Mechanism On Alternative Splicing Of FOXM1 B/cisoforms

Objectives: Alternative splicing is a process in which a pre-mRNA produces different alternative splicing isoforms.About 95% of human multi-exonic genes are alternatively spliced,which increases the diversity of the protein[1].Abnormal alternative splicing is closely related to many diseases such as cancer.The process of alternative splicing is accomplished by the selection of splice sites and recruitment of trans-acting factors such as SR protein,hnRNPs by cis-acting elements such as precursor RNA enhancer,silencer.Forkhead box protein M1(FOXM1)is a member of the transcription factor Forkhead family.FOXM1 has a well-defined role in cell proliferation,cell cycle regulation,DNA damage repair,angiogenesis,apoptosis,oxidative stress,metabolic balance and inflammation[2].FOXM1 produces three mRNA precursors by splicing,encoding three protein subtypes of a,b and crespectively.Thea subtype contains 5a,7a alternative splicing exons,and has no transcriptional activity because of the destruction of the trans-activation domain[3].The b subtype has no 5a and 7a exons,while c subtype contains 5a alternative splicing exons.Both of b subtype and c subtype have transcriptional activity.FOXM1b is one of the effective tumor metabolic activation genes,and is a major subtypes in cancer cells[4].The b subtype has stronger ability of transformation than c subtype in soft agar experiments.In addition,FOXM1 b is an effective activator in tumor metabolism.The inhibition of FOXM1 b by the induction of ARF is the keyto suppress tumor metabolism[5].FOXM1 b activates the Akt-snailI pathway,leading to the up-regulation of tumor metastasis-related genes and promote tumor metastasis[6].Therefore,inhibition of FOXM1 b expression is of great significance in the treatment of tumor metastasis.FOXM1 c is widely expressed in normal cells and cancer cells and can be activated by RAF/MEK/MAPK pathway,which in turn promote proteolysis of FOXM1c[7].FOXM1 c can trans-activate c-myc promoter with universal transcription factor TFIIA,TFIIB by direct combination with the two TATA box or direct combination with TATA-binding protein(TBP).This new mechanism found new target genes of FOXM1 c,such as c-myc,c-fos,hsp70 and histone H2B/a.It strongly supports FOXM1 as evidence of a typical proliferation-related gene[8].The b subtype and c subtype of FOXM1 have different function.Thus,it is very important to study the mechanism of variable splicing of FOXM1b/c subtype,but the mechanism is not clear at present.The purpose of this study is to verify the functional difference of FOXM1b/c in cell proliferation,migration and other aspects,and to explore the mechanism of alternative splicing of FOXM1b/c subtype by explore these cis-acting elements and trans-acting factors involved in regulation of FOXM1b/c expression.Methods:(1)Compare the functional differences between FOXM1b/c subtypes.I n MDA-MB-231 cells,construct the stable cell lines of FOXM1 b and FOXM1 c,respectively.Carry out scratch experiments and plate cloning experiments,to explore cell migration ability and cell reproductive capacity,respectively.(2)Methods for detecting FOXM1b/c subtypes.Extract total RNA from Hela cells,detect the expression of FOXM1 b/c subtype by RT-PCR,and detect the endogenous expression of FOXM1b/c in carcinoma cell lines.(3)Determine the cis-acting elements that can control the FOXM1b/c subtype.At the reporter gene level,remove the sequence of the pyrimidine or purine enrichment sequence and transiently transfect it into Hela cells,and determine the cis-acting elements that can control the FOXM1b/c subtype by RT-PCR.Then knockout the special cis-acting elementin CRISPR-Cas9 technologyendogenously.(4)Determine the trans-acting factors which regulate the FOXM1b/c subtype at reporter gene level.Clone trans-acting factor and transiently transfect it into Hela cells with the reporter gene.Screen the trans-acting factor that regulates the FOXM1b/c subtype by RT-PCR.(5)In endogenous expression levels,verify the regulatory effect of trans-acting factors on FOXM1b/c alternative splicing.Construct cell lines stably expressing trans-acting factors,determine whether or not trans-acting factors can affect the expression of endogenous FOXM1b/c subtypes.Results:(1)The clone formation ability of cells overexpressed FOXM1 b was stronger than FOXM1 c,and the cell migration ability of FOXM1 c was slightly stronger than FOXM1 b.(2)Deletion of pyrimidine enrichment sequence P2 in intron 5aor purine enrichment sequences P3,P5 in exon 5aandintron 5aseparately,significantly increased FOXM1 b expression.Deletion of purine enrichment sequence P4 in intron5 a,significantly increased FOXM1 c expression.(3)Endogenous knockout P2 by CRISPR-Cas9 could significantly increase the expression of FOXM1 b.(4)At the reported gene levels,HNRNPF,HNRNPH significantly increased FOXM1 c expression,and HNRNPA1,HNRNPK,TRA2 A,TRA2B significantly increased FOXM1 b expression.(5)In the Hela stable cell line which overexpressed trans-acting factors,TRA2 B increased FOXM1 b expression.Conclusion: This study confirms that the clone formation ability of FOXM1 b was significantly stronger than FOXM1 c,and the cell migration ability of FOXM1 b was slightly stronger than FOXM1 c.At the reporter gene level,deletion of the pyrimidine or purine enrichment region P2,P3,P4,P5 could significantly alter FOXM1b/c expression,indicating that these pyrimidine/purine enrichment regions are essential for FOXM1b/c expression.Endogenously knockoutpyrimidineenrichment region P2 could significantly increase the expression of FOXM1 b,indicating that P2 is indeed a cis-acting element that regulates FOXM1b/c expression.In Hela cells,overexpression of the splicing factor TRA2 B can increase the expression of FOXM1 b,suggesting that the splicing factor TRA2 B is a trans-acting factor that can regulate the expression of FOXM1b/c.