, 吴宛桦1, 周杰1, 陈勇明1, 李凌峰1, 黄海1, 赖义明1,†()   
  1. 1. 510120　广州，中山大学孙逸仙纪念医院泌尿外科；510120　广州，广东省泌尿系统疾病临床医学研究中心；510120　广州，广东省恶性肿瘤表观遗传与基因调控重点实验室
    2. 510120　广州，广东省恶性肿瘤表观遗传与基因调控重点实验室；510120　广州，中山大学孙逸仙纪念医院病理科
  • 收稿日期:2022-01-04 出版日期:2022-06-01
  • 通信作者: 赖义明
  • 基金资助:
    国家自然科学基金面上项目(81972384，81772733，81974395); 国家自然科学基金青年项目(81802527); 广东省科技计划项目(2021A1515010223); 广东省泌尿系统疾病临床医学研究中心(2020B1111170006); 广东省恶性肿瘤表观遗传与基因调控重点实验室(2020B1212060018)

ASF1B enhances the migration and proliferation of prostate cancers via regulating p53-related signaling pathways

Zhen Lei1, Zhenghui Guo1, Chen Tang1, Shengmeng Peng1, Yanting Ren2, Wanhua Wu1, Jie Zhou1, Yongming Chen1, Lingfeng Li1, Hai Huang1, Yiming Lai1,†()   

  1. 1. Department of Urology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China; Guangdong Provincial Clinical Research Center for Urological Diseases, Guangzhou 510120, China; Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China
    2. Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China; Department of Pathology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China
  • Received:2022-01-04 Published:2022-06-01
  • Corresponding author: Yiming Lai
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引用本文：

雷震, 郭正辉, 唐晨, 彭圣萌, 任艳婷, 吴宛桦, 周杰, 陈勇明, 李凌峰, 黄海, 赖义明. ASF1B通过调控P53相关信号通路促进前列腺癌迁移和增殖的研究[J]. 中华腔镜泌尿外科杂志(电子版), 2022, 16(03): 262-269.

Zhen Lei, Zhenghui Guo, Chen Tang, Shengmeng Peng, Yanting Ren, Wanhua Wu, Jie Zhou, Yongming Chen, Lingfeng Li, Hai Huang, Yiming Lai. ASF1B enhances the migration and proliferation of prostate cancers via regulating p53-related signaling pathways[J]. Chinese Journal of Endourology(Electronic Edition), 2022, 16(03): 262-269.

目的

研究组蛋白伴侣抗沉默功能1B（ASF1B）对前列腺癌（PCa）迁移和增殖能力的影响，探索ASF1B调控P53相关信号通路的具体分子机制。

方法

根据TCGA数据库中的数据分析ASF1B在前列腺癌患者中的生存预后情况，使用细胞小干扰RNA（SiRNA）敲低ASF1B表达后，通过Transwell细胞迁移实验比较细胞的运动能力，通过CCK8实验和平板克隆实验比较细胞的增殖能力，使用细胞凋亡实验比较细胞的凋亡率，通过转录组二代测序比较敲低ASF1B后各个信号通路的改变情况，通过Western-Blot实验及Q-PCR实验比较P53相关信号通路分子的蛋白质和mRNA水平变化，以阐明ASF1B对前列腺癌细胞的迁移及增殖能力的影响。

结果

SiRNA敲低ASF1B表达后，DU145和PC3细胞的运动能力降低，相同时间内穿过小室的细胞数减少。敲低ASF1B表达后，DU145和PC3细胞的细胞活力和集落形成个数及大小降低，细胞增殖能力下降。敲低ASF1B表达后，DU145和PC3细胞的凋亡率增加。敲低ASF1B表达后，PC3细胞的转录组二代测序结果提示P53信号通路受到调控（P<0.01）。敲低ASF1B表达后，PC3细胞内P53相关通路的P21、IGFBP3、BBC3表达量上升，Cyclin D、Snail、Slug表达量下降。

结论

ASF1B以非P53依赖的形式通过P53相关信号通路调控前列腺癌细胞的迁移及增殖，ASF1B有望成为前列腺癌的诊断及治疗的新型靶点。

关键词: 前列腺癌, 组蛋白伴侣抗沉默功能1B, P53信号通路, 细胞迁移, 细胞增殖
Objective

To study the effect of Anti-Silencing Function 1B Histone Chaperone (ASF1B) on the migration and proliferation of prostate cancer (PCa), and to explore the specific molecular mechanism of ASF1B regulating P53-related signal pathway.

