The Role And Mechanism Of NPM1 In The Promotion Of Stemness And Metastasis Of Nasopharyngeal Carcinoma

BackgroundsNasopharyngeal carcinoma(NPC)is a hypodifferentiated epithelial malignant tumor originating from the nasopharyngeal mucosal lining and is increasingly prevalent in southern China.It has a high degree of malignancy,lacks typical early clinical symptoms,and is prone to local recurrence and distant metastasis.The primary treatment for patients who are in the early stage is radiotherapy,whereas for the advanced stage of NPC,chemotherapy combined with radiotherapy is more suitable.However,some patients still suffer local recurrence and even distant metastasis in spite of appropriate treatment.This phenomenon accounts for the death of patients with NPC.Therefore,it is of vital importance to find the key molecule that regulates the development of NPC to verify whether it can act as an effective therapeutic target.Although nucleophosminl(NPM1)is widely expressed in the nucleolar granular region,it is highly conserved and constantly shuttles between the nucleus and cytoplasm.NPM1 is frequently overexpressed,mutated,rearrange,and deleted in human cancer.Mutations in the NPM1 locus were initially identified as an important cause of hematological malignancies,and NPM1 has since been found to regulate tumor progression in solid tumors such as gastric,colorectal,and hepatocellular cancers.However,there’s no report on its expression,biological function,and molecular regulatory mechanism of NPM1 in NPC up till now.Contents and methods1.Investigate the expression of NPM1 in nasopharyngeal carcinoma tissues and NPC cell lines,and then compare them with normal epithelial tissues of the nasopharynx and immortalized epithelial cell line NP69 respectively.2.Explore the function of NPM1 in NPC1)Construction of stable knockdown and transient overexpression of NPM1 in NPC cells respectively;The interference or overexpression efficiency was assessed by qPCR and western blot analysis.2)The influence of NPM1 on the migration of NPC cells was analyzed by wound healing assay,transwell assay,and lung metastasis model in vitro and in vivo.3)The effect of NPM1 on the stemness of NPC cells was investigated by sphere formation test,flow cytometry analysis,immunofluorescence assay,and subcutaneous tumor formation experiment in vitro and in vivo.4)Western blot analysis was adopted to detect the regulatory effects of NPM1 on E-cadherin,N-cadherin,vimentin,CD44,OCT4,Nanog,and SOX2.3.Identify the proteins that interact with NPM11)The underlying interacting proteins of NPM1 were discovered by using CoIP assay in conjunction with mass spectrometry-based quantitative proteomics.2)Co-IP assay combined with western blot analysis confirmed endogenous binding of NPM1 to its interacting proteins.3)The colocalization of NPM1 with its interacting proteins in.NPC cells was testified by immunofluorescence assays.4)qPCR and western blot analysis were used to probe whether NPM1 affected both transcriptional and translational levels of interacting proteins simultaneously.4.Probe into the mechanism of NPM1 in promoting the development of NPC 1)The effect on the half-life of p53 in NPC cells was examined using a cycloheximide(CHX)chase experiment.2)Co-IP was used to track the impact of NPM1 on the ubiquitination of p53.5.Using the functional experiments to investigate whether knocking down p53 can antagonize the biological function and molecular mechanism of NPM1 in nasopharyngeal carcinoma cells.Results1.NPM1 was relatively highly expressed in NPC tissues and correlated with tumor stage and prognosis.2.NPM1 promotes the stemness and metastasis of nasopharyngeal carcinoma in vitro and in vivo.3.NPM1 interacts with p53 to prevent post-transcriptional production of p53.4.NPM1 recruits Mdm2 to induce the ubiquitination-mediated degradation of p53.5.Knocking down p53 antagonizes the action of NPM1.ConclusionsNPM1 promotes the stemness and metastasis of nasopharyngeal carcinoma.Mechanistically,NPM1 recruits the E3 ubiquitin ligase Mdm2 to induce the ubiquitination degradation of p53 and inhibits the p53-related signaling pathway in vitro and in vivo.