Mechanism On Cellular Senescence Regulated By XAB2 Via POLR2A

Background: Cellular senescence is a process that cell proliferation and physiological function decline gradually with the passage of time.It is believed that the shortening of telomeres,the activation of oncogenes,some cytokines,reactive oxygen species(ROS),DNA damage and nucleotide depletion could induce the cellular senescence.It involves a variety of physiological and pathological processes,including development,wound healing,ageing and tumorigenesis.Cellular senescence is characterized by the gradual loss of physiological integrity,which eventually leads to the impairment of biological function.XAB2 is a highly conserved gene that was first reported in 2000.Some studies revealed that XAB2 has been involved in the pre-m RNA splicing,transcription and embryonic development.It has been reported that expression of XAB2 in hematopoietic stem cells of aging mice was significantly down regulated,suggesting that XAB2 may participate in the regulation of cellular senescence.However,the specific mechanism on cellular senescence regulated by XAB2 is not fully understood.In eukaryotic cells,RNA polymerase II protein contains 12 subunits.POLR2A(RNA polymerase II subunit A)is the largest catalytic subunit among them,which plays an important role in the initiation,elongation,pause and termination of transcription.In previous studies of our laboratory,we found that knocking down XAB2 in He La cells can significantly inhibit the expression of POLR2A in at m RNA level.In eukaryotes,RNA polymerase II is responsible for the transcription of all m RNA and some non-coding RNA such as sn RNA,sno RNA,si RNA and mi RNA.RNA polymerase II contains 12 subunits,POLR2 A is the largest catalytic subunit among them.POLR2 A gene is located on human chromosome 17p13.1,and its CTD terminal plays an important role in the initiation,suspension,elongation and termination of transcription.Since POLR2 A is essential to the biological activities of cells,and its absence may lead to the serious imbalance of gene expression in cells,we assumed that XAB2 could promote cellular senescence through POLR2 A.With such hypothesis,we have designed a series of experiments to clarify the mechanism of XAB2 related cellular senescence.Methods:(1)Hydrogen peroxide was used to induce HFF1 and MRC5 cell lines,and oxidative stress cell aging model was confirmed to be well established.(2)Replicative cellular senescence model was also well established.Western blot was also used to explore the expression of XAB2 and POLR2 A.(3)In the natural ageing mouse model,Western blot was performed to compare the expression of POLR2A and XAB2 in the lung,brain,liver and heart tissues of 14 and 11 month old and 1 month old mice.(4)MTT assay was used to detect the effect of XAB2 on the proliferation of HFF1 cells.(5)Flow cytometry was performed to explore the regulation of XAB2 on the cell cycle of HFF1 cells.(6)The cellular senescence caused by XAB2 and POLR2 A knockdown in HFF1 cells were verified by SA-?-Gal staining.Results:(1)The oxidative stress-induced cellular senescence induced by hydrogen peroxide was successfully established in HFF1 and MRC5 cell lines.Compared with the control group,the expression of XAB2 and POLR2 A decreased significantly.(2)Western blot also showed that XAB2 and POLR2 A were down regulated in replicative cellular senescence model.(3)In the natural ageing mouse model,it was found that the expression levels of XAB2 and POLR2 A in the lung,brain and heart decreased with their growth,but there was no significant change in the expression of liver tissue.(4)MTT assay showed that XAB2 knockdown significantly inhibited the proliferation of HFF1 cells.(5)Flow cytometry showed that knockdown of XAB2 in HFF1 cells could induce cell cycle arrest in G2/M phase.(6)SA-?-gal staining showed that depletion of XAB2 and POLR2 A could result in cellular senescence,and the expression of p53 and p21 also increased significantly.(7)The rescue experiment showed that exogenous overexpression of POLR2 A could partially reverse the cellular senescence phenotype induced by XAB2 knockdown in HFF1 cells.Conclusion: In this study,we found that XAB2 and POLR2 A are downregulated in hydrogen peroxide oxidative-induced cellular senescence model,replicative senescence model and lung,heart,brain tissue samples from natural ageing mice.XAB2 also had a certain regulatory effect on cell proliferation and cell cycle.Finally,we concluded that depletion of XAB2 could induce cellular senescence by repressing POLR2A.