NIP7 upregulates the expression of ubiquitin-conjugating enzyme E2 C to promote tumor growth of anaplastic thyroid cancer.

Abstract

Objectives: To investigate the role of nucleolar pre-rRNA processing protein NIP7 in maintaining the malignant phenotype of anaplastic thyroid cancer (ATC) and its molecular mechanisms.

Methods: NIP7expression in ATC tissues and its gene knock-out effects in ATC cells were analyzed using the gene expression microarray (GSE33630), proteome database (IPX0008941000) and the Dependency Map database, respectively. Expression and localization of NIP7 in normal thyroid cells (Nthy-ori 3-1), papillary thyroid cancer cells (Bcpap), and ATC cells (CAL62 and 8505C) were detected by Western blot. 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 blot. 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 NIP7 silencing on ubiquitin-conjugating enzyme 2C (UBE2C) translation. Finally, gene set enrichment analysis was used to identify shared pathways of NIP7 and UBE2C, which were validated by qRT-PCR.

Results: 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 8505C cells and CAL62 cells were significantly higher than those in normal thyroid follicular cells, and it was mainly expressed in the nucleus. NIP7 silencing significantly inhibited the ability of cell proliferation and reduced the number of colony formation. Xenograft tumor model showed that NIP7 knockdown significantly slowed down the growth rate of ATC xenograft tumors, and the tumor volume and weight were significantly lower than those in the control group (P<0.05). Besides, NIP7 silencing downregulated the protein level of UBE2C, but did not affect the expression of UBE2C messenger RNA. Compared to the control group, UBE2C silencing significantly inhibited ATC cell proliferation (P<0.01) and colony formation (P<0.05). Moreover, UBE2C overexpression reversed the proliferation-inhibitory effect induced by NIP7 silencing (P<0.01). Gene set enrichment analysis indicated that NIP7 and UBE2C were both involved in DNA replication. NIP7 or UBE2C silencing could significantly downregulate the expression levels of POLE2 and RFC4 in DNA replication pathway.

Conclusions: NIP7 promotes ATC tumor growth by upregulating UBE2C to mediate DNA replication.