The Role Of Long Non-coding RNA H19 And Their Identification Of Targets In Chronic Myelogenous Leukemia(CML) Cells

Bckground:As with the rockting development of high through-put deep sequencing technology,an increasing number of long non-coding RNAs(lncRNAs)have been discovered in eukarya in recent years.LncRNA is a group of non-coding RNA which is longer than 200 nt,rich in biological function and highly structured.LncRNA could directly interact with protein,microRNA(miRNA)molecules to regulate target gene expression an epigenetic,transcriptional and post-transcriptional levels.Recent years,lncRNA not only can play it's part in different physiological processes of cells,but also it's abnormal expression are closely connected with many diseases including cancers.Numeroius reseachers have reported that lncRNA H19 plays a important role in tumorrigenesisi and it's functional research in CML.But due to lncRNA H19 can only play it's role in mRNA levels and can not encode proteins.So it's increasingly important to explicit it's modulation effect.In our study,we heve done the identification of the targeted proteins and microRNAs of lncRNA H19 and functional reseach of it's target.Objective:Our goal is to investigate the roles of long non-coding RNA H19 and targeted proteins and microRNAs in CML.This study is expected to provide novel insights into the role of long non-coding RNA H19 and the targeted therapeutic in CML.Methods: 1.H19 SiRNA is transfected into K562 cells by Lipofectamine 2000 and Real-time PCR detected the relative expression level of H19 mRNA.2.A stable subline of K562 cells overexpressing H19 is established by H19 lentiviral vector which is transfected into K562 cells by Lipofectamine 3000 and Real-time PCR detected the relative expression level of H19 mRNA post-transfection with H19 lentiviral vector and puromycin screeing.3.The colony-forming activity of H19 in K562 cells was tested by semi-solid culture medium of Methylcellulose.4.P210 cells overexpressing H19 were injected into Subcutaneous.Than the tumor size and survivals were observed.5.NOD/SCID mice were transplanted with the K562 cells overexpressing H19 and K562 cells of negative control.Tumor burden of mice after cell injection was showed by the In-vivo Imaging Systems.6.The H19 lentiviral vector was transcripted into long non-coding RNA H19 in vitro and the agarose gel electrophoresis vertified the lncRNA H19.7.The 3?terminus strand of the long non-coding RNA H19 was attached to a single biotinylated nucleotide using T4 RNA ligase.8.The lncRNA H19 is first bound to the beads to orient the lncRNA H19 for protein binding.lncRNA H19-bound beads are then equilibrated in Protein-RNA Binding Buffer before protein lysate is added.Beads are washed by adding the appropriate buffer,vortexing and separating on a maganetic stand.lncRNA H19-protein complexes were obtained.In the same manner,the RNA-bound beads was added to the total RNA from K562,the lncRNA-RNA complexes were got.9.The lncRNA-protein binding complexes were detected by mass spectrometry followed by electrophoresis and coomassie blue staining and destaining.10.The lncRNA-RNA binding complexes was detected by RNA-seq.11.Mass spectra identified those proteins from RNA pull-down products.The catRAPID and starBase v 2.0 predicted the targeted proteins of long non-coding RNA H19.Specific protein from RNA pull-down products both existed in MS and prediction were tested by western blot.12.The starBase v 2.0 predicted the targeted microRNAs of long non-coding RNA H19.Real-time PCR test miRNAs both existed in RNA-seq and star Base v 2.0 prediction.13.PCBP1-SiRNA and FUS-SiRNA were transfected into K562 cells by Lipofectamine 2000 and Real-time PCR detected the relative expression level of H19 mRNA;The viability of K562 cells of each group was detected by MTT assay.14.The sensitivities of the targeted proteins of lncRNA H19 in K562 cells on TK1 s were detected by CCK8 assay.15.The cell viability of the targeted miRNAs of lncRNA H19 in K562 cells were determined by MTT.16.The colony-forming activity of the targeted proteins and miRNAs of lncRNA H19 in K562 cells were tested by semi-solid culture medium of Methylcellulose.Results: 1.The relative expression of H19 mRNA in K562 cell of transfection with H19 lentiviral vector was higher by Real-time PCR.The over-expression of H19 significant decreased the colony-foming ability in K562 cells.2.We found that the overexpression of H19 inhibit the growth of tumor and improve the survival of CML model mouse.3.The results of In-vivo Imaging Systems showed that the overexpression of H19 improve the survival of CML model mouse.4.The long non-coding RNA H19 was transcriped in vitro and was vetified by agarose gel electrophoresis.5.lncRNA H19-protein complexes and lncRNA H19-RNA complexes was abotained by RNA pull-down and lncRNA H19-protein complexes were detedted by MS and lncRNA H19-RNA complexes were detected by RNA-seq.MS(586)combined with the catRAPID and starBase v 2.0 prediction(9)identified that the lncRNA H19 interact with PCBP1 and FUS protein.RNA-seq(1134)combined with starBase v 2.0 prediction(40)vertified that the lncRNA H19 interact with miR-19a-3p ?miR-106b-5p?miR-148a-3p?miR-148b-3p?miR-454-3p.6.The targeted protein PCBP1 and FUS of lncRNA H19 were vertified by Western Blot.The targeted miRNA-19a-3p and miR-106b-5p of lncRNA H19 were vertified by Real-time PCR.7.The low-expression of PCBP1 and FUS significantly promoted the sensitivities of TKIs and decreased the abilities of colony-formng ability in K562 cells.8.The low-expression of miR-19a-3p and miR-106b-5p decreased the viability colony-formng ability in K562 cells.Conclusion: 1.The overexpression of H19 reduced malignant progression of CML cells and improveed the survival of CML model mouse.2.LncRNA H19 could target PCBP1 and FUS protein in CML;LncRNA H19 could target miR-19a-3p and miR-106b-5p in CML.3.The targeted inhibition of PCBP1 and FUS could reduced malignant progression of CML cells;The targeted inhibition of miR-19a-3p and miR-106b-5p co?Ld reduced malignant progression of CML cells;The low-expression of H19 significantly increased the sensitivities of TKIs;Transcription of t-antimiR-19a-3p and t-antimi R-19a-3p decreased the growth of K562 cells.