Study On The Mechanism Of Naa10p Regulating The Pirh2-p53 Signaling Pathway In The Invasion And Metastasis Of Oral Squamous Cell Carcinoma

Object By studying the expression of Naa10 p in OSCC and its role in OSCC invasion and metastasis,the molecular mechanism of Naa10 p affecting OSCC invasion and metastasis by regulating Pirh2-p53 signaling pathway was elucidated.It also provides some experimental and theoretical basis for the development of novel anti-tumor metastasis drugs,and further improves the treatment and prognosis of OSCC patients.Methods The expression of Naa10 p in tumor and adjacent tissues of OSCC patients was detected by q RTPCR,western-blot and immunohistochemistry,and its correlation with clinicopathological parameters and patient prognosis was analyzed.Lentivirus system was used to establish human OSCC cell lines CAL 27 and SCC-15 cells with stable Naa10 p silencing,and the effects of Naa10 p expression on cell growth,proliferation,migration and invasion were observed.CAL27 cells with Naa10 p silencing and cells of the control group were inoculated under the left rib of 4-6 weeks old nude mice,respectively.The tumor size,volume and weight were observed in vivo,and the survival curve of nude mice was plotted.The expressions of Naa10 p,Pirh2 and p53 in tumor tissues of OSCC patients and tumorigenesis tissues of nude mice were detected by immunohistochemical,and the correlation between the expressions of Naa10 p,Pirh2 and p53 were analyzed.Moreover,the effect of the expression of Naa10 p on the Pirh2-p53 signaling pathway and the downstream migration invasion protein MMP2 and MMP9 was detected by Western-Blot in CAL 27 cells and SCC-15 cells.IP,GST-Pull Down and IF were used to identify Rel A/p65 subunit of NF-κB that interacts with Naa10 p.The luciferase reporter assay examined the effect of Naa10 p and the interacting protein on the transcriptional activation of Pirh2 gene.CHIP-q PCRwas used to detect the ability of Rel A/p65 to target Pirh promoter region and the effect of Naa10 p on the ability of Rel A/p65 to target Pirh promoter region.Finally,OSCC cells were down-regulated Naa10 p and Pirh2,respectively,and down-regulated Naa10 p and Pirh2 at the same time,using small RNA interference technology,to detect the effects on migration and invasion of OSCC cells.Results1.The expression of Naa10 p in OSCC tissues was significantly higher than that in adjacent tissues by immunohistochemistry and q RT-PCR.Moreover,its expression was correlated with TNM stage,lymph node metastasis,degree of tumor differentiation and recurrence,and the expression of Naa10 p in OSCC was positively correlated with OS and RFS;2.Naa10 p inhibited the migration,invasion,growth and proliferation of OSCC cells.Furthermore,Naa10 p silencing promoted tumor growth in nude mice and reduced the survival time of nude mice;3.Naa10 p expression was negatively correlated with Pirh2,and positively correlated with p53 in OSCC tissues and OSCC cells;4.Naa10 p interacted directly with Rel A/p65 of NF-κB and inhibited p65 phosphorylation in OSCC cells;5.Rel A/p65 transcriptionally activated the Pirh2 gene,and Naa10 p inhibited Rel A/p65 transcriptional activation of Pirh2 gene;6.Truncating the promoter of Pirh2 gene,Rel A/p65 transcriptatively activated the promoter region S1 fragment of Pirh2 gene,and was comparable with the full-length Pirh2 promoter.However,the luciferase activity of the truncated S2 had no significant difference with that of the control group,and Naa10 p suppressed the luciferase activity of S1 fragment of Pirh2 gene promoter region activated by Rel A/p65 transcription;7.Rel A/p65 could directly bind to the p65 binding site of S1 fragment in the promoter region of Pirh2 gene,and Naa10 p inhibited Rel A/p65 binding to S1 fragment in the promoter region of Pirh2 gene;8.Naa10 p inhibited the expression of Pirh2,attenuated the degradation of p53 protein induced by Pirh2,and subsequently enhancing the expression of p53 and decreasing the expression of migration and invasion related protein MMP2 and MMP9,and thus inhibiting the migration and invasion of OSCC.Conclusion1.Naa10 p was overexpressed in OSCC and inhibited the migration and invasion of OSCC cells.Meanwhile,Naa10 p attenuated tumor growth in nude mice and prolonged the survival time of nude mice;2.Naa10 p played a role in inhibiting OSCC migration and invasion by regulating the Pirh2-p53 signaling pathway and inhibited the expression of downstream migration and invasion proteins MMP2 and MMP9.3.Naa10 p interacted with Rel A/p65 of NF-κB and inhibited p65 phosphorylation into nucleus,and attenuated Rel A/p65 binding to the Pirh2 gene function promoter region S1 fragment to transcriptionally activate the Pirh2.