Episod 1 The Regulatory Role Of HA117 On Cell Stemness In HL60/ATRA Multi-drug Resistance Episod 2 Dysregulation Of MiRNA Profiling Participate In Hirschsprung's Disease Formation

Background: Complete remission is induced by all-trans retinoic acid(ATRA)in almost all patients with acute promyelocytic leukemia(APL).However,prolonged ATRA treatment can cause drug resistance.Better understanding of the molecular basis of ATRA-induced drug resistance is therefore warranted to exploit the markers and mechanisms underlying this drug-resistant phenotype.Previously,we used ATRA to select drug-resistant HL60 cells,which led to the generation of the multi-drug-resistant cell line,HL-60/ATRA.Suppression subtractive hybridization of differentially expressed sequences HL-60/ATRA cells enabled us identify a highly expressed sequence,which we refer to as HA117(GenBank accession number: CB214920).Bioinformatics analysis of human genomic sequences identified the human gene fragment encoding HA117.The gene is located on the reverse strand of chromosome 14q24.2 in an intergenic region among RGS6,FOXA1,NFKBIA,BMP4,BCL11 B,AKT1 etc..Methods:In order to further study the mechanism of HA117 on the formation of drug resistance in HL60/ATRA cells:(1)we use the bioinformatics means to analyze the sequence of HA117 to identify whether HA117 is a lncRNA;(2)Lnc Tar software was used to predict the interaction zone between HA117 and RGS6;(3)the mRNA profiling array results of bone marrow cells from HA117-overexpressed recurrence / drug resistance group patients and HA117-low-expressed complete remission group patients was analyzed by DAVID database to reveal the possible role of HA117 in drug resistance;(4)In our cell experiments,HL60 cell line,HL60/ATRA cell line,HA117 shRNA transfected HL60/ATRA cell line and HA117 over expression lentivirus transfected HL60 cells were used as cell model.We meared the relative expression level of each domain of RGS6 at mRNA level;the RGS6 protein expression level in total protein,nucleoprotein and plasmosin;the subcellular localization of RGS6 was analyzed by immunofluorescence;the expression level of DNMT1,cancer stem cell markers/ stem cell markers were measured by q PCR and Western Blot;cell methylation level were analyzed by measure the expression level of 5-methylcytosine using immunofluorescence;(5)we repeated the establishment of multidrug resistance HL60 / ATRA cell lines and we measured the proliferation,migration ability,chemosensitivity and tumor stem cell / stem cell marker expression every 5 generation of cell lines.Results:(1)The ORF finder and coding ability score shown that HA117 is a lncRNA;(2)Lnc Tar results shown that HA117 can interact with RGS6 and there two interaction sites,located at 1-301 bp and 769-1108 bp of RGS6 mRNA sequence,respectively;(3)the mRNA profiling array results have shown that HA117 could promote the cell proliferation,survival,cell differentiation resistance and apoptosis resistance;(4)cell experiments results shown that the expression of HA117 can down-regulate the expression of RGS6 GGL-domain,and inhibiting of DNMT1 degradation result in the cell methylation level upregulated and the expression of tumor stem cell / stem cell markers were also upregulated when HA117 expression level goes up.(5)the results showed that the proliferation and migration ability of the cells,the expression of tumor stem / stem cell markers were positively correlated with the expression of HA117,and the sensitivity of chemotherapeutic drugs was negatively correlated with the expression of HA117.Conclusion: For the first time,we revealed that HA117 could regulate RGS6 alternative splicing by down-regulating of GGL-domain,moreover,the down-reglation of GGL-domain caused the inhibiting of DNMT1 degradation,we deduce a conclusion that overexpression of HA117 enhanced the stemness of HL60 cells.Therefore,we hypothesized that the expression of HA117 regulated and maintained the stemness of cells.Background: Hirschsprung's disease(HSCR),the most common congenital malformation of the gut,isregulated by multiple signal transduction pathways.Several components of these pathwaysare important targets for micro RNAs(miRNAs).Multiple miRNAs have been associatedwith the pathophysiology of HSCR,and serum miRNAs profiles of HSCR patients havebeen reported,but miRNA expression in HSCR colon tissue is almost completely unex-plored.Methods:(1)using Exiqon miRCURY LNA? microarray to screen colon tissue to detect miRNAs whose expression profiles were altered in HSCR,following filtering of low-intensity signals,data normalization,and volcano plot fil-tering,we identified 168 differentially expressed miRNAs(104 up-regulated and 64 down-regulated);(2)miRWalk database was used to identify experimentally validated m RNA targets of differentially expressed miRNAs and the results were co-analyzed with m RNAs participating in HSCR formation.Fifty of these m RNAs represent major targets of dysegulated miRNAs and may thus important roles in the pathophysiology of HSCR;(3)Pathway analysis revealed that seven of the miRNA targets encode proteins involved in regulation of cell proliferation and migrationvia RET and related signaling pathways(MAPK and PI3K/AKT);(4)q PCR analysis of miRNAs targeting RET and related pathway shown that the expression of hsa-miR-142-3p,hsa-miR-146b-5p,hsa-miR-429,hsa-miR-938-3p,hsa-miR-369-3p consistence with its expression in miRNA array,moreover,the expression of its target m RNAs negatively correlated with the expression of these miRNAs.Conclusion: Our results identify dysregulation of hsa-miR-142-3p,hsa-miR-146b-5p,hsa-miR-429,hsa-miR-938-3p,hsa-miR-369-3p play key roles in the pathophysiology of the complex multi-factorial disease HSCR through its regulation of RET and its relative pathways.