Identification, Function Studies And Clinical Significance Of A Novel Anti-differentiation Gene HA117

PART ONE IDENTIFICATION AND BIOINFORMATICSANALYSIS OF HA117GENESECTION ONE IDENTIFICATION OF HA117GENEObjective: To establish the multi-drug resistant leukemia cell lineswith all-trans retinoic acid (ATRA), identify the existence of HA117, andobtain the full-length of HA117gene.Methods:(1)Small doses, increasing concentrations of ATRA wasadministrated repeatedly and progressively to induce wild leukemia celllines HL-60, U937and Jurkat, for the sake of establishing the resistant celllines HL-60/ATRA, U937/ATRA and Jurkat/ATRA;(2)MTT assay wasused to analyze the cell proliferation of HL-60and HL-60/ATRA;(3) Flowcytometry was used to detect the changes in cell cycles of HL-60andHL-60/ATRA;(4) Quick Wright-Giemsa staining was used to detect themorphological changes of HL-60and HL-60/ATRA cells;(5) MTT assaywas used to analyze the drug sensitivity of HL-60and HL-60/ATRA cellsto VCR, Ara-C and DNR;(6)Real-Time PCR was employed to detect themRNA expression of human telomerase subunit hTERT;(7)Western blotwas used to detect the proteins expression of HL-60and HL-60/ATRAcells;(8) Real-Time PCR and regular RT-PCR were utilized to detect theexpression of HA117RNA in HL-60and HL-60/ATRA cells;(9) Subcutaneous transplantion was employed to evalue the tumorigenicity ofHL-60/ATRA cells in a subcutaneous nude mice xenograft model;(10)Verify the expression of known HA117sequence by regular RT-PCR;(11)3’ RACE amplification was employed to amplify the3’ end sequence ofHA117.Results:(1)Resistant cell lines HL-60/ATRA were Established;(2)Compared with wild cell line HL-60, proliferation of HL-60/ATRA had notbeen inhibited by ATRA;(3) Cell cycle of HL-60/ATRA had not beensignificantly affected;(4)The morphology of HL-60/ATRA was notsignificantly different from HL-60cells;(5)HL-60/ATRA cell yieldeddifferent levels of resistance to Ara-C, DNR and VCR;(6)The expressionof hTERT mRNA in HL-60/ATRA cell was significantly higher than thatin HL-60(P<0.05);(7) Compared with wild cell line HL-60, theexpressions of CD11b, MDR1, Ube1L protein were lower in HL-60/ATRA,while the expressions of CD133, c-myc, Bcl-2protein in HL-60/ATRA cellwere higher;(8)After treatment with ATRA in a long-term and increasingconcentrations pattern, HA117expression in both acute myeloid leukemiacell lines and acute lymphocytic leukemia cell line displayed higher;(9)Tumorigenicity of resistant cell line HL-60/ATRA had not been abrogatedby a long-term treatment with ATRA;(10)HA117was detectable as atranscript;(11)3’ end sequence of HA117was not obtained with3’ RACEamplification.Conclusions: HA117is an ATRA-inducible transcript and highlyexpressed in leukemic resistant cell line. It is associated with multi-drugresistance of leukemic resistant cell lines, and probably involved in theanti-differentiation and subsequent durg resistance; HA117is a detectablesequence, the detectable length is254bp. Due to the low abundance inleukemic cell lines, and the existence of secondary structure, the full length of HA117is difficult to obtain and difficult to be sequenced. SECTION TWO BIOINFORMATICS ANALYSIS OF HA117Objective: To obtain basic information of HA117gene throughbioinformatic analysis of known HA117sequence.Methods:(1) ORF Finder was utilized to analyze the open readingframe of nucleic acid sequence;(2) Nucleotide sequence alignment andhomology analysis were performed with Blastn online tool of NCBI;(3)Analyze the location of HA117sequence with EMBL and Map Viewer;(4)GENSCAN, Augustus and CPC were used to analyze the protein-codingcapacity of the genome sequence in which HA117located;(5) RNAstructure analysis was performed with UCSC and Vienna RNA web server.Results:(1)HA117did not have a complete open reading frame. It wasjust part of a transcript;(2) The similarity between HA117and thesequence on chromosome14was100%. The sequence of HA117waslocated on the reverse strand;(3) HA117was located in the14q24.2regionon the long arm of chromosome14, and situated between RGS6and DPF3;(4) Theoretically, HA117did not have protein-coding capacity;(5) Nucleicacid sequence of HA117had seconary structure.Conclusions: HA117is located in the14q24.2region on the long armof chromosome14. It is a transcript of a long non-coding RNA, withcomplicated