The Oncogene PIM1 Contributes To Cellular Senescence Via Phosphorylating SND1

Objective:The oncogene PIM1,encoding a constitutively active serine/threonine protein kinase,is involved in the regulation of cell proliferation,survival,differentiation and apoptosis.The up-regulation of its expression is also closely related to the initiation and development of various tumors.Recently,there is increasing body of literatures the role of PIM1 mediated cellular senescence,the precise mechanism remains unclear.In addition,the cellular senescence is increasingly regarded as a protective mechanism against carcinogenesis in subsequent researches.Therefore,exploring new mechanisms of senescence may provide new ideas for the treatment of cancer.Methods:Cell extracts of Flag-PIM1 overexpressing 2BS cells were immunoprecipitated by using FLAG-M2 beads,and then analyzed by SDS-PAGE electrophoresis,and mass spectrometric analysis of PIM1-related proteins was performed after silver staining.Next,the relevant plasmid was overexpressed in 2BS cells,and co-ip and pull-down and immunofluorescence experiments were used to further verify whether the selected target proteins SND1 and PIM1 bind to each other.We then further verified whether SND1 can be phosphorylated by PIM1 using kinase assays and western-blot experiments.Since PIM1 can phosphorylate SDN1,will it cause cell senescence after that? What is the specific mechanism? We then knocked down SND1 and used immunofluorescence,immunohistochemistry,CCK8,EDU,and ?-gal experiments to validate our conjecture.Finally,we knocked down SND1 in 2BS cells and sequenced them to see what aspects of the study caused aging changes and to verify the results of sequencing using RT-PCR.Results:First,we used mass spectrometry and co-immunoprecipitation to find that PIM1 and SND1 can directly interact.Next,we used in vivo phosphorylation experiments and in vitro kinase experiments to confirm that PIM1 can phosphorylate SND1 and promote its degradation.Then we did related functional experiments,such as ?-gal experiment,EdU,CCK8,etc.,which confirmed that SND1 is involved in the cellular senescence caused by the oncogene PIM1.Finally,we also found that SND1 is also involved in the regulation of the senescence-associated secretory phenotype(SASP)by RNA-seq and qPCR.Conclusion:The oncogene PIM1 is a serine/threonine kinase,which holds an important role in the regulation of cellular senescence.Many literatures have published the role of PIM1 in regulating the growth and transformation of malignant cells,while the function of PIM1 in senescence has only recently been reported.In present study,we provided evidences that PIM1 phosphorylates SND1 and promoting its degradation resulting in cellular senescence,and that SND1 is also engaged in the regulation of SASP.This discovery may be a novel mechanism for PIM1 to drive cellular senescence.To sum up,we believe that SND1 can promotes PIM1-induced senescence by regulating SASP.Notably,SASP has a two-way turnover,which depends mainly on the biological environment where it is located.Therefore,this should be taken into consideration when PIM1 inhibitors or SND1 inhibitors are used for cancer treatment in the clinic.