| Acute leukaemia is a form of malignant cloned disease involvingmultiple chromosomal translocations and genetic alterations.Malignantclonal proliferation is the necessary factors of leukemia patients havingabnormal hematopoietic and organ infiltration, but the intermediate stepsof leukemia cell proliferation is not entirely clear. It has been reportedthat a high mobility group protein group A2,which is widely involved inthe occurrence of many human Malignant tumors, including leukemia,andthe expression of HMGA2was significantly increased on the lever ofmRNA and protein in leukemia, but the role of HMGA2in leukemiacarcinogenesis isn’t well known. Exploration of signal molecule mediatedby HMGA2and comprehension of the role of HMGA2in leukemiacarcinogenesis are very important to know the mechanism of leukemia,which contribut to identify the target protein for antitumors drug and treatcolorectal carcinoma.The human HMGA2gene (also known as HMGI-C gene), is a of high mobility protein family member, because of its member of smallermolecular weight, can be in polyacrylamide gel electrophoresis in rapidmigration.It located on human chromosome12q15district, consists of5exons and4introns, exons1-3encodes a3DNA binding region, exon4encodes the interval between DNA binding region and acid carboxylterminal area, exon5coding acid carboxyl terminal.The HMGA2protein is a kind of non histone nuclear chromatinbinding protein,which located within the nucleus generally, composed of109amino acid residues. The protein helium end containing3shortfoundation repeat sequence coding structure domain, and theDNA-binding domains specifically bind to the minor groove of anAT-rich DNA,hence referred to as AT hooks. HMGA2itself withoutactivation or repression of transcription activity, but can be used as aconstruction factor, through AT-hooks and with DNA binding andchanges chromatin structure, through a complex protein-protein,proteinDNA interaction network, co-ordinating nuclear protein complexassembly, thereby regulate the expression of target genes positively ornegatively.HMGA2proteins plays an important role in early embryonicdevelopment and cell differentiation proliferation. Research shows thatalmost all tissues there are the expression of HMGA2in addition to braintissue, but there is virtually undetectable to the transcription product in adult tissue and differentiated mature tissues (3). In recent years, moreand more research report that, there are HMGA2overexpression orshowing the HMGA2gene rearrangement characteristics result of12chromosomes balanced translocations in most benign and malignanttumor,indicate that there is a close relationship between HMGA2andtumor development.However,there is less research on hematologicneoplasm．Initially some scholars checked HMGA2expression werepositive in CD34positive stem cells from healthy donors and the acuteand chronic myeloid leukemia patients samples while all peripheral bloodsamples from healthy volunteers were negative,they havec concludedthat the expression of the HMGA2gene in leukemia seems to be asecondary effect due to abnormal stem cell proliferation and might be asensitive tumor marker for particular types of leukemia．In addition, FISHanalysis showed that the rearrangements concerning HMGA2rearrangements,following translocation involving the chromosome band12q13-15,which have been reported that in the transformation of Richterchronic lymphocytic leukemia, acute lymphoblastic leukemia,myelofibrosis with myeloid metaplasia, myelodysplastic syndrome, acutemyeloid leukemia patients．It is further observation to note that most ofthe patients showing HMGA2rearrangements did not have a longsurvival,suggesting that HMGA2expression might indicate a poorprognosis. RNA interference technology can effectively and specially block theexpression of endogenous genes, through dsRNA and some nuclearproteins forming complexes RISC (RNA-induced silencing complex),which complementary binding to mRNA, then RISC degradates mRNAand blocks the protein translation, closing the gene function, thus makescells show specific phenotype with the loss of some gene. RNAinterference technology is a powerful tool of blocking down theexpression of a particular gene in cells, compared with the traditionalantisense technology, it can be a promising tool of gene therapy as resultof its more powerful function, which makes it play an important role inmany diseases, such as tumor,dominant heredity diseases or infectiondiseases.RNA interference technology provide a powerful force forfurther revealling the pathogenesis of leukemia and gene targeting drugsbased on the RNA interference technology and has wide applicationprospect. The chemical synthesis siRNA only function a relatively shorttime in intracellular. In order to be able to maintain target genessustainable suppression, Brummelkamp reported siRAN of synthesisintracellular. The general is a promoter+siRAN+corresponding seriestermination code form, which forming hairpin short chain interferenceRNA (small hairpin RNA)after intracellular transcription.The siRANcan sustainably inhibit the target gene expression intracellular,and thiseffection can last up to7days or more, and significantly reduce the cost of preparation of siRNA.Recently researches have indicate that21-ntsiRNA can effectivelyand specially block the expression of endogenous genes in mammals, butin term of the specific introduction of siRNA, on the basis of DNAplasmid siRNAs when introducted into mammalian cells result intransient expression of siRNAs,while methods involving cationic lipidand liposome-mediated delivery of DNA or physical methods such aselectroporation result in low transfection efficiency, lossing of cellviability, and the difficulty of obtaining stable transfection effection. Incontrast, lentiviral is an ideal carrier of carrying the interference RNAwith high infection efficiency and weak efficiency of stimulating theimmune response. Viral vectors can effectively transduct siRNA into cellsand tend to provide stable silence of genes interference, in particular,lentiviral vectors can be used for siRNA expression result of its hightransduction ability on various types of cells and stable gene transductionfor relative long-term RNAi efficiency.Acute myeloid leukemia HL-60cell line was used in this study toinvestigate