The Effection And Mechanism Of Shlnc-EC6/miR-451 Regulating Pathway On Enucleation In Erythrocyte Mature And Erythroid Cells Treated With Caulis Spatholobi

It is a key step that erythroblasts enucleate cell karyon nucleolus and become reticulocytes, then chang into mature red blood cells. Because red blood cells have obstacle of enucleation In vitro culture and various types of anemia and dysplasia, it is the current scientific urgent to research enucleation of red blood cells to discovery new blood source and cure of related anemia caused by red cell diseases and treat red blood disease. It has important physiological and pathological significance to find some diagnostic and treatment approaches on dysplasia of erythrocyte.Ras related C3 botox substrate 1 (Rac1) is a type of Rac and belongs to the GTPases family. Racl have many functions include promoting cell proliferation, participating in the formation and rearrangement of the cytoskeleton, enhancing the survival rate of cells, and playing a key role on cell adhesion and metastasis. Research shows that down-expression of Rac GTPases blocked enucleation of red blood cell in the late stage of blood cells but did not affect the proliferation and differentiation of red blood cells. It is unclear that the mechanism and expression of Rac1 in red blood cells of mouse.Shlnc-EC6 is a kind of Long noncoding RNA (Lnc-RNA), the RNA transcription is more than 200 nt molecules in length without the ability to encode proteins. Lnc-RNAs regulate epigenetic of in encoding gene expression, become a part of the transcriptome, can directly regulate gene transcription and protein degradation and genomic imprinting, etc. Previous research showed that more than 400 Lnc-RNAs have important biological functions in the development of red cell, including shlnc-EC6 associated with enucleation of red blood cells. It is necessary to detect whether shlnc-EC6 affects enucleation of red blood cells by targeting Rac1 or not.MicroRNAs (miRNAs) are endogenous, small noncoding single-stranded RNAs that regulate body’s almost all important physiological process, including growth, development, differentiation, metabolism and death. miRNAs can act as either oncogenes or suppressor genes by interacting with mRNAs 3’ untranslated region(3’UTR) and inducing either translation suppression or degradation of mRNA at the post-transcriptional level, and might represent promising biomarkers for human cancer. Emerging evidence suggests that miR-451 might be involved in red cells mature, and bioinformatics analysis predicts that Rac1 3’UTR and miR-451 have conservative binding sites in mouse, then whether miR-451 can target Rac1 affecting enucleation of red blood cells deserves further study.Recent studies have pointed out that long noncoding RNA has the function of competition or cooperation with endogenous RNA can combine with the miRNA recognition element of mRNA 3’UTR region, to regulate expression of mRNA and protein of miRNA-target genes by blocking the degradation or restraint of the target mRNA. Existing researches show long noncoding RNA and miRNA had interacted with each other. lncRNA-HULC abnormal high expression and has the typical structure of mRNA samples in liver cancer tissue, but do not encode proteins, so the researchers think the HULC RNA may play a role of molecular bait and natural miRNA sponges, HULC RNA can absorb miR-372, inhibit the expression of miR-372, miR-372 target PRKACB, phosphorylation of PRKACB active CREB, then enter the nucleus and combining to the HULC promoter regions to further activate the HULC expression. In enucleation of red blood cells, whether long noncoding shlnc-EC6 and miR-451 is cooperate with each other and regulate the expression of target gene Rac1 or not, it is unclear.Caulis spatholobi was used one thousand years of traditional Chinese medicine (TCM) for activating blood circulation and producing new red blood cells. Caulis spatholobi in modern medicine cure diseases of blood cells such as blood cells were decreased in number caused by radiation and chemotherapy and blood system diseases. Caulis Spatholobi Total Flavonoids (CSTF) are an effective active ingredient extracted from the stem of Caulis Spatholobi, and its fuction include promote the production of blood, anti-inflammatory and anti-tumor. Research reported CSTF can contribute to the proliferation of red blood cells in mice and treatment of anemia caused by a variety of causes, but whether Caulis spatholobi promote mature of red blood cells in mice, especially nuclear or not, it is unclear.In the present study, we choose to detect red blood cells to study the expression and significance of Rac1 in mice, and to study whether shlnc-EC6 and miR-451 can infect enucleation of red blood cells in mice by targeting Racl, and to study whether CSTF can change expression of shlnc-EC6 and Racl to promote enucleation of red blood cells by inhibitting Racl. The research