seconary structure, and situates between RGS6and DPF3. PART TWO DRUG RESISTANCE AND MECHANISMRESEARCH OF HA117SECTION ONE DRUG RESISTANCE RESEARCH OF HA117IN LEUKEMIA CELL LINESObjective: To investigate the drug resistance function of HA117genein leukemic cell line and the affection toward its flanking protein-codinggenes. Methods:(1) ATRA0.5μM was used to treat HL-60cell for72h,and then Real-Time PCR was used to detect the mRNA expression ofDPF3, RGS6and HA117;(2) Real-Time PCR was utilized to detect themRNA expression of DPF3, RGS6in different passages of HL-60/ATRAcells;(3) The lentiviral vectors of HA117-RNAi and empty lentivirus wereused to infect the drug resistant cell line HL-60/ATRA;(4) Real-Time PCRwas used to detect the expression of DPF3mRNA after interference ofHA117;(5) Western Blot was employed to analyze the protein expressionof DPF3in the drug resistance cell line HL-60/ATRA, and the proteinexpression of DPF3after interference of HA117;(6)MTT assay wasemployed to analyze the sensitivities of leukemic cell lines to Ara-C, ADM,DNR, VCR and CTX.Results:(1) After treatment with0.5μM ATRA for72h, DPF3b mRNAin HL-60cell was significantly reduced (P<0.05), while the expression ofHA117RNA increased significantly (P<0.05);(2) In the10th,20th,30th,40th,50th passages of HL-60/ATRA cells, DPF3mRNA levels were allsignificantly lower than that in untreated HL-60cells (P<0.05);(3)Lentiviral vectors of HA117-RNAi and empty LV-vector infected theHL-60/ATRA cells successfully;(4) The mRNA expression of DPF3b inHL-60/ATRA cell infected by HA117-RNAi was significantly higher than that in HL-60/ATRA and in HL-60/ATRA cells infected by LV-vector (P<0.05);(5) The protein expression of DPF3in wild cell line HL-60, as wellas in the HL-60/ATRA infected with HA117-RNAi, was not significantlydifferent from that in HL-60/ATRA cells;(6) After the interference ofHA117, the resistance of HL-60/ATRA to Ara-C, ADM, DNR, VCR andCTX was reduced (P<0.05).Conclusions: HA117is a LncRNA involved in the multi-drugresistance of leukemia. Regulation of the mRNA expression of itsneighbouring gene DPF3b, possibly, is one of the mechanisms of themulti-drug resistance of HA117. The mechanisms of the multi-drugresistance of HA117is partially different from the mechanisms of classicmulti-drug resistance genes. SECTION TWO RESEARCH ON THE EXPRESSION OF HA117IN CLINICAL LEUKEMIA PATIENTSObjective: To investigate the clinical significance of HA117expression in leukemia patients.Methods: Bone marrow isolated from100cases of childhoodleukemia patients and41cases of adult leukemia patients were divided into3groups: newly diagnosed group, remission group, and refractory/relapsed group. Bone marrow isolated from12cases of childhood ITPpatients and5cases of adult IDA patients were set as control. And acuteleukemias was divided into standard risk, middle risk, and high risk.Real-Time PCR was utilized to detect the expression of HA117RNA andBcl-2, c-myc, RARα, PU.1, DPF3b, bcrp mRNA in bone marrow mononuclear cells.Results:(1) In childhood leukemia cases, the positive expression ratesof HA117in newly diagnosed group, remission group, refractory/relapsedgroup and control group were76.00%,71.64%,100.00%,33.33%,respectively. In adult leukemia cases, the positive expression rates ofHA117in newly diagnosed group, remission group, refractory/relapsedgroup and control group were68.42%,56.25%,85.71%,80.00%,respectively;(2) The expression of HA117was not associated with thegender, age and leukemia classification of patients;(3) In newly diagnosedchildhood AML patients, HA117expression in high risk group wassignificantly higher than that in standard risk (P<0.05) and middle risk(P<0.05) group. In newly diagnosed childhood ALL patients, HA117expression in high risk group and middle risk group was significantlyhigher than that in standard risk (P<0.05), respectively;(4) In childhoodALL patients of remission group, HA117expression in high risk group andmiddle risk group was significantly higher than that in standard risk group(P<0.05), respectively. In childhood AML patients of remission group,HA117expressions in high risk group and middle risk group were bothhigher than that in standard risk group;(5) In refractory/relapsed childpatients, HA117expression in high risk group was significantly higher thanthat in middle