the role of HMGA2in leukemia carcinoma carcinogenesis.With cell biology technology, molecular biology technology and RNAinterference, the relation between HMGA2and leukemia biologicalbehavior of malignant clonal proliferation leukemia cells may beinvestigated, which can reveal the role of HMGA2and provides therapeutic and experimental basis to the gene therapy of leukemia.Firstly HMGA2double-stranded oligodeoxynucleotides DNAfragments and negative control sequence which did not direct against anymRNA DNA fragment were designed and synthesized, then recombinantform of shRNA expression vectors with virus vector plasmidPGCSIL-PUR, PGCSIL-GFP respectively,which served as a expressionvector was transfected into the293T cells to produce recombinantlentivirus(HMGA2-RNAi-LV,NC-GFP-LV) by Lipofectamine TM2000inthe presence of the mixture of pHelper1.0and pHelper2.0.HL-60cellswere transduced with each lentiviral construct, stably transduced cellswere selected by complete culture medium containing the appropriateamount of puromycin. The protein expression change of HMGA2wasexamined by western-blot and RT-PCR analysis, and the effect of thelentivirus on the cell proliferation inhibiting rate and clonogenic capacityand cell cycle was analyzed by CCK-8method and soft agar clonogenicassay and FCM,respectively. The protein and mRNA expression of cyclinA2and cyclin B2were also examined by Western-blot and RT-PCR.The main results and conclusions are as follows:1. Recombinant eukaryotic expression plasmids HMGA2siRNA andLV-HMGA2shRNA and LV-non-specific were constructedsuccessfully. The recombinant plasmids were indentified with agaroseelectrophoresis and DNA sequencing assay. Then the recombinant vectors served as a expression vector was transfected into the293Tcells to produce recombinant lentivirus(HMGA2-RNAi-LV,NC-GFP-LV) by Lipofectamine TM2000in the presence of the mixture ofpHelper1.0and pHelper2.0.The transduced cells with each lentiviralconstruct at an MOI of200were selected by complete culturemedium containing10ug/ml of puromycin on the time of48hourspost-transduction. After15days of selection, puromycin-resistantcolonies were picked,and each clone was expanded to culture inorder to assay for knockdown efficiency of the target gene byWestern blot and RT-PCR. Three kinds of selectedpuromycin-resistant colonies were respectively named as:(1)LV-HMGA2shRN、(2)LV-HMGA2shRNA、(3)LV-HMGA2shRNAand LV-non-specific construct.Group of infected with recombinantvirus of LV-HMGA2shRNA virus was detected that the HMGA2expression level was significantly inhibited by Western-blot andRT-PCR analysis, compared with normal HL-60cells and the negativecontrol group. The results showed that: we constructed HMGA2siRNA eukaryotic expression recombinant plasmid and preparation ofthe recombinant lentivirus, selected a stable at interferencingHMGA2expression cell lines successfully. This laid sound basis forthe following research of HMGA2.2. Reporter gene analysis indicates that the expression of reporter gene LamininA/C hadn’t been affected by stable silence of HMGA2.These results indicate that in HL-60cells expressing shRNAtargeting HMGA2, non specific interference with the function of areporter gene is not a problem. The results also show thatlentiviral-mediated RNAi is capable of silencing endogenous genesdriven by a strong promoter.3. The high expression of HMGA2significantly increased leukemia cellproliferationability. The role of HMGA2in cell appreciation abilitywas determined by the in vitro CCK-8method. The appreciation ofcells infected lentivirus of LV-HMGA2shRNA showed to be asignificantly greater slowed down, compared with non-specificconstruct lentivirus infected HL-60cells and normal HL-60. Theresults showed that silencing of endogenous HMGA2resulted inefficient inhibition of the cellular proliferative activity, startingseparation after24hours culture,low and flat of the cell growthcurve and the lack of typical character of exponential growth, thedifference was statistically significan(tF=17.982,P=0.00)The resultindicates that increased expression of HMGA2play an abvious rolein acute leukemia HL-60cell proliferative activity.4. Increased expression of HMGA2may significantly contribute to cellclonogenic capacity. The role of HMGA2in cell colony formationwas determined by the in vitro soft agar clonogenic assay.The formation clones of HL-60cells from (1) and (3) were less andsmaller after long-term culturing in vitro, and the clone formationrate were (42.7+2.8)%,(37.2+4.1)%respectively. while cloneformation rate of the negative control group and blank groupwere(70.9+1.8)%,(73.5+2.1)%respectively; the experimentalgroup clone formation rate decreased obviously compared withthe negative control group and blank control group, resulted insignificant difference(F=20.227,P＜0.001).5. LV-HMGA2shRNA leads to HL-60cell cycle redistribution. TheG2/M phase of the cells from experimental groups(1) and(3)increased obviously, the percentage were increased to (27.2+3.81)%,(30.1+5.78)%respectively, compared with the negativecontrol group cells and blank group resulted in significantdifferences(P <0.05, n=3), the result of flow cytometry revealedsignificant cell cycle G2／M arrest in stable HL-60cell linedeficient in HMGA2gene expression as control. Compared with thecontrol group, cyclin B2mRNA and protein relative expressionlevels were significantly decreased(P <0.01), the inhibition rate ofmRNA transfected with shRNA1group and shRNA3group were(57.31+7.11)%and (67.55+7.69)%, and protein expressioninhibition rates were (51.77+4.81)%and (55.29+3.99)%respectively due to RNA interference silencing HMGA2in HL-60 cells, while the expression of cyclin A2had no significant changeduring groups.Taken together,as a carry of siRNA,Lentivirus not only silentspecific gene expression level but also exert its advantage andinfluence on gene function research,and provide a more powerfulresearch tool. Recombinant eukaryotic expression plasmids HMGA2shRNA which can inhibit HMGA2expression efficiently wereconstructed successfully,which would not only provide the basis forthe further study in the mechanisms of leukemia cells malignantclonal proliferation but also suggest that signal pathway mediated byHMGA2could be targeted for therpeutic intervention in leukemia. |
PDF Full Text Request