will be described in four parts as follows:Part Ⅰ expression and mechanism of shlnc-EC6 in process of erythroid enucleationObjective:To Study the expression of shlnc-EC6 between erythroblast and reticulocyte, and to explore whether its enucleation mechanism of red blood cells is related to shlnc-EC6 by targeting Rac1.Methods:Erythroblast(before enucleation) and reticulocyte(after enucleation) were sorted By flow cytometry (FCM) in mice bone marrow, then shlnc-EC6 RNA expression is detected between erythroblast and reticulocyte using real-time fluorescent quantitative PCR (q-RTPCR); Through short hairpin RNA (shRNA) technology shlnc-EC6-shRNA plasmids were made artificially retroviruses of shlnc-EC6-shRNA, the retroviruses infected mice fetal liver erythroid precursors and hematopoietic stem cells (FLEPHSCs), erythroid enucleation was observed using flow cytometry and smear instrument; Constructed Racl-PGL3BS plasmid following predicting that Racl is the potential target of shlnc-EC6 by bioinformatics, and then detected that whether Racl can be derectly regulated by shlnc-EC6 through Dual Luciferase Reporter Kit in 293T cells. Detected expression of mRNA and protein of Rac1 and PIP5K in Mel cells and G1E cells infected with shlnc-EC6 by qRT-PCR and Western blot analysis. Erythroid enucleation were observed after low-expression of Racl to normal levels using FLEPHSCs infected with retroviruses of shlnc-EC6-shRNA, then Racl and PIP5K mRNA and protein expression were detected through the q-RTPCR and Western blotting; Further explored the relationship between signal pathway of shlnc-EC6-Racl-PIP5K and erythroid enucleation.Results:1. The expression of shlnc-EC6 increased obviously in reticulocyte compared to erythrocyte (p<0.05)2. The number of erythroid enucleation decreased in FLEPHSCs of low-expression compared to erythrocyte (p< 0.05)3. Racl 3’UTR have 2 binding sites with shlnc-EC6 in mouse cells by bioinformatics, and then verified that shlnc-EC6 can be negatively regulated through biding with Racl 3’UTR by Dual Luciferase Reporter Gene methods in 293T cells.4. The mRNA and protein expression of Racl and PIP5K is obviously increased in red cells infected with virus of shlnc-EC6-shRNA. (p< 0.05)5. Expression of Rac1 reduced in red blood cells infected retroviruses of Rac1-shRNA artificially from Racl-shRNA plasmids constructed by researchers. (p<0.05)6. The mRNA and protein level of the Rac1 and PIP5K decreased and the number of erythroid enucleation increased in FLEPHSCs of low-expression shlnc-EC6 infected retroviruses of Rac1-shRNA. (p<0.05)Conclusions:Low-expression of shlnc-EC6 inhibited erythroid enucleation through targeting Rac1, to clarify that Racl and shlnc-EC6 were involved in the mature process of red blood cells, which may be new molecular targets for interventing the prognosis and treatment of red blood cells diseases.Part Ⅱ expression and mechanism of miR-451 in process of erythroid enucleationObjective:To study the expression of miR-451 between erythroblast and reticulocyte, and to explore whether its enucleation mechanism of red blood cells is related to miR-451 by targeting Rac1.Methods:Erythroblast(before enucleation) and reticulocyte(after enucleation) were sorted By flow cytometry (FCM) in mice bone marrow, then miR-451 expression is detected between erythroblast and reticulocyte using real-time fluorescent quantitative PCR (q-RTPCR); Constructed miR-451-MSCV-PIG plasmid; Breeding and identification of miR-451 knockout mice (miR-451 KO mice) and C57BL/6 mice. Observed enucleating in FLEPHSCs of miR-451 knockout and over-expression of miR-451 using magnetic bead separation through FCM and smear method in vitro differentiation culture. Constructed Racl-PGL3 plasmid following predicting that Racl is the potential target of miR-451 by bioinformatics, and then detected that whether Rac1 can be derectly regulated by miR-451 through Dual Luciferase Reporter Kit in 293T cells. Detected expression of mRNA and protein of Racl and PIP5K in Mel cells and G1E cells infected with miR-451 virus by qRT-PCR and Western blotting.Results:1. identified the three types of miR-451 mice:wild type (WT) and homozygous type (KO) and heterozygous type (Het).2. The expression of miR-451 increased obviously in reticulocyte compared to erythrocyte (p<0.05)3. The number of erythroid enucleation decreased in FLEPHSCs of miR-451 KO compared to miR-451 WT. (p<0.05)4. Racl 3’UTR have binding seed sequence of miR-451 in mouse cells by bioinformatics, and then verified that miR-451 can be negatively regulated through biding with Rac1 3’UTR by Dual Luciferase Reporter Gene methods in 293T cells.5. The mRNA and protein expression of Racl and PIP5K is obviously increased in erythroblast of miR-451 KO compared to miR-451 WT. (p<0.05)6. The expression of Racl decreased obviously in red blood cells of over-expression-miR-451 compared to control. (p<0.05)7. The mRNA and protein level of the