The Mechanism Of STAT3/MZF1 In PKM Gene Alternative Splicing Regulation

Malignant tumor is a major threat to human health.The change of gene expression existed in multiple links in tumor development,including the transfer of normal genes to the oncogenes,or the inactivation of tumor suppressor genes.Alternative splicing is an important process in gene expression regulation,which can produce different genotypes,closely related to cell physiological,pathological process.Alternative splicing play a vital role in the development process of tumor.It is crucial to reveal the mechanism of alternative splicing for tumorigenesis and tumor targeted therapy.The metabolism of tumor cells has remarkable characteristics which is different from normal cells.The metabolism in normal cells is given priority to mitochondrial pathway of aerobic oxidation,whereas even under the condition of sufficient oxygen,tumor cells can adopt aerobic glycolysis manner to decomposit glucose and gain energy,which is known as the Warburg effect.Among them,as a key enzyme involved in the metabolism of tumor Warburg effect,the Pyruvate Kinase M2 subtypes(PKM2)is of great significance to the tumor metabolism and regulatory mechanism of tumor growth.Pyruvate kinase(PKM)gene,via alternative splicing of precursor mRNA,produces two different subtypes,PKM1 and PKM2.PKM1 retains the ninth exon,while PKM2 keeps the tenth exon.Although the only one exon differs in the structure of genes,but the position in the organization and function of genes are significantly different.PKM1 expressed in normal tissues that need a lot of energy,such as in brain and muscle tissue,while PKM2 expressed in the tissues synthesized a large number of nucleotides,such as in tumor tissues.PKM2 is the key enzymes to the tumor metabolic in the Warburg effect.However,how the PKM gene is regulated to prompt PKM2 isoform expression in tumor tissues is still not clear.This paper used a variety of methods including bioinformatics,cell and molecular biology,and RNA alternative splicing technology,aiming to reveal and discuss this issue.The discovery of novel RBPs(RNA Binding Proteins)is the core to characterize the mechanisms of the alternative splicing regulation.In this thesis,we used bioinformatics method to analyze the genetic structure of PKM,and found that in the intron area which each located in the upstream of 9 exon and10 exon,has the binding sites of STAT3(Signal Transducer and Activator of Transcription 3)or MZF1(myeloid zinc finger protein 1),individually.STAT3 signaling pathway is sustained activated in a wide variety of tumor cells and STAT3 is demonstrated major as a important transcription factor in tumor cell proliferation.Whether STAT3 can act as a new type of RBP protein to control alternative splicing in RNA level remains unclear.MZF1 belongs to Kroppel type related(C2H2 zinc finger)family of transcription factors,contains 13 C2H12 type zinc finger,which structure is suggested to be able to bind to the specific sequences of target DNA and RNA.However,the study on MZF1 gene just begun,the evidence whether RNA-binding function of MZF1 exists or not is not clear.STAT3 and MZF1 can be or not can be,as a new type of RBP to regulate alternative splicing of PKM genes,may become an important issue to reveal occurrence of Warburg effect.Subsequently in this paper,we used kinds of tissues and cells to compare the gene expression links between STAT3/MZF1 and different gene types of PKM.Meanwhile,we used breast cancer cells and normal mammary epithelial cells as the research object,verified different PKM genotypes regulation by STAT3 or MZF1.Notably,we performed EMSA and RIP experiments to prove that STAT3 and MZF1 attributed with PKM RNA genes binding ability.We steadily revealed STAT3/MZF1 as RBP target to regulate the process of alternative splicing of PKM,and analyzed the lactic acid metabolism.Our study provided a new molecular mechanism in the regulation of alternative splicing of PKM gene.Firstly,we tested the expression of pyruvate kinase subtypes of PKM1 and PKM2 in various cells and tissues.We found that PKM1 only expressed in brain,muscles,heart tissue,normal mammary epithelial cells and embryonic kidney epithelial cells,while PKM2 specificity expressed in human breast cancer MCF7/MDA-MB-231 cells,and human prostate cancer LNCaP/DU145 cells.Moreover,we detected the correlation of STAT3/MZF1 with PKM1/PKM2 at mRNA level and protein level.Our investigations showed that the STAT3 was expressed consistent with PKM2 in tumor cells or in the proliferation organizations;MZF1 was expressed consistent with PKM1 in normal tissues and cells.Further,we