ZC3H15 Regulates The Ubiquitination Of PTEN Via Recruitment Of TRIM56 And Promotes Malignant Progression Of Non-small-cell Lung Cancer

Objective: 1.Lung cancer is a cancer with high incidence and mortality in China,with non-small cell lung cancer(NSCLC)accounting for 80-85% of all lung cancer cases.Risk factors for lung cancer include tobacco use,family history,radiation exposure and chronic lung disease.Most patients with early stage NSCLC miss the best time for treatment due to the lack of clinical symptoms.NSCLC is often associated with mutations in oncogenic driver genes,a mutational phenomenon that plays an important role in the tumor progression of lung cancer.Zinc finger CCCH domain-containing protein 15(ZC3H15)is a member of the CCCH zinc finger protein family,also known as the erythropoietin-inducible gene(likely ortholog of mouse immediate early response erythropoietin 4,LEREPO4)or DRG Family-Regulatory Protein 1(DFRP1),located on chromosome 2q32.1,is a highly conserved eukaryotic protein involved in a variety of cellular processes and tumorigenesis.ZC3H15 contains a conserved DFRP structural domain in addition to the CCCH zinc finger structural domain.Previous findings suggest that ZC3H15 is highly expressed in both glioblastoma and gastric cancer and is associated with poor prognosis.In glioblastoma,ZC3H15 can act as a transcription factor to enhance the stability of epidermal growth factor receptor Gene(EGFR)protein by repressing CBL transcription,thus promoting the proliferation,migration and invasion of glioblastoma cells.ZC3H15 promotes the proliferation,migration and invasion of glioblastoma cells by repressing the F-box containing ZC3H15 promotes the proliferation,migration and invasion of gastric cancer cells by suppressing the transcription of F-box and WD repeat domain containing 7 Gene(FBXW7),thereby regulating the stability of c-Myc protein.However,the functions and molecular mechanisms of ZC3H15 in lung cancer need to be further explored.Methods: 1.Firstly,the online database was applied to analyze the expression of ZC3H15 in NSCLC tissues and paraneoplastic tissues and the relationship with T-stage and N-stage.2.Eight pairs of NSCLC tissues were clinically collected,total proteins were extracted and the expression of ZC3H15 protein was detected by the experimental method of protein immunoblotting(Western blot).Next,immunohistochemical staining was performed on paraffin sections of 164 NSCLC tissues and 40 paraneoplastic tissues to analyze the expression of ZC3H15 and the relationship between it and clinicopathological features.The statistical method of cox regression was applied to analyze the relationship between the expression of ZC3H15 and the prognosis of patients in 1149 clinical samples through an online database.3.The effect of ZC3H15 on the proliferation ability of NSCLC cells in vitro was investigated by CCK-8 assay,plate clone formation assay and Ed U cell proliferation assay.The effect of ZC3H15 overexpression on cell proliferation ability in vivo was investigated by xenograft tumor assay.4.Wound healing assay,Transwell migration and invasion assay to investigate the effect of ZC3H15 on the migration and invasion ability of NSCLC cells.5.ZC3H15 was found to be enriched to AKT-mTOR signaling pathway by gene enrichment analysis,the changes in expression levels of key proteins of this pathway were detected by Western blot method.6.Protein profiling and Bio GRID online database were used to screen the protein phosphatase and tensin homolog(PTEN)that could bind to ZC3H15.The subcellular localization of ZC3H15 and PTEN was investigated by cellular immunofluorescence assay and further investigated by immunoprecipitation assay.7.AKT inhibitor(LY294002)was added to cells overexpressing ZC3H15 to see if it could reverse the promoting effect of ZC3H15 on proliferation,migration and invasion of NSCLC cells.PTEN inhibitor(VO-Ohpic trihydrate)was added to cells knocking down ZC3H15 to see if it could reverse the inhibiting effect of down-regulated ZC3H15 on proliferation,migration and invasion of NSCLC cells.8.Real-time quantitative polymerase chain reaction(RT-q PCR)was used to detect the changes in m RNA levels of PTEN after overexpression and knockdown of ZC3H15.Western blot was used to detect the changes in m RNA levels of PTEN after overexpression and knockdown of ZC3H15.The changes of PTEN and p-PTEN protein levels after overexpression and knockdown of ZC3H15 were detected by Western blot.9.Knockdown of ZC3H15 was accompanied by the addition of actinomycin(CHX),and the changes