Function And Mechanism Of Splicing Factor PCBP1 On STAT3 Exon 23 In Oral Squamous Cell Carcinoma

Background:STAT3(signal transducer and activator of transcription 3)plays very important roles in the initiation and development of tumors.Oral squamous cell carcinoma(OSCC)is the most common type of head and neck squamous cell carcinoma.STAT3 is significantly upregulated and constitutively activated in head and neck squamous cell carcinoma.Constitutive activation of STAT3 was also found to be an early event in head and neck carcinogenesis.However,there are few effective therapeutic methods to inhibit STAT3 in the treatment of OSCC.Despite of extensive studies in repressing its activation and function via multiple ways,so far,there are few effective therapeutic methods to inhibit STA T3 in the clinic.STA T3 has two isoforms generated by alternative splicing of exon 23.STAT3a is the longer isoform and encodes the full-length oncogenic STAT3a protein.STAT3? is shorter and encodes the truncated and tumor-suppressive STAT3? protein.It is essential to control the alternative splicing of STAT3 exon 23 during tumorigenesis.However,so far,it remains unknown how the alternative splicing of STAT3 exon 23 is regulated.AimThis study aimed to investigate the mechanism of STAT3 alternative splicing and its function during the development of oral cancer.MethodsGenomic DNA sequence from exon 22 to exon 23 was amplified from the CAL-27 genome and cloned into pEGFP-N1.The resulting minigene plasmid carried genomic sequence of STAT3 from exon 22 to exon 23.To map potential regulatory motifs,exon 23 in minigene was serially mutated.Twenty-six alternative splicing factors were transiently transfected into 293 cells,and the key factors regulating the alternative splicing of STAT3 exon 23 were screened.RNA pulldown experiments were performed to verify the key sequences binding to the RNA splicing regulatory protein PCBP1.The lentiviral plasmid expressing PCBP1 was constructed and transfected into oral cancer cells to observe cell growth,alternative splicing and protein expression of STAT3 gene,and expression of target gene of STAT3.The lentiviral plasmid expressing STAT3? was constructed,transfected into oral cancer cells,and the growth of cells was observed to study the function of STAT3?.Results:1.PCBP1 regulates the alternative splicing of STAT3 exon 23 and increases the level of STAT3?,which is correlated with its tumor suppressive function.PCBP1 interacts with an exonic splicing suppressor(ESS)in STAT3 exon 23 and inhibits the expression of STAT3a.2.PCBP1 inhibits the expression of STAT3a in oral cancer cells,thereby inhibiting the growth and the proliferation of OSCC cells.3.STAT3? inhibits the growth and proliferation of OSCC cells.Conclusions:PCBP1 is the key splicing factor that regulates the alternative splicing of STAT3 exon 23 and promotes the switch from oncogenic isoform STAT3a to tumor-suppressive isoform STAT3?.Our results may initiate new methods for anti-STAT3 treatment in cancers.