Role And Mechanism Of RBPJ In Adult T Lymphocytic Leukemia

Human T-cell leukemia virus type 1(HTLV-1)is the pathogenic pathogens of adult T-cell leukemia(ATL).It has been reported that the virus protein is involved in the regulation of multiple signaling pathways in host cells,resulting in the proliferation and migration of cells and the occurrence of ATL.Clinical reports demonstrated that Notch1 pathway was activated in ATL,and inhibition of Notch1 mediated signaling pathway could inhibited the cell proliferation and formation of ATL significantly.As an important transcription factor in Notch1 signaling pathway,RBPJ,plays an important role in the activation of Notch1 signaling pathway.However,the roles and mechanisms of RBPJ in the pathogenesis of ATL remains unclear.To explore the expression and function of RBPJ in ATL,the aim of the present study intended to explore the roles of RBPJ in the proliferation and migration of ATL cells at the molecular,cellular,and animal levels.That would reveal the molecular mechanisms of RBPJ in the proliferation and migration of ATL cells.The main contents of this study were as follow:Part I.To investigate the expression and regulation mechanism of RBPJ in HTLV-1 infected cells.Firstly,the Real-time PCR and Western blot were applied to detect the expression of RBPJ in HTLV-1(un)-infected cells.The data showed that RBPJ was highly expressed in HTLV-1 positive cells.Furthermore,the expression of RBPJ was positively correlated with the expression of HTLV-1 viral protein p30,implying the over-expression of RBPJ in HTLV-1 positive cells might be related with the expression of p30.Part II.To ascertain the role of RBPJ in the proliferation and migration of ATL cells.A RBPJ-silenced stable cell line was established first,while the HTLV-1 positive cell line ATL-T as the research material.Real-time PCR and Western blot were carried out to detect the silencing effect of RBPJ and the expression of the viral core antigen.The evidences from the experiments revealed that the expression of viral core antigen remained unchanged after knockdown of RBPJ in ATL-T cells.Next CCK-8 assay and flow cytometer were used to detect the proliferation and cycle distribution of the cells.The results showed that cell proliferation was inhibited and the cell cycle was mainly arrested in the G1 phase after knockdown of RBPJ in ATL cells.The results of migration assay further displayed that the ability of cell migration decreased significantly after silencing RBPJ in ATL cells.Moreover,silencing the expression of RBPJ could inhibit the tumor formation of ATL cells in tumor-based mice significantly.Part III.To verify the mechanisms of RBPJ in promoting the proliferation and migration of ATL cells.We used the Real-time PCR and Western blot to detect the expression of genes in cells cycle monitoring point and the EMT pathway of the cells.The results showed that the expression of cell cycle-dependent kinase 4/6,cyclinD2/D3/E1/A2 and p-Rb decreased while knockdown RBPJ in ATL cells.The expression of E-Cadherin in EMT pathway was elevated,whereas TCF-8,Snail,N-Cadherin and ?-Catenin decreased.Similarly,these results above have also been reconfirmed in the animal models.In summary,we for the first time demonstrated that HTLV-1-p30 activated the RBPJ-mediated Notch1 signaling,thereby promoted the gene expression of cell cycle monitoring-point and EMT pathway,leadin to the formation of ATL.This study revealed the roles and mechanisms of RBPJ in the oncogenesis of ATL.Our finding provided a better understanding of the pathogenesis of ATL and a new targeting site for the treatment of HTLV-1 infected disease.