studied the relationships between STAT3/MZF1 and the different PKM subtypes transformation,by means of the over-expression or knockdown STAT3 in breast cancer cells.We detected expression conditions of PKM1 and PKM2 respectively from the level of protein and mRNA.Our experimental results showed that the over-expression of STAT3 could raise PKM2 mRNA and protein expression levels,reduce PKM1 mRNA and protein expression levels.Whereas the knockdown of STAT3 can reduce PKM2 and raise PKM1 gene expression.In normal mammary epithelial cells,after over-expression or knockout MZF1,we surprisingly found that MZF1 could promote PKM1 mRNA and protein expression,reduce PKM2 mRNA and protein expression;while the knockdown of MZF1 could decrease PKM1 and promote PKM2 gene expression.The results implied that STAT3/MZF1 modulates PKM1/PKM2 subtypes transformation at mRNA level,indicating that STAT3MZF1 participate in the PKM alternative splicing process at mRNA level.To confirm the RNA binding capacity of STAT3 and MZF1 as novel RBPs,we first constructed prokaryotic expression protein of STAT3/MZF1 DBD(DNA binding domain),which was subjected to EMSA experiments to prove the binding activity of STAT3/MZF1 to the RNA precursor of PKM in vitro.The results showed that the DBD of STAT3 specifically bound to 19 bp length of RNA intron sequences located in the upstream of exon 9 of PKM gene,and MZF1 specificity bound to 21 bp length of RNA intron sequences located in the upstream of the exon 10 of PKM gene.Then,RIP experiments further confirmed STAT3 was capable of binding to the upstream of PKM exon 9,MZF1 was capable of binding to the upstream of PKM gene exon 10 in vivo.These results proved that the STAT3/MZF1 functions as novel RBP,has the capacity of RNA binding.We secondly analyzed whether STAT3/MZF1 can recruit splicesome by immunoprecipitation.The results suggested that STAT3/MZF1 have an interaction with U1SnRNP/U2 SnRNP belongs to splicing complex and U2AF35/U2AF65 belongs to serine/arginine-rich related proteins.In addition,using U2AF35 antibodies to conduct RIP experiment,we found that STAT3/MZF1 recruited splicesome to the upstream area of PKM precursor mRNA extron 9 or 10,determining the occurrence of alternative splicing of PKM gene.These results systematically explained STAT3/MZF1 bound to RNA and recruit splicesome,in the regulation of PKM1/PKM2 gene transformation by alternative splicing.Finally,we discussed the physiological significance of PKM gene selective spliced by STAT3/MZF1.As the key enzyme of Warburg effect,PKM2 is beneficial to cohesion of each reaction step in glycolysis,to accelerate the transformation of glucose into lactic acid.We found that overexpression of STAT3 promoted tumor cell growth,at the same time promoted the lactic acid level in the tumor cells.The overexpression of MZF1 inhibited tumor cell growth and lowered the lactic acid level in the tumor cells.Furthermore,we proved that STAT3 and PKM2 both highly expressed in breast cancer MCF7 cells;MZF1 and PKM1 both highly expressed in normal breast epithelium MCF10 A.In normal mammary epithelial cell line MCF10 A cell,the overexpression of STAT3 raised the lactic acid level,and this effect can be interrupted by PKM2-shRNA.Finally,we found that STAT3/MZF1 antagonist each other in the process of alternative splicing of PKM gene.The overexpression of STAT3 could promote the expression of PKM2 and inhibit the expression of PKM1,and increased the intracellular accumulation of lactic acid,which effect could be reversed by MZF1.MZF1 could promote the expression of PKM1 and inhibit PKM2 expression,down-regulated the lactic acid level,and this effect can be reversed by STAT3.These results indicated that STAT3/MZF1 controled the transformation of PKM1/PKM2 to modulate the lactic acid level in cells.In conclusion,we found two new types RBPs of STAT3 and MZF1 in PKM gene isoform transformation regulation,through alternative splicing mechanisms.STAT3 protein can regulate the alternative splicing of PKM precursor mRNA at transcription level,which makes PKM2 subtypes increased in tumor cells,thus enhances the aerobic glycolysis and promotes the growth of tumor cells.We also demonstrate that MZF1 protein can regulate the alternative splicing of PKM precursor mRNA at transcription level,to make PKM1 subtypes increased in tumor cells,inhibits aerobic glycolysis and the growth of tumor cells.Finally our study illustrated a new tumor related signaling pathways of the alternative splicing of PKM gene,and provided the possible novel strategies and ideas for the regulation of tumor metabolism targets and the effective treatment of tumors.