Regulation Of MYC On Splicing Factors PQBP1 And SF3B1 In Ovarian Cancer

BackgroundMYC,a transcription factor belongs to the bhlh-lz family,often interacts with Max as a dimer that binds specific DNA sequences(CACGTG)to transcriptionally regulate many genes.It is known as a classical oncoprotein because it mediates more than 30%of tumorigenesis.MYC amplification and abnormal activation exist in most of the tumors,such as gastric cancer,cervical cancer or breast cancer.MYC is a very important transcription factor which regulates about 15%of genes transcription in human genome,such as E2F2,CDKN1A,CCND1,etc.MYC plays an important role in angiogenesis as well as promoting tumorigenesis or maintaining the proliferation and differentiation of tumor cells by regulating the transcription of many genes.Recent studies have shown that MYC can regulate tumor cells metabolism.Although a huge amount of studies have analyzed the biological function of MYC in tumors and many target genes have been discovered,many mechanisms still have not been illuminated.In 2015,Nature reported that the survial of MYC-driven cancer depends on spliceosome.MYC interacts with splicing factor BUD31 would lead to synthetic lethal.targeting spliceosome may effectively kill MYC-driven tumors.However,the mechanism of MYC-dependent spliceosome survival has not been clarified in that paper.Here,we hypothesis that MYC may promote the survival of cancer cells by transcriptionally regulating the expression of spliceosomeRNA splicing refers to the process of removing introns and retaining exons of pre-mRNA in nucleus.snRNP nuclear picorribosin and other protein complexes can recognize Pre-mRNA splicing sites,after which,mature mRNA was obtained though two transesterification reactions.RNA splicing can be classified as constitutive splicing and selective splicing,both catalyzed by the similar molecular mechanism.Alternative splicing(AS)refers to the process that pre-mRNA generates different transcripts(splicing isomers)through the selective sequence combination of different splicing sites.Alternative splicing,as the key to expand human proteome diversity,is often regulated by RBP(RNA binding protein)trans-splicing factors.These splicing factors can affect the alternative splicing process by targeting specific Motif sequence.In tumor,not only did different transcripts amplification of the same gene varies enormously campared with normal tissue,but also new splicing variants are produced.The occurrence of aberrant alternative splicing can promote tumor cells' malignant behaviors such as proliferation,invasion,migration,angiogenesis and immune escape.Alternative splicing,as a new symbol of cancer,participates oncogene activation by producing splicing isomers with aberrant proliferation or anti-apoptotic properties that are beneficial to tumor cell survival.Through preliminary experiment and database analysis,the transcriptome analysis,our laboratory found that splicing factor PQBP1(Polyglutamine Binding Protein 1)and SF3B1(Splicing factor 3B subunit 1)is highly expressed in ovarian cancer and may be transcriptional regulated by MYC.ObjectiveThis project aims to explore the transcriptional regulation of MYC on splicing factors PQBP1 and SF3B1,determines whether MYC influences its downstream splicing events though regulating splicing factors.MethodsqPCR,Western blotting were used to detect the expression of MYC in original ovarian carcinoma cell lines;TCGA database,GSE database and GO analysis were used to analyze the influence of MYC towards alternative splicing events and to find downstream splicing factors;constructing lentiviral knockdown and overexpression lentiviral vectors to build MYC up-regulated or down regulated stable cell lines;qPCR,Western blotting were used to detect the expression of PQBP1 and SF3B1 in MYC high-expression and low-expression stable cell lines;PQBP1 and SF3B1 wt/mutant promoter vectors were constructed,which are used in Luciferase assay and ChIP to identify whether MYC can bind to both promoter as well as identify the binding sites;clonogenic assay,CCK-8 and rescue experiments demonstrated the regulation of MYC on them;JQ1 was used as MYC upstream Brd4 inbitor in ovarian cancer cells to observe the expression of PQBP1 and SF3B1 and ovarian cancer cells proliferation;qPCR detected the effect of MYC towards Bcl-x gene alternative splicing events through regulating PQBP1/SF3B1.Results1.MYC amplification and over-activation exist in ovarian cancer.MYC amplification was found in 34%of high-grade serous ovarian cancer.MYC highly expression is associated with poor prognosis.2.MYC tanscriptionally activates the expression of splicing factors PQBP1 and SF3B1 in ovarian cancer.The expression of PQBP1 and SF3B1 up-regulated distinctly in MYC overexpressed cell lines,while the results were reversed in knockdown cell lines.The results of Luciferase assay showed that compared with the mutant vector,MYC over-expression could significantly increase luciferase activity of PQBP1 and SF3B1 promoter region.Using JQ1 inhibitor BRD4 to deal with ovarian cancer cells leads to a decline of PQBP1 and SF3B1 expression,which further demonstrated the regulatory role of MYC towards PQBP1 and SF3B1.3.PQBP1 and SF3B1 were involved in the promotion of MYC on the proliferation of ovarian cancer cellsKnocking down PQBP1/SF3B1in MYC overexpression cell lines,the malignant proliferation ability of cells was significantly reduced compared with that before the knockdown.4.MYC affects Bcl-x splicing by regulating PQBP1/SF3B1 transcription.It has been demonstrated that PQBP1 and SF3B1 can influence the alternative splicing of apoptosis-related gene Bcl-x,and Bcl-x minigene plasmid was constructed to verify it in ovarian cancer cell lines.Transient transfect Bcl-x minigene plasmid at the same concentration and MYC overexpression plasmid at different concentrations.qPCR results showed that with the increase of MYC content,the expression of apoptotic transcription Bcl-xS declined,while apoptotic transcription Bcl-xL showed a reverse consequenceConclusion1.MYC amplification and over-activation exist in ovarian cancer.2.MYC transcriptionally activates the expression of splicing factors PQBP1 and SF3B1 in ovarian cancer3.PQBP1 and SF3B1 were involved in the promotion of MYC on the proliferation of ovarian cancer cells4.MYC affects bcl-x splicing by regulating PQBP1 and SF3B1 transcription...