Role Of CAPZA1 3'UTR Mutation And Overexpression In The Aggressiveness Of Esophageal Squamous Cell Carcinoma(ESCC)

Esophageal cancer is one of the most aggressive cancers.China,as a high incidence of esophageal cancer country and most of which has been diagnosed as ESCC histopathological type.Lacking of better therapeutic methods lead to poor prognostic effects and low survival rates.For long-term outcome of this cancer is still dismal,the study at molecular level has become one of research hotspots.In our study,according to the previous study about WGS and WES on ESCC,we identified that CAPZA1 significantly had copy number losses and single nucleotide variations in patients with ESCC.The biological functions and molecular mechanisms of CAPZA1 have never been associated with ESCC,let alone its SNVs in 3'UTRs.We have found that CAPZA1 displayed 5.84%copy number losses and only 0.65%copy number gains in the ESCC sequencing cohort.We further constructed stably overexpressed wild-type and mutant plasmids and transfected in ESCC cell lines(KYSE180 and KYSE510).Comprehensive study in vitro and vivo demonstrated that stably ectopic expression of CAPZA1 greatly suppressed cell proliferation,colony formation,migration,invasion,xenograft tumor formation and lung metastasis abilities,while the mutant cells attenuated the suppressive abilities.Additionally,we found that knockdown of CAPZA1 promoted the malignancy of ESCC.Further studies revealed the underlying mechanism that wild-type CAPZA1 suppressed cell malignancy of ESCC by increasing mRNA stability,while mutant CAPZA1 promoted mRNA decay.Furthermore,we found that the secondary structure of mutant CAPZA1 transcript was rich in more chain and stem-loop structures,relative to wild-type CAPZA1.Biotin-RNA pulldown assays,mass spectra analysis and RIP assays were performed to reveal that wild-type CAPZA1 targeted hnRNP K and PTBP1,while UPF1 targeted mutant CAPZA1 mRNA to promote mRNA decay.IP assays indicated that PTBP1,hnRNP K and UPF1 can interact with each other to influence mRNA stability.3'UTRs of eukaryotes play important roles in the regulation of gene expression at post-transcriptional levels.Recent studies have demonstrated that 3'UTR function as an independent RNA molecule,whose molecular mechanism is still dismal,let alone CAPZA1 3'UTR in ESCC.According to the results of RACE PCR and northern blot,we characterized a new transcript of CAPZA1 3'UTR,which was unannotated previously.RT-PCR and western blot assays showed that the expression level of 3'UTR won't impact the full mRNA of CAPZA1.Deeply,comprehensive study demonstrated that wild-type 3'UTR played oncogenic roles in accelerating cell proliferation,colony formation,migration and invasion and reducing apoptosis induced by UV irradiation,while the mutant 3'UTR attenuated the effects.RNA secondary structure is essential for the function of RNA transcripts,especially of IncRNAs.Furthermore,we discovered wild-type and mutant 3'UTR transcripts were incapable to encode proteins or peptides and secondary structure of mutant 3'UTR transcript contained more chain and cervical-loop structures.Further studies unmasked the underlying mechanism of 3'UTR in malignancy of ESCC.Biotin-RNA pulldown,RIP and IP assays were performed to test the RBPs involved in the crosstalk.We found IGF2BPs bound to wild-type 3'UTR particularly.Given the explicit function of IGF2BPs in the progression of tumor malignancy,we assumed that the IGF2BPs may exert their functions to 3'UTR or 3'UTR influence IGF2BPs by upregulating the expression level of their downstream protein C-Myc.Above all,our study comprehensively demonstrated that wild-type CAPZA1 targeted hnRNP K and PTBP1 to promote mRNA stability,leading to suppressive function.In contrast,mutant CAPZA1 presented loss of function.Interestingly,we provided an update and perspective on 3'UTR and its emerging role in ESCC.CAPZA13'UTR acts as a separate RNA molecule and plays a role as a specific IncRNA in the progression of ESCC.This scenario offers a new dimension to the crosstalk between 3'UTR and RBPs,which may help us identify novel biomarkers for cancer diagnosis and present new opportunities for 3'UTR based targeted therapy.