The Function And Mechanism Of Splicing Factor SRSF3 In Hypoxia-induced Autophagy

Background: Autophagy is an evolutionarily conserved cellular catabolic process.Dysfunction in the autophagy pathway has been demonstrated to be associated with many human diseases,including cancer.Alternative splicing of pre-m RNA is also an evolutionarily conserved regulatory mechanism of gene expression,leading to the production multiple isoforms of mature RNA from a single primary transcription.Aberrant alternative splicing is often linked to various pathological condition in human including numerous types of cancer.However,the association between these two cellular conserved processes is unclear.Splicing factor SRSF3(also called SRp20)plays an important role in regulation of alternative RNA splicing.SRSF3 has been reported as an oncogene and overexpressed in multiple cancers to promote cell proliferation and transformation.Objective: The aim of this study was to uncover the relationship between autophagy and SRSF3 in oral squamous cell carcinoma(OSCC).Methods: 1.The expression of a number of splicing factors was screened under hypoxia treatment by RT-PCR.The expression of splicing factors was verified by western blot.2.SRSF3 was knocked down or overexpressed by si RNA or T7-SRSF3 plasmid.The autophagic level was analyzed by western blot.The expression of GFPLC3 was observed by confocal microscope.3.The expression levels of BECN1,Fox O1 and p65,were analyzed by western blot and RT-PCR.The T7-SRSF3 plasmid was co-transfected with BECN1,Fox O1 or p65 plasmids into cells,and the change of autophagy was detected by western blot and RTPCR.Results: 1.Oncogene SRSF3 was significantly downregulated under hypoxia in CAL 27,Fa Du and SCC-9 cells.SRSF3 was negatively correlated with autophagy.2.Knockdown of SRSF3 increased autophagic activity,while overexpression of SRSF3 inhibited hypoxia-induced autophagy.3.Knockdown of SRSF3 significantly increased the m RNA levels of p65,Fox O1,BECN1.Then gain-of-function assays further showed that SRSF3 inhibits the expression of p65 and Fox O1,and their downstream target gene BECN1.The overexpression of BECN1,Fox O1,or p65 rescued autophagy inhibited by SRSF3.Conclusion: SRSF3 inhibits autophagy through p65,Fox O1 and BECN1.