of PTEN protein at 0 h,4 h,8 h and12 h were detected by Western blot.The effect of ZC3H15 on PTEN ubiquitination was detected by the addition of proteasome inhibitor(MG132)48 h after co-transformation of ZC3H15 and ub-HA plasmids.10.Protein profiling was performed to screen the E3 ubiquitin ligase protein containing tripartite motif containing 56(TRIM56)that can bind to ZC3H15,and TRIM56 can also bind to PTEN.Cellular immunofluorescence assays were used to determine the subcellular localization of TRIM56 to ZC3H15 and TRIM56 to PTEN,and the binding relationship was further determined by immunoprecipitation assays.11.Knockdown of TRIM56 was accompanied by the addition of CHX,and the changes of PTEN protein at 0 h,4 h,8 h and 12 h were detected by Western blot.TRIM56 alone and in ZC3H15 stable knockdown cells were transfected with ub-HA plasmid after knockdown of TRIM56,and the level of PTEN ubiquitination was detected by the addition of MG132 at 48 h of action.Results:1.The TCGA database found that ZC3H15 was highly expressed in lung cancer tissues and was associated with poor patient prognosis.8 NSCLC paired tissues showed significantly higher ZC3H15 protein expression levels than those of paraneoplastic tissues.Immunohistochemical staining of paraffin sections from 164 NSCLC tissues and40 paraneoplastic tissues revealed that high expression of ZC3H15 was associated with tumor size,TNM stage and lymph node metastasis.ZC3H15 was mainly localized in the cytoplasm,which was consistent with the results of immunofluorescence experiments on NSCLC cell lines.2.ZC3H15 promotes the proliferative ability of NSCLC cells.CCK-8,plate clone formation,Ed U cell proliferation assays and xenograft tumor assays in A549 and NCI-H1299 stable-transformed cell lines revealed that overexpression of ZC3H15 was able to promote the proliferative capacity of NSCLC cells in vitro and in vivo.3.ZC3H15 promotes the migratory and invasive ability of NSCLC cells.Wound healing and Transwell experiments showed that overexpression of ZC3H15 promoted the migration and invasion ability of NSCLC cells in vitro.4.Gene enrichment analysis suggested that ZC3H15 was associated with AKT-mTOR signaling pathway.Western blot experiments confirmed that p-AKT and p-mTOR protein expression levels increased after overexpression of ZC3H15 and decreased vice versa.The addition of AKT inhibitor LY294002 to the cells overexpressing ZC3H15 was able to reverse the proliferative,migratory and invasive effects of ZC3H15 on NSCLC cells.5.By using protein profiling and Bio GRID database,we found that ZC3H15 was able to interact with PTEN and both co-localized in the cytoplasm.Addition of the PTEN inhibitor VO-Ohpic trihydrate to knockdown ZC3H15 cells was able to reverse the inhibitory effects of knockdown ZC3H15 on NSCLC cell proliferation,migration and invasion.6.RT-q PCR assay found no significant changes in PTEN m RNA levels after overexpression or knockdown of ZC3H15.Western blot assay found that the protein levels of PTEN and p-PTEN were down-regulated after overexpression of ZC3H15,and the protein levels of PTEN and p-PTEN were up-regulated after knockdown of ZC3H15.7.The results of actinomycin assay showed that the half-life of PTEN after knockdown of ZC3H15 was significantly longer than that of the control group.The results of ubiquitination assay showed that overexpression of ZC3H15 increased the level of ubiquitination of PTEN.8.Combined with the results of protein profiling,immunoprecipitation and cellular immunofluorescence experiments,it was found that TRIM56 was able to interact with ZC3H15 and PTEN,respectively,and co-localize in the cytoplasm.9.The results of actinomycin assay showed that the half-life of PTEN in the knockdown TRIM56 cell group was longer than that of the control group.The ubiquitination assay showed that the ubiquitination level of PTEN was significantly reduced after knockdown of TRIM56 compared with the control group,after knockdown of TRIM56 along with overexpression of ZC3H15,the ubiquitination level of PTEN was reduced and the protein level of PTEN was increased after knockdown of TRIM56 compared with the cell line overexpressing ZC3H15 alone.Conclusion: 1.ZC3H15 is highly expressed in NSCLC and associated with poor prognosis.2.ZC3H15 promotes the proliferation,migration and invasive ability of NSCLC cells.3.ZC3H15 interacts with PTEN to activate the AKT-mTOR signaling pathway,which in turn promotes the proliferation,migration and invasive ability of NSCLC cells.4.ZC3H15 promotes the ubiquitinated of PTEN by recruiting TRIM56.