The Role Of CT45-5in DNA Damage Response

CT45is a cancer/testis gene family that Chen identified by massively parallelsignature sequencing (MPSS).20CT or CT-like genes were identified,one of thesegenes is a member of a CT gene family that were designated as CT45. The CT45family comprises six highly similar (>98%cDNA identity) genes that are clustered intandem within a125-kb region on Xq26.3. The full length of CT45-5gene is567bp,which encodes189amino acids. We previously found when FHIT gene isoverexpressed in HeLa cells, the expression of CT45-5is increased by usinggene-Chips, and this differentiation was verified by RT-PCR and Western blotting.Then CT45-5was expressed and purified, the polyclonal antibody against CT45-5was prepared.We previous reported that Fhit-/-cells are more resistant to IR-induced killingthan Fhit+/+cells, in order to study the relationship of CT45-5and Fhit, and the role ofCT45family in DNA damage response, siRNAs targeting CT45-5gene werepredicted by using Bioinformatics, two pairs of CT45-5-siRNA were selected andinserted into the pSilencer2.1-U6Hygro vector. After confirmation of the constructsby DNA sequencing, only one is correct. Human cervical cancer HeLa cells andHeLa/Fhit cells overexpressing FHIT gene (3-18) were transfected withCT45-5-siRNA expression vectors, the interfering effect of CT45-5-siRNA wasidentified by Western blot, the results showed that CT45-5-siRNA could effectivelyinhibit the expression of CT45-5and FHIT gene, the CT45-5and Fhit may have somefunctional links.Next, the function of siRNAs targeting CT45-5gene was studied. The resultsshow that introduction of CT45-5-siRNA expression in human cells resulted insignificant G2phase arrest following ionizing radiation (IR) by the flow cytometry,and overexpression of CT45-5-siRNA protects cells from DNA damage inducedkilling in HeLa/Fhit cells by MTT assay. All these results proved that siRNAstargeting CT45-5gene improved cell tolerance to DNA damage by repressing theexpression of Fhit. In this study, we get one CT45-5-siRNA, which provide a basis on the furtherstudy of the function of CT45-5in DNA damage response.