Aberrant Expression Of Splicing Factors In Newly Diagnosed Acute Myeloid Leukemia

Background:Alternative splicing is the process by which a single gene may produce manydifferent transcripts that can show a wide range of activities, and is responsible formuch of the diversity of the human proteome. Recent studies suggest that up to94%of all human genes undergo alternative splicing, and aberrant pre-mRNA splicing isa critical component of the pathogenesis of neoplasia.Plentiful evidence has revealed a close connection between AML andalternative splicing. Now it seems that the majority of splicing alterations in AMLare due to modifications in the concentration, localization, composition, or activityof RNA-binding proteins acting as splicing regulatory factors. The functionalproperties of splicing factors have recently received increased attention, but to date,no study has examined the expression of splicing factors in AML patients.Caspase-8, encoded by the CASP8gene, is a critical proteolytic enzyme in thesignaling cascade of apoptosis. As a result of alternative splicing, at least7isoformsof caspase-8have been reported. Previous studies showed that a splice variant namedCASP8L, which contains exon8b, is the predominant CASP8mRNA form in humanperipheral blood lymphocytes, and the caspase-8L protein was also expressed inCD34+stem cell-derived leukemic blasts from AML-M0patients and exhibited ananti-apoptotic function.In this study, we analyzed the mRNA expression of SR proteins (SRSF17) inpatients with newly diagnosed AML. Moreover, HNRNPA1, which mainly binds tosequences of splicing silencers and acts as a splicing repressor, was also examined.The potential association between splicing factors and the abnormal splicing ofCASP8was also investigated. A significant correlation was observed betweensplicing factors and abnormal CASP8pre-mRNA splicing.Methods:Using quantitative real time PCR we measured expressions of seven splicingfactors belonging to the serine/arginine-rich (SR) protein family, SRSF1(SF2/ASF), SRSF2(SC35), SRSF3(SRp20), SRSF4(SRp75), SRSF5(SRp40), SRSF6(SRp55)and SRSF7(9G8), and one non-SR factor, heterogeneous nuclear ribonucleoproteinA1(HNRNPA1) mRNA, in peripheral blood mononuclear cells of26patients withnewly diagnosed AML, and26healthy controls. In addition, the relationshipbetween splicing factors and the mRNA splicing patterns of the caspase-8gene(CASP8) was investigated.Results:1. Compared to healthy controls, the expression of splicing factors wasobviously aberrant in newly diagnosed AML patients. The expression of SRSF1,SRSF3and SRSF4mRNAs was significantly decreased. Both of the SRSF1vs.HNRNPA1and the SRSF1vs.SRSF3ratios were also decreased in AML patientswhen compared with the healthy controls.2. Abnormal patterns of alternative splicing of CASP8pre-mRNA wasobserved in AML patients. CASP8L was highly expressed in AML patients, whileCASP8A was highly expressed in healthy controls. The ratio of CASP8L/CASP8Awas significantly high in PBMC from AML patients.3. There were significant correlations of mRNA expression in the AML groupbetween SRSF3and CASP8L, SRSF3and the CASP8L/CASP8A ratio, and SRSF4and CASP8L. No correlation was found in healthy controls.Conclusions:1. The expression patterns of splicing factors were altered in newly diagnosedAML patients. In addition, the altered patterns were potentially linked to abnormalexpression of oncogenes, which may lead to the pathogenesis of AML.2. The levels of SR proteins in cancers are disease-specific. The aberrantexpression of splicing factors in AML may be useful for early diagnosis, prognosis,and therapy of AML.