The Effect Of SF3B1 Deficiency On Human Erythropoiesis

Background and ObjectiveSplicing is an important modification process after gene transcription.The main function is to remove the introns in premature mRNA and align the exons.Splicing occurs mainly in the nuclear spliceosome which contains 5 small nuclear ribonucleoprotein(U1,U2,U4,U5 and U6 snRNP)and more than 150 proteins.In the process of splicing,the recognition of exons is mainly mediated by splicing sites,so the splicing process is flexible,and auxiliary splicing factor can promote the removal or including the exons by the identification of splicing sites.This is known as the mechanism of alternative splicing.Therefore,the identification of exons has become very important and more and more attention has been paid to it.Splicing factor SF3B1 is the core component of U2-snRNP,it can identify the splice site in the 3' end upstream brand of pre-mRNA,and stabilize the combination between U2-shRNP and the splice sites,and play an important role in the process of splicing.In recent years,with the development of High-throughput DNA sequencing of sequencing,a large number of genes related to splicing have been observed in many cancers with recurrent mutations.The highest one among which is SF3B1,and it has been reported that up to 80%of patients with refractory anaemia with ring sideroblasts harbor SF3B1 mutation.The main biological character of RARS is the impaired erythropoiesis in bone marrow and the clinical manifestations is refractory anemia.The selective specity high frequency of SF3B1 mutation in RARS made it an important candidant of the role in erythropoiesis.Erythropoiesis is a complex process involving two stages:early stage erythropoiesis and erythroblasts terminal differentiation.The early stage erythropoiesis is refered to the differentiation of CD34+ cells to erythroid progenitor cells BFU-E and CFU-E.Erythroblasts terminal differentiation start from the morphologically recognizable proerythroblasts,which go throgh several mitosis to become basophilic,polychromatic,and orthochromatic erythroblasts.After enucleation the orthochromatic erythroblasts become reticulocytes and then become mature erythrocytes.Any problems during the process will affect the normal erythropoiesis and cause anemia.In order to study the relationship between SF3B1 and erythropoiesis,and further to observe the mechanism of SF3B1 in the pathogenesis of RARS,we performed SF3B1 knockdown in human peripheral blood derived CD34+ cells by lentiviral mediated transfection.During the culturer in three phases medium,we observe the cell proliferation,differentiation,enucleation and apoptosis.The molecular mechanism underlying the phenotypes is observed by bioinformatics analysis of RNA sequencing results.At last we studied the target genes picked after the analysis of RNA sequencing to confirm the phenotypes in SF3B1 knockdown.Part 1 The Effect of SF3B1 Knockdown On the Proliferation and Differentiation of ErythroblastsMethods1.Analyzed the previous RNA sequencing database in our lab to study the expression of SF3B1 mRNA during erythropoiesis.CD34+ cells were isolated from human peripheral blood and cultured in vitro by using the three-phase medium,and collected cells on different culture days to observe the protein level of SF3B1 during erythropoiesis by using Western Blotting.2.SF3B1 knockdown in human peripheral blood derived CD34+ cells by lentiviral mediated transfection was performed on the culture d2.The proliferation of erythroid progenitors was observed by colony forming assay,the cell cycle was studied by EdU and cell apoptosis was detected by flow cytomety.For the erythroid terminal differentiation,the proliferation,differentiation,enucleation and apoptosis were detected by flow cytometry and DNA ladder was used to cofirm the apoptosis during the erythroid terminal differentiation.At the same time,the effects of SF3B1 knockdown on erythroid terminal differentiation cells morphology was studied by microscope.3.Distinct stage cells of SF3B1 knockdown groups and Luciferase control groups during erythropoiesis were isolated by using FACS-based cell sorting,and colony forming assay was performed using the pure populations of BFU-E and CFU-E.The morphology of the sorted cells were observed and the abnormal nuclear cells were quantified by microscope.Results1.The analysis of RNA-seq indicated that SF3B1 mRNA showed high expression in each stage of erythroblasts.The results of Western Blotting also showed that the protein of SF3B1 expressed in every stage