Construction Of CIA2A Recombinants And The Function Study Of CIA2A On Apoptin Selectively Inducing The Apoptosis Of Hela Cells

Human CIA2A(cytosolic Fe/S protein assembly machinery member 2A,Fam96A) consists of 160 amino acids. Its molecular weight is 18.4KD and locates on chromosome 15q22.31. We have known very little about CIA2 A so far. The existed research shows that CIA2 A locates in the cytoplasm as monomers or dimers and it was found to interact with Ciao 1 both in vitro and in vivo, which functions as a cytoplasmic Fe-S assembly protein. CIA2 A also may play a role in chromosome segregation by establishing the sister chromatid cohesion and may participate in cell growth, cell differentiation, etc by adjusting the biological function of WT1(Wilms tumor suppressor protein).Apoptin is a small protein encoded by chicken anemia virus(chicken anemia virus, CAV). It contains 121 amino acids and weights 13.6kd. Apoptin only induce tumor cells and transformed cells to apoptosis, but not do to normal cells. Its nuclear localization is one of the critical factors of Apoptin specifically induces tumor cells to apoptosis, it localizes in the nucleus in the tumor cells, whereas it localizes in the cytoplasm in normal cells.Our preliminary laboratory research finds and verifies that CIA2 A can interact with Apoptin in both tumor cells and normal cells. The study of clinical tissues shows that: the expression of CIA2 A in tumor cells is significantly lower than that in normal cells, as Apoptin only induce tumor cells and transformed cells to apoptosis, but not do to normal cells. Therefore, doing deeper research about CIA2A’s function and its interaction with Apoptin will make an important biological significance.Objective: To explore the impact of over-expressed CIA2 A on proliferation of Hela cells(cervical cancer cell line) and find out the preliminary molecularmechanism, to explore the impact of over-expressed CIA2 A in Hela cells on Apoptin inducing tumor cells apoptosis specifically, as well as on the sub-cellular localization of Apoptin, and find out the preliminary molecular mechanism.Methods: We constructed the eukaryotic expression vector and the recombinational segments of CIA2 A with myc tag by molecular cloning techniques depending on the DUF59. The recombinants of CIA2 A are: CIA2A△1(its deletional amino acid sequences are from No.1-38), CIA2A△2(its deletional amino acid sequences are from No.39-119), CIA2A△3(its deletional amino acid sequences are from No.120-160). Then we transfected these vectors respectively into Hela cells and detected whether CIA2 A and its recombinants can affect the apoptosis of Hela cells by Flow Cytometry. And we transfected these vectors respectively with pcDNA3.1-flag-Apoptin plasmid into Hela cells and detected whether CIA2 A and its recombinants can affect the tumor-specific cell apoptosis activity of Apoptin in Hela cells by Flow Cytometry. Also we detected the effect of over-expressed CIA2 A and its recombinants on sub-cellular localization of Apoptin in Hela cells by Immunofluorescence.Results: We constructed three truncated CIA2 A recombinants with myc tag successfully and all of them could express when they are transfected into Hela cells(individually or with pcDNA3.1-flag-Apoptin plasmid) successfully. The results of Flow Cytometry showed that the over-expressed full-length CIA2 A, CIA2 A △ 1could induce Hela cells apoptosis(apoptosis rates were 46.5% and 40.1% respectively,P < 0.01), while CIA2A△2 and CIA2A△3 didn’t have this effect(apoptosis rates were 15.5% and 13.8% respectively, ns). Furthermore, the results of Flow Cytometry showed that the over-expressed full-length CIA2 A, CIA2A△2 and CIA2A△3 could inhibit the tumor-specific cell apoptosis activity of Apoptin in Hela cells(apoptosis rates were 38.4%, 36.8% and 32.5% respectively, P < 0.01), but CIA2A△1 didn’t have this effect(apoptosis rate was 42.6%, ns). Immunofluorescence found that theover-expressed full-length CIA2 A, CIA2A△2 and CIA2A△3 could make Apoptin re-locate in the cytoplasm instead of nucleus in Hela cells, while CIA2A△1 didn’t have the above effect.Conclusion: 1. The over-expressed CIA2 A could induce Hela cells apoptosis,and the corresponding function domain is the missing area of CIA2A△2 and CIA2A△3(the corresponding amino acid sequences are from No.39-160). 2. The over-expressed CIA2 A could inhibit the tumor-specific cell apoptosis activity of Apoptin in Hela cells, and the corresponding function domain is the missing area of CIA2A△1(the corresponding amino acid sequences are from No.1-38). 3. The over-expressed CIA2 A could change the localization of Apoptin from nuclear to cytoplasm in Hela cells, and the corresponding function domain is the missing area of CIA2A△1(the corresponding amino acid sequences are from No.1-38).