Construction Of YWHAZ Overexpression Vectors And Exploration Of Their Effects On Tumor Migration

Background and Objective: YWHAZ belongs to14-3-3genefamily, plays important role in signal transduction, cell cycle control andapoptosis. Previous work showed that YWHAZ was highly expressed insome tumors, such as lung cancer, breast cancer, etc. Recently, scientistsrealized that YWHAZ served as a oncogene and contributed totumorigenesis and development. The latest research on cell adhesionrevealed that it was even involved in tumor metastasis. In this article weplanned to construct the YWHAZ adenoviral and retroviral vectors. Bydetecting the efficiency of virus on target cells and overexpressedYWHAZ, we investigated whether overexpressed YWHAZ would affecttumor migration. Our experiment provided the basis for furtherinvestigation of YWHAZ in tumor metastasis.Methods:1. Preparation of YWHAZ adenovirus: YWHAZ wassub-cloned into adenoviral shuttle plasmid(pacAd5CMV-IRES-GFP),then verified by enzyme identification, PCR amplification and genesequencing. The correct recombinant adenoviral plasmid was transfectedinto293T cells with the backbone plasmid.293T cells were constantlyused to amplify YWHAZ adenovirus.2. Preparation of YWHAZretrovirus: YWHAZ gene was sub-cloned into retroviral plasmid(pLXSN-IRES-GFP), then confirmed by enzyme digestion, PCR andgene sequencing. YWHAZ retrovirus was made by transfecting the recombinant retroviral plasmid into Ampho293cells. G418was used toscreen cells to get the stable transfected Ampho293cells.3. Analysis ofYWHAZ virus infection efficiency:293T cells were infected withYWHAZ adenovirus and retrovirus respectively. The infectionefficiency was observed by fluorescence microscope after24hours,48hours and72hours.4. Analysis of YWHAZ gene expression: TheYWHAZ mRNA expression between293T cells infected by YWHAZvirus and uninfected293T cells was detected by RT-PCR.5. Analysis ofcell migration ability: Scratch test was conducted on YWHAZadenovirus infected MDA-MB-231breast cancer cells and uninfectedMDA-MB-231cells to evaluate the cell migration ability. The same testwas done on A549lung cancer cells. Cell migration was observed after12hours and24hours.Results:1. Preparation of YWHAZ adenovirus: PacAd5CMV-GFP-YWHAZ recombinant plasmid was confirmed by enzyme digestion,PCR and gene sequencing, a700bp amplified PCR product proved thatthe construction was correct. YWHAZ adenovirus were producedthrough homologous recombination, then amplified, and purified. A titerof (4×108)unit/ml virus was achieved on measurement.2. Preparationof YWHAZ retrovirus: PLXSN-GFP-YWHAZ recombinant plasmidwas confirmed by enzyme digestion, PCR and gene sequencing, a700bpamplified PCR product proved that the construction was correct.Ampho293cells could pack YWHAZ retrovirus, stably retrovirus-producing Ampho293cells was screened by G418.3. Analysis of YWHAZ virus infection efficiency:293T cells were infected withrecombinant YWHAZ adenovirus and retrovirus respectively. After72hours, the strong green fluorescence was visible in cells. In adenovirusinfected cells, nearly90%cells expressed GFP; in retrovirus infectedcells, nearly70%cells expressed GFP. It demonstrated that these twoYWHAZ virus could infect target cells effectively.4. Analysis ofYWHAZ gene expression: RT-PCR result showed that the YWHAZadenovirus infected293T cells expressed YWHAZ mRNA much higherthan normal293T cells. The similar result was observed in YWHAZretrovirus infected293T cells.5. Analysis of cell migration ability:Compared with non-virus infected MDA-MB-231cells, MDA-MB-231cells infected by YWHAZ adenovirus migrated faster. A549cellsshowed a similar phenomenon.Conclusion:1. We successfully constructed YWHAZ adenovirusand retrovirus.2. YWHAZ adenovirus and retrovirus could infect targetcells effectively and highly expressed YWHAZ mRNA.3. YWHA genecould promote migration of MDA-MB-231breast cancer cells and A549lung cancer cells, which proved that YWHAZ was associated with tumorcell migration.