| Objective: Lung cancer is a main cause of cancer-related death,with 85% of cases involving non-small-cell lung cancer.Deubiquitinases and noncoding RNAs have been the subjects of recent extensive studies regarding their roles in lung cancer,but the mechanisms involved are largely unknown.In our study,we used The Cancer Genome Atlas(TCGA)dataset and bioinformatics analyses and identified USP21,a DUB,as a potential contributor to oncogenesis in non-small-cell-lung cancer(NSCLC).We further demonstrated that USP21 was highly expressed in NSCLCs.We then conducted a series of in vitro and in vivo assays to explore the effect of USP21 on NSCLC progression and the underlying mechanism involved.Methods:(1)We analyzed the expression levels of USP21 in LC patients using the LUAD and LUSC data set of TCGA.(2)NSCLC cells lines(A549,H1299,NCI-H460,NCI-H520)and 16 HBE cell line were cultured.(3)We constructed a USP21-overexpressing plasmid,two USP21 si RNAs,a YY1-overexpressing plasmid and a YY1 si RNAs respectively transfected into A549 cells and NCI-H460 cells.(4)We measured the m RNA and/or protein expression levels of USP21,YY1,E-cadherin,N-cadherin,SNHG16 and miR-4500 in 42 paired NSCLC samples and their adjacent normal tissues by RT-PCR and/or western blotting analysis and/or IHC,all of which were obtained from Xin Hua Hospital.(5)The roles of USP21 in NSCLC cell proliferation were investigated by performing the MTT assay and the roles of USP21 in NSCLC migration and invasion were then investigated by performing transwell assays(6)We examined whether USP21 could directly interact with and deubiquitinate YY1 by co-IP assays and GST pull-downs.A549 cells were treated with cycloheximide to inhibit protein synthesis and were then transfected with USP21 si RNA and the protein expression levels of YY1 was measured by the western blotting analysis.Moreover,YY1 levels were then assessed in wild-type(wt)USP21 or a catalytic site mutant USP21(USP21 C221A)-transfected cells after added MG132.(7)With the aid of JASPAR,we predicted that there was a binding site for YY1 in the promoter region of SNHG16.The interaction between YY1 and SNHG16 was then investigated by the dual-luciferase reporter assays,Ch IP and EMSA.(8)With the aid of Starbase,we predicted that miR-4500 was one of the possible ce RNAs of SNHG16 and it also interacted with USP21.To confirm our previous results,we performed luciferase reporter assays and RIP assays.(9)To investigate tumor formation in vivo,USP21 and YY1 overexpressing plasmids were packaged into a lentiviral vector,and si-USP21 and si-YY1 were independently inserted into recombinant adenoviruses.Results:(1)USP21 was overexpressed in lung cancer tissues when compared to adjacent normal tissues.Moreover,USP21 was also overexpressed in A549,H1299,NCI-H460 and NCI-H520 cell lines in comparison to what was observed in the 16 HBE normal lung cell line.(2)USP21 promotes NSCLC cell proliferation in vitro and in vivo.(3)USP21 also promotes cell migration and invasion in NSCLC cells.(4)USP21promoted cell proliferation through deubiquitinating and stabilizing protein levels of YY1 in NSCLC cells.(5)YY1 transcriptionally activates SNHG16 via binding to its promoter region.(6)SNHG16 regulates USP21 expression by targeting miR-4500.Conclusions: In summary,the USP21/YY1/SNHG16 axis plays a role in promoting the progression of NSCLC.Therefore,the USP21/YY1/SNHG16/miR-4500 axis may be a potential therapeutic target in NSCLC treatment. |