Methods

The survival and prognosis of ASF1B in prostate cancer patients was analyzed according to the data in TCGA database. After the expression of ASF1B was knocked down by small interference RNA (SiRNA), the mobility of cells was compared by Transwell cell migration test, the ability of cell proliferation was compared by CCK8 test and plate cloning test, and the apoptosis rate was compared by apoptosis test. The changes of signal pathways after ASF1B knockout were compared by transcriptome second generation sequencing, and the changes of protein and mRNA levels of P53 related signal pathway molecules were compared by Western-Blot test and Q-PCR test, in order to clarify the effect of ASF1B on the migration and proliferation of prostate cancer cells.

Results

After SiRNA knocked down the expression of ASF1B, the motor ability of DU145 and PC3 cells decreased, and the number of cells passing through the chamber decreased at the same time. After knocking down the expression of ASF1B, the number and size of cell viability and colony formation of DU145 and PC3 cells decreased, and the ability of cell proliferation decreased. After knocking down the expression of ASF1B, the percentage of apoptosis of DU145 and PC3 cells increased. After knocking down the expression of ASF1B, the second generation sequencing results of transcriptional group of PC3 cells showed that the P53 signal pathway was regulated. After knocking down the expression of ASF1B, the expression of p21, IGFBP3 and BBC3 of P53-related pathways in PC3 cells increased, while the expression of Cyclin D, Snail and Slug decreased.

Conclusion

ASF1B regulates the migration and proliferation of prostate cancer cells through P53-related signaling pathways in a P53-independent manner. ASF1B is expected to be a new target for the diagnosis and treatment of prostate cancer.

Key words: Prostate cancer, Anti-Silencing Function 1B Histone Chaperone, P53 signaling pathway, Cell migration, Cell proliferation
图2 敲低ASF1B表达后，DU145和PC3细胞的迁移能力下降（Transwell小室×200）
图3 敲低ASF1B表达后，DU145和PC3细胞的增殖能力下降注：a，b为CCK8实验检测敲低ASF1B后DU145细胞和PC3细胞的活力；c为集落形成实验，检测敲低ASF1B后DU145细胞和PC3细胞形成的集落大小和数目；*表示P<0.05，柱状图中各数据均以（±s）表示
图4 敲低ASF1B表达后，DU145和PC3细胞的凋亡增多注：细胞凋亡实验检测敲低ASF1B后DU145细胞和PC3细胞的凋亡情况；*表示P<0.05，柱状图中各数据均以（±s表示
图5 敲低ASF1B表达后，PC3细胞的P53相关信号通路受到调控注：a为KEGG数据富集分析提示敲低ASF1B表达后，PC3细胞内P53相关信号通路等信号通路受到影响；b为气泡图提示，敲低ASF1B表达后，PC3细胞内P53相关信号通路受调控最为明显
图6 敲低ASF1B表达后，PC3细胞内P53相关信号通路被激活注：a为在PC3细胞中敲低ASF1B表达后P53相关信号通路内分子的蛋白水平的变化。b为在PC3细胞中敲低ASF1B表达后P53相关信号通路下游分子的mRNA水平变化；*表示P<0.05，柱状图中各数据均以（±s）表示
表1 细胞小干扰RNA（siRNA）序列
siRNA 序列（5’-3’）
Si-NC UUCUCCGAACGUGUCACGUTT
Si-ASF1B-1 GUGGAAGAUCAUUUAUGUUTT
Si-ASF1B-2 UGGGCUACUACGUCAACAATT
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