Study On The Transcription Mechanism Of RNA Splicing Factor SF3B1

Cancer is a major type of disease threatening public health.The incidence and mortality of cancer in China rank first in the world.Exploring the mechanism underlying the initiation and progression of cancer provides basis for the development of new treatment that could reduce the incidence and mortality of cancer.The abnormal splicing of precursor RNA is highly associated with the occurrence and development of a variety of cancers.Alternative splicing is mediated by the splicing machinery.SF3B1(Splicing factor 3 subunit 1)is an important component of the RNA splicing complex U2 sn RNP(small nuclear ribonucleoproteins).SF3B1 plays a vital role in the assembly of U2 sn RNP and stablizes the interaction between the U2 sn RNP and branch sites sequence(BPS).Previous studies have shown that SF3B1 has a high mutation rate in myelodysplastic syndrome,chronic lymphocytic leukemia,uveal melanoma,breast cancer and pancreatic cancer,and the mutation of SF3B1 causes abnormal splicing of a variety of genes,which participate in the occurrence and development of cancers,such as myelodysplastic syndrome,chronic lymphocytic leukemia,and uveal melanoma.Studies have shown that inhibiting the activity of SF3B1 or reducing the expression of SF3B1 can significantly inhibit the proliferation and metastasis of breast cancer as well as other types of cancer cells.However,transcriptional regulation mechanism of SF3B1 is still unclear.Therefore,this study aims to find the transcriptional regulatory factors of SF3B1 and reveal the transcriptional regulatory mechanism of SF3B1 at the molecular level.pGL4 fluorescein expression vector with SF3B1 promoter fragments of different lengths was constructed.Then the constructed p GL4-SF3B1 promoter vector was co-transfected with the Renilla vector into 293 T cells,and the expression of the corresponding Luciferase was detected using the dual luciferase reporter gene.Luciferase results showed that the expression level of Luciferase transfected with p GL4-SF3B1 promoter-200 vector in 293 T cells was 33 times higher than that transfected with p GL4-SF3B1 promoter-100 vector.The expression level of Luciferase transfected with p GL4-SF3B1 promoter-200 vector was 850 times higher than that of transfected p GL4 empty vector.The biggest difference of expression level of Luciferase in the SF3B1 promoter is between the-200 ?-100 bp region.Therefore,there should be transcription factor binding sites that regulate the expression of SF3B1 in this region.Deletion mutations were made every 10 bp in the-200 ?-100 bp region of the SF3B1 promoter.When the-110 ?-101 bp region of the SF3B1 promoter was deleted,the amount of fluorescein expression was about 10 times lower than that of p GL4-SF3B1 promoter-200 which was transfected with the fluorescein expression vector without deletion,indicating that there is a transcription factor binding site that regulates the expression of SF3B1 in this region.The sequence CGTTTGGCGC of the-110 ?-101 bp region of the SF3B1 promoter was analyzed by the PROMO database,and two potential transcription factors that could bind to this region were predicted: FOXP3 and CEBPB.Q-PCR results showed that when FOXP3 was knocked down,the expression of SF3B1 decreased by about 2 times(n = 3,*P < 0.05),indicating that FOXP3 had a positive regulatory effect on the expression of SF3B1 at the m RNA level.However,when CEBPB was knocked down,the expression of SF3B1 did not change significantly,indicating that CEBPB had no regulatory effect on the expression of SF3B1.Western Blot was used to further verify whether FOXP3 can regulate the expression of SF3B1 at the protein level.When FOXP3 is knocked down,the expression of SF3B1 at the protein level is also significantly reduced,which confirms that FOXP3 can regulate the expression of SF3B1.FOXP3 is a member of the forkhead box protein family(FOX)and a member of the forkhead/wing helix family of regulatory factors.This gene is expressed in many tumor cells and can interact with other transcription factors,histone acetylases and deacetylases as transcription activators or transcription repressors.This discovery provides a significant foundation for the tuture study that investigate the role of the transcriptional regulation mechanism of SF3B1 in the initiation and progression of cancer.