The Role Of LncRNA CASC2/MiR-124-3p/MKL-1/NFATC1 Pathway On Megakaryocytic Maturation And Differentiation

Purpose:The maturation and differentiation of megakaryocytes are closely related to the occurrence of platelet-related diseases,and transcription factors play a very important role in the maturation and differentiation of megakaryocytes.Studies have shown that the expression of transcription factor MKL-1 is upregulated during the differentiation of megakaryocytes in mice.NFATC1 is another important transcription factor involved in the regulation of megakaryocyte maturation and differentiation.The specific molecular mechanism of how the transcription factors MKL-1 and NFATC1 affect megakaryocyte development is currently unclear.This study aims to find a breakthrough point for the basic theory of diseases related to the maturation and differentiation of megakaryocytes to be solved urgently.The interaction between MKL-1 and NFATC1 regulated by LncRNA CASC2 and hsa-miR-124-3p at the cellular and molecular levels will be discussed.Its signaling pathway in turn affects the role and mechanism of megakaryocyte maturation and differentiation.Method:In this thesis,RNAi technology was used to construct megakaryocyte cell lines with stable knockdown of endogenous genes;qPCR technology was used in combination with western blot experiments to detect the mRNA and protein levels of the target gene;Detection of the effect of target gene on the transcriptional activity of luciferase reporter gene plasmid by luciferase activity assay;Analysis of the combination of MKL-1 and the megakaryocyte maturation and differentiation marker gene promoter using chromatin immunoprecipitation technology;Detection of the distribution of MKL-1 and NFATC1 in megakaryocytes by immunofluorescence colocalization technology;Truncation mutation technology and protein immunoprecipitation technology and detection of the interaction area of MKL-1 and NFATC1 protein;Use RNA IP experiment to detect the binding of target protein to hsa-miR-124-3p;Use Giemsa staining to analyze megakaryocytes Morphology and number;Flow cytometry analysis of megakaryocyte DNA ploidy and detection of apoptosis rate;Using CCK-8 technology to detect the effect of target gene expression on megakaryocyte proliferation.Through the above experimental techniques,the role and mechanism of LncRNA CASC2,hsa-miR-124-3p,MKL-1 and NFATC1 in megakaryocyte maturation and differentiation are demonstrated from the cellular and molecular levels.Result:The following results were obtained through the above studies: 1: Interfering with endogenous MKL-1 and blocking the RhoA-MKL-1 signaling pathway can significantly reduce the transcription and expression of megakaryocyte maturation and differentiation marker genes,thereby affecting the megakaryocyte maturation and differentiation.MKL-1 promotes its transcription and expression by binding to the CAr G box of the megakaryocyte maturation and differentiation marker gene CD41 / CD61 / C-KIT promoter.NFATC1 can promote the proliferation of megakaryocytes.2: MKL-1 and NFATC1 are co-localized in the megakaryocyte nucleus and there is a direct protein-protein interaction;NFATC1 inhibits MKL-1 nucleation and promotes MKL-1 nucleus transfer.MKL-1 does not affect the transcription level of NFATC1,but inhibits the protein level of NFATC1;The interaction between MKL-1 and NFATC1 weakens the ability of MKL-1-SRF to bind to the CAr G box of the CD41 / CD61 / C-KIT promoter;Phosphorylation of MAPK1 and NFATC1 in turn affects the maturation and differentiation of megakaryocytes.3: Hsa-miR-124-3p inhibits the proliferation of megakaryocytes.Hsa-miR-124-3p binds to the 3’UTR of NFATC1 and MAPK1 promoters and inhibits the expression of NFATC1 and MAPK1.MKL-1 promotes the transcriptional activation of the hsa-mi R-124-3p promoter by binding to the CAr G box of the hsa-miR-124-3p promoter.4: The expression of LncRNA CASC2 in megakaryocyte cell line is negatively correlated with hsa-miR-124.LncRNA CASC2 promotes the transcription and expression of megakaryocyte maturation and differentiation marker gene CD41 / CD61 / C-KIT.Lnc RNA CASC2 combines with hsa-miR-124-3p to play a sponge role in inhibiting hsamiR-124-3p.Hsa-miR-124-3p inhibits the expression of LncRNA CASC2.MKL-1 promotes the transcriptional activation of LncRNA CASC2 by binding to the CAr G box of the LncRNA CASC2 promoter.Conclusion:This paper proves that the feedback loop of LncRNA CASC2 / miR-124-3p / MKL-1 / NFATC1 regulates the maturation and differentiation of megakaryocytes,and provides theoretical support for diseases related to abnormal megakaryocyte maturation and differentiation.