The Role Of IASPP-SV In Tumorigenesis And Development

Background and Objective: Inhibitor of apoptosis-stimulating protein of p53(iASPP)is one of the ASPP family.It binds to p53 to inhibit the transcriptional activity of apoptosis-related target genes and p53-mediated cell apoptosis,which is associated with tumor formation.Previously,we found a new subtype of iASPP,iASPP splice variant(iASPP-SV),which is a nuclear protein containing 407 amino acid residues and can bind to p53,inhibiting transcriptional activity of p53 on the promoters of both Bax and p21.However,the expression lever of iASPP-SV in tumor lines and the relationship of iASPP-SV and tumor cell proliferation are still obscure.Therefore,the purpose of this research was to identification of 5’-end of iASPP-SV m RNA to identify the transcription initiation region of iASPP-SV and the structure of the iASPP gene,and detect the endogenous iASPP-SV and iASPP(828)expression in 8 types of human cancer cell lines.To detect the function of iASPP-SV and iASPP(828)on the proliferation of cell for studying the role of iASPP-SV and iASPP(828)on tumorigenesis and progression.Then dectect the relationship between iASPP-SV,iASPP(828),p53 and NF-κB/p65.Methods: 5’-rapid amplification of c DNA ends(RACE)was used to identify the 5’-end of iASPP-SV m RNA in MCF-7 cells.Established expression plasmid for p FLAG-iASPP-SV and p FLAG-iASPP(828),and then transfected HEK 293 cells with p FLAG-iASPP-SV and p FLAG-iASPP(828)were used as positive control.Then Western blot was used to identify whether endogenous iASPP-SV and iASPP(828)were expressed in 8 types of human tumor cell lines.Transfected NIH 3T3 cells with p FLAG-iASPP-SV and p FLAG-iASPP(828)and established the stable clones of NIH 3T3 expressing iASPP-SV and iASPP(828).Cell proliferation assay and colony formation were used to identify whether iASPP-SV and iASPP(828)can promote cell proliferation and is associated with tumor formation.Luciferase assays were used to identify the relationships between iASPP-SV,iASPP(828),p53 and NF-κB/p65.Results: The result of 5’-rapid amplification of c DNA ends(RACE)identified that 5’-end of iASPP-SV was almost identical to RAI(AF078037.1),thus iASPP-SV and iASPP(828)were transcribed from the same gene and iASPP-SV was encoded by “correct” RAI sequence(DQ986418.1).iASPP-SV was endogenously expressed in many human cancer cell lines.Analysis of cell proliferation and colony formation assay identified that similarly to iASPP(828),iASPP-SV promoted cell proliferation and iASPP-SV exhibited more oncogenic activity than iASPP(828).Luciferase assays showed that iASPP-SV,iASPP(828),and p53 could bind to NF-κB/p65 and suppress its transcriptional activity.Conclusion: The only difference between iASPP-SV and iASPP(828)was in their N-terminal sequence and the 5’-end of iASPP-SV was almost identical to RAI(AF078037.1).These findings indicate that iASPP-SV and iASPP(828)were transcribed from the same gene,and iASPP-SV was encoded by “correct” RAI sequence(DQ986418.1).Thus iASPP can encode iASPP(828 amino acids)and iASPP-SV(407 amino acids)subtypes by selectively splicing.iASPP-SV which palys important role in the development of cancer,is expressed in a variety of tumor cells and promotes cell proliferation.iASPP-SV was endogenously expressed in many human cancer cell lines.Both iASPP(828)and iASPP-SV promoted cell proliferation,and iASPP-SV exhibited more oncogenic activity than iASPP(828).Therefore,iASPP-SV and iASPP(828)may be a potential target for cancer therapy.However,iASPP-SV and iASPP(828)not only inhibit apoptosis by inhibiting the transcriptional activation of p53-dependent target genes,but also promote the apoptosis by inhibiting the transcriptional activity of NF-κB.Thus iASPP-SV may be bifunctional proteins that interact with different proteins and play different functions that depend on different cell backgrounds.