SRSF10 Mediated IL1RAP Alternative Splicing Regulates Cervical Cancer Oncogenesis

Objective: HR-HPV oncoproteins E6 and E7 are the major etiological factors of CC but are insufficient for malignant transformation of CC,since CC is a multifactorial,multi-stage pathological process.Dysregulated Alternative splicing,mainly ascribed to aberrant splicing factor levels and activities,contributes to most cancer hallmarks.However,does AS acts as an “accomplice” of E6E7 to promote CC progression? Considering the significance of HPV infection and AS,we try to explore the relationship among HPV infection,AS and CC oncogenesis providing an attractive therapeutic target for the treatment of CC.Contents and Methods: 1)We used siRNAs targeted against E6E7 in HPV 16/18 positive CC cells and performed a microarray analysis to explore whether E6E7 regulate the expression of splicing factors;PCR was used for verification and to screen the most significantly downregulated splicing factor.2)The public databases(Oncomine,etc.)were applied to analyze the mRNA levels of SRSF10 in human CC tissues,which was validated in protein level in a CC tissue array by IHC;we further investigated the role of SRSF10 in CC in a xenograft mouse model by establishing CC cells which stably express SRSF10 shRNA.3)To explore the molecular mechanism of E6E7 regulating SRSF10 expression,ChIP and dual luciferase reporter assay were used to verify the transcription factor which binds the SRSF10 promoter;RNAi and WB were utilized to examine the regulatory capacity of transcription factor on SRSF10 expression.4)TCGAspliceseq and CLIP-seq were applied to analyze the AS events related to SRSF10.PCR,RNA-seq and CLIP were used to validate the position-dependent AS regulation of SRSF10.5)Dual luciferase reporter assay,WB,ChIP and IHC were applied to explore whether SRSF10-mediated mIL1 RAP promote CC progression by regulating IL1?-induced NF-?B activation and the expression of the immune checkpoint molecule CD47.Results: 1)Knockdown of E6E7 downregulates the expression of 19 core splicing factors,especially SRSF10.2)SRSF10 is increased in CC and related to HPV infection.Knockdown of SRSF10 significantly decreases the tumorigenesis of CC.3)E2F1 binds to the SRSF10 promoter,upregulating the expression of SRSF10 via transcriptional activation in HPV16/18-positive CC.4)SRSF10 promotes the production of mIL1 RAP via regulating the AS of IL1 RAP exon 13.Dysregulated mIL1 RAP participates in the tumorigenic potential of SRSF10 in CC.5)SRSF10-mediated mIL1 RAP upregulates the “don't eat me” signal CD47 via promoting IL1?-induced NF-?B activation in CC.Conclusion: HR-HPV oncoproteins E6E7 increase the expression of SRSF10 via E2F1 transcriptional activation.SRSF10-mediated IL1 RAP alternative splicing upregulates the “don't eat me” signal CD47 by regulating IL1?-induced NF-?B activation,which inhibits the phagocytosis of macrophages to promote CC progression.