risk(P<0.05) and standard risk group(P<0.05);(6)Inchildhood ALL patients, HA117expression in refractory/relapsed groupwas significantly higher than that in newly diagnosed group (P<0.05) andremission group (P<0.05). In childhood AML patients, HA117expressionin refractory/relapsed group was significantly higher than that in newlydiagnosed group (P<0.05) and remission group (P<0.05);(7) In adult AMLpatients, HA117expression in refractory/relapsed group and newlydiagnosed group was higher than that in remission group, respectively. In adult ALL patients, HA117expression in refractory/relapsed group wassignificantly higher than that in newly diagnosed group and remissiongroup (P<0.05), respectively;(8) In adult chronic leukemia patients,HA117expression in refractory/relapsed group was significantly higherthan that in newly diagnosed or remission group (P<0.05), respectively;(9)In childhood acute leukemia patients, Bcl-2expression in newly diagnosedgroup and refractory/relapsed group was higher than that in remissiongroup, respectively;(10)In childhood patients, c-myc expression in newlydiagnosed group and refractory/relapsed group was higher than that inremission group, respectively;(11) In childhood patients, there was nosignificant difference betweeen newly diagnosed, remission and refractory/relapsed group in PU.1expression (P>0.05);(12) In childhood patients,the average values of RARα expression in remission and refractory/relapsed group were higher than that in newly diagnosed group;(13) Inchildhood patients, there was no significant difference among newlydiagnosed, remission and refractory/relapsed group in DPF3b expression(P>0.05);(14) In childhood patients, there was no significant differencebetweeen newly diagnosed and refractory/relapsed group in bcrpexpression (P>0.05). Average values of bcrp expression in the two groupswere higher than that in remission group.Conclusions: HA117is both involved in the primary resistance ofleukemia， and chemotherapy-induced acquired resistance, but mainlyacquired resistance. The expression of HA117in leukemia is associatedwith the severity and risk, and HA117may be an indicator of severity andrisk of leukemia. PART THREE INVESTIGATION ON THE EXPRESSION OFHA117IN HIRSCHSPRUNG’S DISEASEObjective: To investigate the expressions of ATRA-related LncRNAHA117and its neighbouring gene DPF3in different segments ofHirschsprung’s disease (HD).Methods: Colon tissues of25cases of HD were divided into3groups:spasmic segment, dilated segment and proximal anastomosis, seven casesof colon tissues resected from non-HD patients were set as reference.(1)Real-Time PCR was utilized to detect the mRNA expression of RARα,DPF3b and HA117RNA in different segments of HD;(2) Fluorescence insitu hybridization (FISH) was employed to detect the expression of HA117RNA in different segments of HD;(3) Western Blot was employed toanalyze the expression of RARα, Bcl-2protein in different segments of HD;(4) Immunohistochemistry (IHC) was utlized to detect the proteinexpression of DPF3in different segments of HD, calretinin (CR) was set asa reference.Results:(1) HA117RNA expression in spasmic segment of HD wassignificantly higher than that in dilated segment and anastomosis (P<0.05).The mRNA expressions of DPF3b and RARα in dilated segment andanastomosis were significantly higher than that in spasmic segment(P<0.05);(2) FISH detection of HA117showed that the amount offluorescencein in intestinal mucosa from spasmic segment of HD was morethan dilated segment and anastomosis. In tunica muscularis, spasmicsegment was also the segment containing the most amount offluorescencein, while anastomosis had the least distribution offluorescencein;(3) The protein expression of RARα in spasmic segmentwas significantly lower than that in dilated segment or anastomosis (P<0.05); Expression of Bcl-2protein in spasmic segment was significantlyhigher than that in dilated segment or anastomosis (P<0.05);(4) IHCshowed that positive DPF3staining in spasmic segment was not observed,while it was strong positive in anastomosis. The trend of DPF3expressionin segments of HD was cosistent with CR.Conclusions: By the analysis of25cases of HD, it is concluded thatHA117may exert an anti-differentiation effect in the genesis of HD.HA117may be one of the important factors in the genesis of HD.