Gong Yingying, Fang Ziwen, Wang Yixuan, Ge Minghua, Pan Zongfu
Otolaryngology & Head and Neck Center, Cancer Center, Department of Head and Neck Surgery, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou 310014, China.
Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou 310014, China.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2025 May 25;54(3):372-381. doi: 10.3724/zdxbyxb-2024-0708.
To investigate the role of nucleolar pre-rRNA processing protein NIP7 (NIP7) in maintaining the malignant phenotype of anaplastic thyroid cancer (ATC) and its molecular mechanisms.
NIP7 expression in ATC tissues and its gene knock-out effects in ATC cells were analyzed using gene expression microarray (GSE33630), proteome database (IPX0008941000) and the Dependency Map database, respectively. Expression and localization of NIP7 in normal thyroid cells, papillary thyroid cancer cells, and ATC cells were detected by Western blotting. Small interfering RNA (siRNA) was transfected into ATC cells, and the knockdown efficiency of NIP7 was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting. Cell proliferation was assessed by CCK-8 assay, colony formation was evaluated by colony formation assay, and tumor growth was assessed by xenograft tumor model in nude mice. SUnSET (surface sensing of translation) assay combined with co-immunoprecipitation were employed to evaluate the effect of silencing on ubiquitin-conjugating enzyme E2 C (UBE2C) translation. Finally, gene set enrichment analysis was used to identify shared pathways of NIP7 and UBE2C, which were validated by qRT-PCR.
Compared with normal tissues and papillary thyroid cancer, NIP7 was significantly upregulated in ATC tissues, and had a gene knock-out fitness effect on different ATC cell lines. The relative protein levels of NIP7 in ATC cells were significantly higher than those in normal thyroid follicular cells, and the protein was mainly expressed in the nucleus. silencing significantly inhibited cell proliferation and reduced colony formation. Xenograft tumor model showed that knockdown significantly slowed down the growth of ATC xenograft, and the tumor volume and weight were significantly lower than those in the control group (all <0.05). silencing downregulated the protein level of UBE2C, but did not affect the expression of mRNA. Compared to the control group, silencing significantly inhibited ATC cells proliferation (<0.01) and colony formation (<0.05). UBE2C overexpression reversed the proliferation-inhibitory effect induced by silencing (<0.01). Gene set enrichment analysis indicated that NIP7 and UBE2C were both involved in DNA replication. or silencing could significantly downregulate the expression levels of DNA polymerase epsilon, catalytic subunit 2 and replication factor C4 in DNA replication pathway.
NIP7 promotes ATC tumor growth by upregulating UBE2C to mediate DNA replication.
探讨核仁前体rRNA加工蛋白NIP7在维持间变性甲状腺癌（ATC）恶性表型中的作用及其分子机制。
分别利用基因表达微阵列（GSE33630）、蛋白质组数据库（IPX0008941000）和依赖性图谱数据库分析ATC组织中NIP7的表达及其在ATC细胞中的基因敲除效应。通过蛋白质免疫印迹法检测NIP7在正常甲状腺细胞、甲状腺乳头状癌细胞和ATC细胞中的表达及定位。将小干扰RNA（siRNA）转染至ATC细胞中，通过定量逆转录聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测NIP7的敲低效率。采用CCK-8法评估细胞增殖，通过集落形成试验评估集落形成情况，并通过裸鼠异种移植瘤模型评估肿瘤生长。采用SUnSET（翻译表面传感）试验结合免疫共沉淀法评估沉默对泛素结合酶E2C（UBE2C）翻译的影响。最后，利用基因集富集分析确定NIP7和UBE2C的共同通路，并通过qRT-PCR进行验证。
与正常组织和甲状腺乳头状癌相比，NIP7在ATC组织中显著上调，并且对不同的ATC细胞系具有基因敲除适应性效应。ATC细胞中NIP7的相对蛋白水平显著高于正常甲状腺滤泡细胞，且该蛋白主要在细胞核中表达。沉默NIP7可显著抑制细胞增殖并减少集落形成。异种移植瘤模型显示，敲低NIP7可显著减缓ATC异种移植瘤的生长，肿瘤体积和重量均显著低于对照组（均<0.05）。沉默NIP7可下调UBE2C的蛋白水平，但不影响其mRNA表达。与对照组相比，沉默NIP7可显著抑制ATC细胞增殖（<0.01）和集落形成（<0.05）。UBE2C过表达可逆转沉默NIP7诱导的增殖抑制效应（<0.01）。基因集富集分析表明，NIP7和UBE2C均参与DNA复制。沉默NIP7或UBE2C可显著下调DNA复制途径中DNA聚合酶ε催化亚基2和复制因子C4的表达水平。
NIP7通过上调UBE2C介导DNA复制促进ATC肿瘤生长。
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