during the erythropoiesis,but the protein level decreased with the the differentiation.2.Lentivirus mediated SF3B1 knockdown efficiency of CD34+ cells derived from human peripheral blood was about 60%and SF3B1 knockdown severly impaired the erythroid progenitors proliferation.The colony forming assay showed that when seeded 400 cells on d6,the Luciferase control groups can give us 37 of BFU-E and 102 of CFU-E,but SF3B1 KD groups do only 8 and 14,respectively.There is significant difference between the two groups.We then performed colony forming assay by using the sorted BFU-E and CFU-E,and we got almost the same results,the numbers of the colonies in SF3B1 KD groups were only 1/10 of those in Luciferase control groups.The aopotosis was dected on d6 and the percentage of apoptosis in SF3B1 KD group was 20%,and that in Luciferase control group was only no more than 5%.3.Knockdown of SF3B1 severly impaired the erythroid terminal differentiation.On d7,GPA,the landmark of erythroid terminal differentiation,was about 40%in the control group but that was only no more than 20%in SF3B1 knockdown groups.In the subsequent culture days,Band3 and a4-intetrin were chose to detect the differentiation.From the results we can see that,the cells in SF3B1 KD group delayed differentiation during the culture d9-d13 compared the Luciferase control cells and the cells catght up during the culture dl5-d17.4.SF3B1 knockdown inhibited the proliferation of erythroblasts and the growth curve showed that in Luciferase control group,the cell munber was expanded from 1 million to about 90 million,but the number in SF3B1 KD group was expanded only from 1 million to 20 million.5.The results of flow cytometry and DNA ladder both showed that knockdown of SF3B1 led to cell apoptosis,and the EdU test showed that SF3B1 knockdown caused cell cycle arrest.6.SF3B1 knockdown resulted in bi/multinuclear cells or nuclear fragmentation,and the morphology observation of the sorted cells showed that the phenotype occurred only in the stage of Poly and Ortho and the percentage of each stage was 30%and 40%,respectively.But in Luciferase control group,the percentage was below 5%.Summary1.Knockdown of SF3B1 severly impaired the proliferation of erythroid progenitor cells and significantly affected the proliferation,differentiation,enucleation and apoptosis of terminal differentiated erythroids.2.The impaired proliferation in SF3B1 KD was caused by the increase of cell apoptosis and cell cycle arrest.3.SF3B1 knockdown induced bi/multinuclear cells or nuclear fragmentation in the stage of Poly and Ortho.Part 2 Construction of Tetracycline Inducible Lentiviral Vector and the Application of SF3B1 Knockdown In Human ErythroblastsMethods1.By using the helper plasmmid pTRIPZ,the SF3B1-shRNA and scramble-shRNA were cloned into the vector of pTRIPZ-EF1?-GFP respectively to form the construct of iGFP-shRNA-EFla-pTRIPZ and iGFP-scramble-EFla-p TRIPZ.2.Package the lentiviral particles by co-tranduction CMV,MDG and iGFP-scramble-EFla-pTRIPZ into 293T cells.Lentiviral transfection was performed in human peripheral blood derived CD34+ cells.After added Doxycycline,the cells were observed on the following time point:6h,12h,24h,36h,48h,72h by fluorescence microscopy and flow cytometry and at the same time the cells were collected at each time point for cytospin and for the detection of cell differentiation.The results were compared with those of Luciferase control and the feasibility of the new inducible vector was assessed.3.Prepare the lentivirus containing inducible SF3B1 shRNA,and perform the transfection.6h,12h,24h,36h,48h,72h after induction using Doxycycline,cell pellets were collected to check the dynamic changes of SF3B1 mRNA by Real Time RT-PCR,and the dynamic changes of protein through Western Blotting.4.After transfection,SF3B1 shRNA was induced on different culture days,and from 48h after induction,cells were collected every 2 days to check the apoptosis by flow cytometry.Results1.GFP expression was observed by fluorescence microscopy and flow cytometry,and the results showed that GFP expressed 6h after Doxycycline induction,,and the fluorescence increased to the peak 24h after induction.2.The transfection of the new inducible construct did not affect the morphology differentiation and enucleation of erythroblasts.3.The mRNA of SF3B1 decreased 6h after induction,and reached to the platform stage 24h after induction.This was consisitant with the expression of GFP.But the decrease of the protein delayed 40h compared with the expression of mRNA.4.The