Construction Of TLP Expression Vector And The Research Of Its Effect On Cell Cycle Reaulation In HeLa Cells

TLP (TATA-box binding protein like protein), as an important member of the TBP (TATA-box binding protein) family, is an extensive transcription factor. Its structure is similar to the CCD of TBP with39%identity and76%similarity, so it is probably diverged from an ancestral TBP gene through a process of gene duplication and point mutations. Studies show that TLP is required for embryogenesis, spermiogenesis and apoptosis. In this study, we investigate how TLP regulate the growth of HeLa cells. It shows that TLP can inhibit the viability of HeLa cells as well as prolong G2phase of cell cycle.Firstly, we constructed the pEGFP-N1expression vector by inserting the coding sequence of TLP which was cloned from LO2cells into pEGFP-Nl expression vectors encoding a red-shifted variant of wide-type GFP which has been optimized for brighter fluorescence and higher expression in mammalian cells. Then, the expression vector was amplified in E.coli and was sequenced. The gene is completely consisted with the sequence in the NCBI, so it can be used in the following transfection exprements.Then, in order to examine whether plasmids of pEGFP-TLP and pEGFP-N1vectors were efficiently transfected into HeLa cells and the expression level, the fluorescence in transfected cells was detected with LCSM. After24h transfection, HeLa cells exposed in exciting light exhibitd significant green fluorescence indicating that TLP gene successfully expressed in HeLa cells. Besides, transfection efficiency of TLP was detected by flow cytometry. These results indicated that exogenous plasmids were successfully transfected and expressed in HeLa cells.And then, the viability of the HeLa cells, HepG2cells, LO2cells, Hek293cells and HL60cells, transfected by pEGFP-TLP and pEGFP-Nl vectors were detcted by MTT assay, respectively, which showed that HeLa cells were the most sensitive to the effect of TLP. Moreover, the above-mentioned conclution was identified through the assay of nuclear staining showing up by deeper nuclear staining and shrivelled karyotheca. Besides, the inhibiting effect of TLP was relieved when we silencd the expression of TLP by siRNA.At last, we examined the number of tansfected HeLa cells in each phase of cell cycle by flow cytometry which showed that TLP caused cell cycle arrest in G2phase. And we also detect the transcription level and expression level of the cell cycle-related genes (CDK1, CDK2, CDK4, CyclinBl, CyclinDl and CyclinEl) in Hela cells using RT-PCR and Western blot assay. These results show that TLP down regulated transcription and expression levels of CDK1and CyclinBl gene but there is no obvious change in control (HeLa cells transfected with EGFP-N1vector).In this study, the expression vector of TLP gene was constructed and was transfected into HeLa cells. It demonstrated that over expressing TLP can significantly inhibit the growth of HeLa cells which is confirmed by morphological changes, cell cycle arrest in G2phase. Our research showed that the mechnism of this change was the lower transcription and expression levels of cell cycle relevant proteins:CDK1and CyclinB1.