<i>PLK3</i>基因Y318H罕见突变促进视网膜母细胞瘤的生长

doi:10.3877/cma.j.issn.2095-5782.2023.02.009

目的

运用全外显子测序（whole exome sequencing，WES）技术检测我国视网膜母细胞瘤（retinoblastoma，Rb）患者的关键突变基因，并通过生物学功能研究探索该基因在Rb进展中发挥的功能作用。

方法

对我国82例RB1基因突变阴性（RB1-/-）的Rb患者血液样本行WES筛选罕见突变基因；进一步检测基因罕见突变位点所引起的编码蛋白表达改变情况；构建敲低和过表达上述基因的体外、体内模型，观察其对Rb细胞增殖、迁移功能和肿瘤大小的影响。

结果

运用WES和罕见突变富集分析[SNP-set（sequence）kernel association test，SKAT]，我们在RB1-/-的Rb患者中发现PLK3基因的罕见突变（Y318H），Y318H是一种致病性突变。转染突变质粒（Y318H）的Rb细胞株PLK3蛋白表达量减低。敲低PLK3促进Rb细胞的增殖和迁移，而过表达PLK3抑制Rb细胞的增殖和迁移。同时，PLK3表达改变可调控小鼠Rb皮下瘤的生长。

结论

PLK3基因罕见突变（Y318H）是我国患者Rb进展的重要致病因素，Y318H突变可介导PLK3蛋白表达减低，继而促进Rb细胞增殖和迁移，最终导致Rb的生长。上述研究表明PLK3基因突变可作为Rb早期筛查的分子标志物，为疾病的诊疗提供新靶点。

关键词: 视网膜母细胞瘤, 全外显子测序, PLK3

Objective

This study aimed to investigate the crucial mutant gene of Chinese patients with retinoblastoma (Rb) by whole-exome sequencing (WES), and explore the functional role of this genetic variant in the development of Rb using biological experiments.

Methods

Blood samples of 82 Rb patients with negative RB1 gene mutation (RB1-/-) in China were screened for rare variations via WES analysis. Moreover, the expression of protein encoded by selected rare variants was detected by Western blot. We further observed the effect of selected gene knockdown or overexpression on the proliferation, migration of Rb cells and xenograft tumor size in vitro and in vivo.

Results

We identified a rare variation Y318H of PLK3 gene in RB1-/- patients by WES and SKAT analysis, and Y318H substitution was a pathogenic mutation. The PLK3 protein of Rb cell lines transfected with Y318H mutation plasmids was reduced compared to the control. PLK3 knockdown promoted the proliferation and migration potentials of Rb cells, while PLK3 overexpression inhibited the proliferation and migration potentials of Rb cells. Additionally, the transition of PLK3 expression regulated Rb growth of mouse subcutaneous xenograft model in vivo.

Conclusions

Our findings provide evidence that the rare variation Y318H of PLK3 gene is an significant pathogenic factor for Rb progression in Chinese patients. The mutation of Y318H can reduce PLK3 protein expression. PLK3 knockdown promoted the proliferation and migration of Rb cells, thereby resulting in the growth of Rb. The study suggests that PLK3 rare variation Y318H can be used as a new molecular marker for the early genetic screening of Rb, and may provide a novel target for diagnosis and treatment of the disease.

Key words: Retinoblastoma, Whole-exome sequencing, PLK3

图1 PLK3罕见突变（Y318H）对其编码蛋白表达和Rb细胞功能的影响1A：RB1-/-的Rb患者存在PLK3基因突变及相关罕见突变位点；1B：Western blot检测Y79细胞转染PLK3过表达质粒（PLK3-OE）和不同罕见突变位点质粒（P299H、L323P、Y318H和R610H）后的PLK3蛋白的表达情况（左图）。对上述目的蛋白条带用Image J软件进行灰度检测和统计学分析（右图）；1C：Western blot检测Y79（左图）和WERI-RB1细胞（右图）转染PLK3过表达质粒（PLK3-OE）和突变质粒（Y318H）72 h后，PLK3蛋白的表达情况；1D：CCK8实验检测Y79（左图）和WERI-RB1细胞（右图）不同转染处理后的细胞活力情况；1E、1F：Transwell细胞迁移实验检测Y79（上图）和WERI-RB1细胞（下图）不同转染处理后的细胞迁移能力（E），对细胞迁移数量的进行技术和统计学分析（F）。以上结果以均数±标准差表示，实验结果为3次独立重复实验所得出，比例尺：200 μm，GAPDH作为内参，^*P < 0.05，^**P < 0.01，^***P < 0.001，^****P < 0.000 1。

图2 敲低PLK3对Rb细胞增殖和迁移的影响2A：人Rb细胞株Y79和WERI-RB1感染shRNAs和对照shNC慢病毒，利用荧光倒置相差显微镜观察荧光表达强度，比例尺：500 μm；2B、2C：qPCR检测Rb细胞系PLK3 mRNA水平的敲低效果；2D、2E：Western blot实验检测Rb细胞系PLK3蛋白水平的敲低效果，GAPDH作为内参；2F、2G：CCK8实验检测敲低PLK3后Y79和WERI-RB1细胞的细胞活力；2H、2I：Transwell细胞迁移实验检测敲低PLK3后，细胞迁移能力的影响（2H），比例尺：200 μm。对细胞迁移数量的进行计数和统计学分析（2I）。以上结果以均数±标准差表示，实验结果为3次独立重复实验所得出，^*P < 0.05，^**P < 0.01，^***P < 0.001，^****P < 0.000 1。

图3 过表达PLK3对Rb细胞增殖和迁移的影响3A：Y79和WERI-RB1细胞感染PLK3-OE和对照Vector慢病毒，利用荧光倒置相差显微镜观察荧光表达强度，比例尺：500 μm；3B、3C：利用qPCR检测Rb细胞系PLK3 mRNA水平的过表达效果；3D、3E：Western blot实验检测Rb细胞系PLK3蛋白水平的过表达效果，GAPDH作为内参；3F、3G：过表达PLK3后，CCK8实验检测细胞活力；3H、3I：通过Transwell细胞迁移实验检测过表达PLK3对Rb细胞迁移能力的影响（3H），比例尺：200 μm。对细胞迁移数量的进行计数和统计学分析（3I）。以上结果以均数±标准差表示，实验结果为3次独立重复实验所得出，^*P < 0.05，^**P < 0.01，^***P < 0.001，^****P < 0.000 1。

图4 敲低和过表达PLK3对Rb皮下移植瘤生长的影响4A：分别将Y79 shNC、shRNA-1、shRNA-2稳定株行皮下注射到BALB/c裸鼠肩胛区构建皮下移植瘤模型，约1个月后取出不同分组肿瘤进行形态学观察；4B：测量不同分组肿瘤体积变化情况；4C：测量不同分组肿瘤重量情况；4D：分别将Y79 Vector、PLK3-OE稳定株行皮下注射到BALB/c裸鼠肩胛区构建皮下移植瘤模型，约1个月后取出不同分组肿瘤进行形态学观察；4E：测量不同分组肿瘤体积变化情况；4F：测量不同分组肿瘤重量情况。n = 5，计算公式为：（长度×宽度×高度）/ 2 mm³，^*P < 0.05，^**P < 0.01，^***P < 0.001，^****P < 0.000 1。

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PLK3 rare variation Y318H promotes the development of retinoblastoma

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PLK3 rare variation Y318H promotes the development of retinoblastoma