expression of SF3B1 shRNA could be induced at any stag of the erythropoiesis and even though induction on different culture day,simillar knockdown efficiency could be achieved.But for apoptosis,only shRNA induced on d4 and d7,the apoptosis could be detected and when induced on d9 and dll,the phenotype of apoptosis couldn't be detected.Summary1.Ideal transfection efficiency and knockdown efficiency could be achieved by transfecting the tetracycline inducible vector in human primary hematopoietic stem cells.The expression of shRNA and the expression of GFP were driven by the same tetracycline inducible promotor,so the knockdown could be monitored easily by the expression of GFP.2.Simillar knockdown efficiency could be achieved even though the shRNA were inducied on different culture days.But only the shRNA was induced on progenitor cells,the apoptosis could be detected.Part 3 The Relationship Between Impaired Erythropoiesis Inducied by SF3B1 Knockdown and the Activation of p53 PathwayMethods1.Total RNA was extracted from the sorted distinct stage cells and cDNA library was created by Illumina TruSeq kit to prepare for the RNA sequencing.2.The RNA seq results were analyzed by using the software of Cummerbund R package.The expression of each gene was calculated,differentially expressed genes were defined,and the analysis of splicing was performed using spliceR software.3.Screening the results of bioinformatics analysis,we then focused on the gene of MKRN1 and p53 pathway.The expression the genes was checked by Real Time RT-PCR and Western Blotting and specific primers were designed to check the abnormal splcied genes in CFU-E cells after SF3B1 knockdown.4.By using MSCV-IRES-GFP and pcDNA3-Flag-MKRN1 helper plasmids,the fragment containing human full-length MKRN1 was cloned into pRRLSIN.cPPT.PGK-GFP.WPRE to construct MKRN1 overexpression lentivirual vector.5.Observe the phenotypes after overexpression MKRN1 in SF3B1 knockdown eryrhtoblasts.6.PLK1 inhibitors were added during the culture of the erythroblasts to observe the effects of the inhibitor on the nuclei ant to detect the mitosis/cytokinesis pathway in erythroid terminal differentiation.Results1.The results of RNA sequencing showed that there were 255 genes differential expressed at the stage of CFU-E,of which 167 genes were down regulated,88 genes were up-regulated.Analysis by GSEA showed that the top up-regulated pathway in SF3B1 knockdown CFU-E was p53 pathway.The bioinformatics analysis revealed that the mRNA levels of genes related to the p53 pathway,such as p21,Bax and PUMA were significantly elevated in the SF3B1 knockdown CUF-E,and this was confirmed by Real Time RT-PCR.2.The analysis of splicing showed that about 288 genes were differentially spliced in SF3B1 knockdown CFU-E.The most types of outcomes after SF3B1 KD were exon skipping/increased alternertive transcription start site,alternertive transcription termination site.The top 15 differentially spliced genes were detected by using Real Time PCR and the results were consisitant with the results of RNA seq.3.We focused on the top differentially expressed genes MKRN1,and found that mRNA level and protein levels decreased in SF3B1 knockdown group.MKRN1 overexpression in the SF3B1 knockdown cells partially rescued the cell growth.4.Bioinformatics analysis revealed that mitosis/cytokinesis pathway was downregulated in both Poly and Ortho cells,and we focused on one of the genes invovled in this pathway-PLK1.We found that treatment with PLK1 inhibitors in the late stage of erythroid differentiation induced the generation of abnomal nuclear cells and this was consistent with the results of SF3B1 knockdown.Summary1.Apoptosis and cell cycle arrest is due to the activation of p53 pathway caused by alternative splcing of MKRN1,an E3 ligase.2.The generation of abnnomal nuclear cells in SF3B1 KD in the stage of Poly and Ortho was due to the the downregulation of mitosis/cytokinesis pathway.Conclusion1.Knockdown of SF3B1 leads to cell apoptosis,cell cycle arrest,and the generation of bi/multinuclear cells or nuclear fragmentation in the stage of Poly and Ortho.2.Apoptosis and cell cycle arrest is due to the activation of p53 pathway caused by alternative splcing of MKRN1,an E3 ligase.3.The generation of abnnomal nuclear cells in SF3B1 KD in the stage of Poly and Ortho is due to the the downregulation of mitosis/cytokinesis pathway.4.The tetracycline inducible lentiviral vector works well in human primary CD34+ cells,and the apoptosis caused by SF3B1 mainly occours in the stage of CFU-E.