PSMD2 Promotes Tumor Cell Proliferation And Invasion By Targeting PTEN Protein In Head And Neck Squamous Cell Carcinoma

Objective:To investigate the role of proteasome 26 S submit,non-ATPase2(PSMD2)in the genesis and development of head and neck squamous cell carcinoma(HNSCC),and to explore the target of PSMD2 and the possible molecular mechanism.Methods:(1)The differential expression and prognosis correlation of 804 ubiquitin proteasome system-related genes(UPSGs)were analyzed using the Hi Seq data of 498 HNSCC samples and 44 adjacent samples in TCGA database to screen out the differentially expressed UPSGs related to prognosis of HNSCC.(2)Kaplan-Meier survival analysis and t-test were used to evaluate the prognostic value of PSMD2 in HNSCC and its correlation with other clinical parameters.(3)The si RNA and overexpression plasmid of PSMD2 were used to transfect HNSCC cells.The influence of PSMD2 on the proliferation,invasion and metastasis of HNSCC cells were evaluated by CCK8 cell proliferation experiment,colony formation experiment,scratch healing experiment and Transwell cell invasion experiment.(4)KEGG enrichment analysis of RNA sequencing data and protein protein interaction(PPI)network in STRING database were used to find the possible downstream target of PSMD2(PTEN protein)and its possible regulatory signal pathway(MAPK pathway).Whether PTEN protein and key molecules of MAPK signal pathway expression changes following the change of PSMD2 expression was measured through Western Blot.To further prove the results above,CCK8 cell proliferation experiment,colony formation experiment,scratch healing experiment and transwell cell invasion experiment were used to determine whether PTEN knockdown can change the effect of PSMD2 knockdown on the proliferation,invasion and metastasis of HNSCC cells.(5)Subcutaneous tumorigenesis experiment and immunohistochemistry were used to verify the effect of PSMD2 on the genesis and development of HNSCC and its specific mechanism in vivo.Results:(1)114 of 804 UPSGs were differentially expressed in HNSCC tissues and adjacent tissues,among which 8 USPGs were related to the prognosis of HNSCC.Among the 8 USPGs,PSMD2 had the most significant prognostic correlation(P=0.0104).The expression of PSMD2 was significantly higher in HNSCC tissues,and its elevated expression was significantly involved in worse prognosis.(2)After knockdown of PSMD2 in Fadu,Cal-27 and HN8 cell lines,CCK8 cell proliferation experiment and other phenotypic experiments showed that the cell growth,colony number,scratch healing width and number of penetrating cells in PSMD2 knockdown group reduced significantly compared with the control group.(3)After overexpression of PSMD2 in Tca8113 cell line,CCK8 cell proliferation experiment and other phenotypic experiments showed that the cell growth rate,colony formation number,scratch healing width and number of penetrating cells in PSMD2 overexpression group were significantly increased compared with the control group.(4)After knocking down PSMD2,KEGG enrichment analysis of RNA sequencing data was carried out.The results showed that MAPK signal pathway had the highest enrichment.PPI network analysis showed that there were 28 proteins interacting with PSMD2,including 10non-ubiquitination-related proteins.Furthermore,only PTEN protein and RELA protein were involved in regulating MAPK signal pathway,but only PTEN protein was located upstream of MAPK signal pathway.(5)Western blot showed that after overexpression of PSMD2 in Tca8113 cell lines,the expression of PTEN protein decreased,and the proportion of phosphorylation forms of ERK and P38,the key molecules of MAPK signaling pathway,increased significantly,indicating that they were significantly activated.At the same time,when PSMD2 was knocked down in Fadu and Cal-27 cell lines,the expression of PTEN protein increased,and the proportion of phosphorylation forms of ERK and P38 decreased significantly,indicating that their activation was inhibited.(6)In the rescue experiment,PTEN was knocked down at the same time when PSMD2 was knocked down in Fadu cell line.Compared with the group that PSMD2 was knocked down alone,the activation of ERK and P38 was enhanced in the group that both PSMD2 and PTEN were knocked down.Moreover,CCK8 cell proliferation experiment and other phenotypic experiments showed that the growth rate of cells,the number of cell colonies,the width of cell wound and the number of penetrating cells were also increased.(7)The subcutaneous tumorigenesis experiment showed that the growth rate of tumor volume and weight in PSMD2 knockdown group was slower.Immunohistochemical of tumor tissue indicated that the expression of PTEN protein elevated along with the PSMD2 knockdown,which further verified the target protein of PSMD2 and its impact of promoting the genesis and development of HNSCC in vivo.Conclusion:PSMD2 can promote the proliferation,invasion and migration of HNSCC cells probably by inhibiting its target protein PTEN.PSMD2/PTEN axis may promoting HNSCC cells by activating MAPK signal pathway,leading to poor prognosis of HNSCC patients.