Racl and PIP5K decreased and the number of erythroid enucleation decreased in FLEPHSCs of over-expression miR-451 in C57BL/6 mice, (p <0.05)Conclusions:High-expression or low-expression of miR-451 inhibited erythroid enucleation through targeting Rac1, Rearrangement disorder of actin caused by high expression Rac1 can’t form a resultant force to extrude off cell nucleus result in anomalies of erythroid enucleation, and insufficient power of enucleation due to lower-expression-Rac1.To clarify that Rac1 and miR-451 were involved in the mature process of red blood cells, which may be new molecular targets for interventing the prognosis and treatment of erythroid cells diseases.Part Ⅲ The interaction relationship between shlnc-EC6 and miR-451 in process of erythroid enucleationObjective:To study the interacted relationship between shlnc-EC6 and miR-451 in the process of mouse erythroid enucleation, to explore whether shlnc-EC6 and miR-451 work together to suppress the expression of target gene Racl.Methods:Detected s the expression correlation of shlnc-EC6 and miR -451 by the qRT-PCR; Explored miR-451 and shlnc-EC6 exist in Ago2 complex using RNA-protein co-immunoprecipitation technology; The two RNA of shlnc-EC6 and miR-451 have a direct or indirect relationship by dual-color fluorescence in situ hybridization in MEL cells of over-express miR-451.Results:1. Expression of shlnc-EC6 is decreased after MiR-451 knockout (p< 0.05), and expression of shlnc-EC6 is increased after MiR-451 of excessive expression (p< 0.05).2. The expression of miR-451 decreased obviously after knockdown of shlnc-EC6 in red blood cells. (p< 0.05)3. miR-451 and shlnc-EC6 exists commonly in Ago2 immune complexes, association of miR-451 and shlnc-EC6 may be direct or indirect.4. Overlap of miR-451 and shlnc-EC6 in the same cell area by double-color FISH experiments suggests that direct association play same effective role.5. miR-451 cooperate with shlnc-EC6 reciprocally to regulate erythroid enucleation in post-transcription level though Racl-PIP5K signaling pathways.Conclusions:The expression of miR-451 and shlnc-EC6 expression are related positively, and vice versa, these results show that there is a certain positive feedback regulating pathways between them; In Ago2 immune complexes, miR-451 and shlnc-EC6 have a direct link in post-transcription level to regulate Racl and PIP5K and transform red blood cells mature, especially erythroid enucleation, these data provides new theoretical basis as soon as possible about mechanism ofenucleationt of red blood cells.Part Ⅳ The mechanism of CSTF promote erythroid enucleation through Racl pathwayObjective:To investigate the effect of CSTF on erythroid enucleation and to explore the possible mechanism.Methods:FLEPHSCs of miR-451 knockout from 14.5 day fetal liver of mice using magnetic bead separation; The FLEPHSCs were cultivated and divided into control group and CSTF group (concentration respectively 0.5ug/ml,1.0 ug/ml.1.5ug/ml,2.0 ug/ml,2.5 ug/ml). The effect of CSTF on cell apoptosis cultivated after 24 h were studied using apoptosis kit; The effect of CSTF on erythroid enucleation of miR-451KO mice cultivated after 24 h were based on cell W-G dyeing by FCM and smear instrument; When FLEPHSCs were treated by CSTF after 24 h, the mRNA and protein expression of Rac1 and shlnc-EC6 RNA were assessed by western blot and quantitative real-time PCR analysis.Results:CSTF promoted erythroid enucleation of FLEPHSCs of miR-451KO in a dose-dependent manner. The number of erythroblast decreased and enucleated red cells increased in group of low-toxic doses CSTF compared with control group. (P< 0.05); Cell apoptosis rate increased with the increase of concentration of CSTF, of which 2.0 ug/ml,2.0 ug/ml CSTF can obviously promote cell death. (P<0.01) The mRNA and protein level of Rac1 are down-regulated and shlnc-EC6 RNA level is up-regulated in FLEPHSCs of miR-451 KO treated by CSTF after 24 h in a time-dependent manner.Conclusions:CSTF promoted erythroid enucleation in a dose-dependent manner through targeting Rac1, its mechanism may be provides a new theoretical basis for traditional Chinese medicine treatment of various types of anemia.In summary, down-regulation of shlnc-EC6 suppressed erythroid enucleation, and the results suggest that erythroid enucleation need shlnc-EC6.Racl is one direct target genes of shlnc-EC6 and miR-451, shlnc-EC6 and miR-451 regulate erythroid enucleation by targeting Rac1; Shlnc-EC6 cooperate with miR-451 reciprocally to regulate erythroid enucleation; CSTF promoted FLEPHSCs enucleation of miR-451 KO though shlnc-EC6-Rac1 signaling pathways.In this study, we investigated the effection of shlnc-EC6/miR-451/Rac1 signaling pathways to regulation of erythroid enucleation and afforded new molecular target for diagnosis and treatment of red blood cells diseases, trying to treat these diseases by combining traditional Chinese and western medicine.