The Relationship Of Hedgehog Signaling Pathway Activation And HHIP Gene Methylation In Leukemia And Demethylation Mechanism Of Arsenic Trioxide

BackgroundThe etiology and pathogensis of leukemia is very complex, and it is not entirely clear. Epigenetic regulation is a kind of control mode which based on non sequential genetic alterations induced heritable gene expression level changes, including DNA methylation, histone acetylation, small RNA (miRNA) silence. In leukemia development process, it plays a very important role. Epigenetic regulation has led to the tumor suppressor gene expression of silence is one of the important factors inducing leukemia. In recent years, some studys have proved that DNA methylation was a common phenomenon in human leukemia, and closely related to the onset of leukemia.Demethylation therapy as a new antitumor method has been shows a good application prospect in leukemia treatment, especially opened up a new way in the treatment of refractory and relapsed leukemia.The clinical application of demethylation drugs have begun to hematological malignancies such as acute leukemia and myelodysplastic syndrome(MDS) treatment, such as5-aza-2’-deoxycytidine (deoxyeytidine) etc. As2O3is a kind of traditional Chinese medicine, initially for the treatment of acute promyelocytic leukemia and achieved good results. The main mechanism of As2O3in treatment of acute leukemia is inducing leukemia cell apoptosis. Recent studies found that, As2O3can reverse the methylation of certain tumor suppressor gene in myeloma cells and lymphoma cells, so in induction to apoptosis, As2O3may be has other mechanisms of action.The Hedgehog signaling pathway plays an important role in human and animal normal embryonic development and organ formation.Its channel members including hedgehog, PTCH, SMO, and Gli.The channel member mutations or error expression will activate the pathways, ultimately leading to cancer occurrence and development. Recent research demonstrated that the pathways relevant to tumors including basal cell carcinoma, pancreatic cancer, colon cancer, stomach cancer, small cell lung cancer, melanoma, breast cancer, ovarian cancer and other. In the tumors that Hh pathway is activated, approximately half did not find the channel member mutation. so we hypothesized that Hh pathway activation may have other regulation mechanism. Although the existing research has proved that Hh pathway has close relationship with tumor, but Hh pathway through what mechanisms contribute to tumorigenesis remain unclear. Recent studies found that some tumor in the presence of Hh signal pathway members promoter methylation. There are also reports indicate that Hh pathway is activate in some leukemia.For the further study of Hh signaling role in leukemia and activation mechanism, Whether aberrant methylation of tumor suppressor genes are involved in the Hh pathway activation and the occurrence of leukemia, we detect the channel member from the RNA levels and protein level before and after As2O3treatment, detect Hh pathway tumor suppressor gene HHIP methylation status by methylation specific PCR. The study will be have important significance on the further elucidation of the pathogenesis and the discovery of new therapeutic targets in leukemia.Objective The study was designed to investigate whether Hh pathway is activate in acute leukemia, and whether the activation of Hedgehog pathway is related to HHIP methylation. To explore the effect of DNA methylation on the pathogenesis of leukemia. Through the research of arsenic trioxide’s demethylation effect mechanism on HHIP gene, to explore whether Hh pathway exists target of demethylation drugs.MethodsSelection of acute myeloid leukemia cell line HL60, chronic myeloid leukemia cell line K562, acute T lymphocytic leukemia cell lines Jurkat, B cell acute lymphoblastic leukemia cell line BALL-1, cell lines containing10%calf serum RPMI1640culture medium, at37degrees and5%C02culture box culture.1、 The activity level of Hh pathway in leukemia cell lines1.1using reverse transcription polymerase chain reaction (RT-PCR) to detecte Hedgehog pathway members SHH, PTCH, Gli, HHIP gene expression of the leukemia cell lines and leukemia patients.1.2Using Western-blot to detect the corresponding protein expression.2、 Methylation status of HHIP gene in leukemia cell lines and its influence on the expression of Gli2.1Using DNA Extraction Kit by silica gel column extraction of genomic DNA, Select specimen of purity between1.70-1.90.Using DNA CpGenome DNA Methylation kit sample kit for DNA was modified, then using methylation-specific PCR (MS-PCR) to detect Hedgehog signal pathway of HHIP gene promoter region methylation of4leukemia cell lines.2.2Using siRNA closed HHIP gene expression, then use RT-PCR to detect the expression of Glil HHIP gene.3、 Demethylation mechanism of arsenic trioxide3.1Treat BALL-1cell line With different concentrations of As2O3, in accordance with the concentration of As2O3is divided into0.0,2.5,5.0, 10.0umol/L group, after treatment for24,48,72,96hours, add lOul CCK-8solution, the microplate reader on450nm single wavelength measuring absorbance0D value, calculate the inhibition rate, and take the time as the horizontal axis,0D value for the cell growth curve.3.2As203treatment after48hours, collecte the cells, washing2times by PBS, add cold ethanol fixation for more than24hours, washing cells by PBS,37℃magazine incubated for30minutes, PI staining, avoid the light for30minutes, detect changes of cell cycle after arsenic trioxide treatment by flow cytometry.3.3As2O3influence HHIP gene methylation pattern of BALL-1cell.BALL-1cells were treated with As2O3for24hours,48hours,72hours, DNA Extraction Kit by silica gel column extraction of genomic DNA, using DNA CpGenome DNA Methylation kit to modify1g DNA sample, then using MS-PCR to detect HHIP gene promoter region methylation of BALL-1cell line.3.4Gli is not only the Hh pathway members, but also the target genes of the pathway, therefore may reflect the activity of the pathway. As2O3act on BALL-1cells after48h, Total RNA extracting reagent RNAiso extracted from BALL-1cell line, using RT-PCR to detect the expression of Gli mRNA.4.5How does As2O3influence DNA methyltransferase dnmt1、 dnmt3b mRNA and mbd2mRNA expression:We detect dnmt1、 dnmt3b and mbd2mRNA expression by RT-PCR.Result1、 The activity level of Hh pathway in leukemia cell linesThe important components of Hh signaling pathways such as signal peptide SHH, membrane receptor PTCH, downstream of the transcription factor Gli and HHIP mRNA have different levels of expression. In the majority of acute leukemia cell lines, Gli or PTCH mRNA expression were increased(P<0.05), the expression of SHH had no obvious change, except for K562cell lines, the rest of leukemic cell lines HHIP expression is reduced (P<0.05). The expression of HHIP protein in BALL-1、Jurkat、 HL60was lower than that of control group, and protein expression of Gli in BALL、HL60is higher than that of Control group.2、 Methylation status of HHIP gene in leukemia cell lines2.1BALL-1、 Jurkat cells showed hypermethylation,HL60、 K562showed partial methylation, and HHIP gene were not detected methylation in normal lymphocyte.2.2Incubation BALL-1by HHIPsiRNA for48h, HHIP mRNA transcription is inhibited, HHIP/GAPDH decreased from0.983to0.409; GlimRNA transcription levels are elevated, Gli/GAPDH increased from1.045to1.383. This indicate that HHIP is probably the upstream negative regulatory factor of Glil.3、Demethylation mechanism of arsenic trioxide3.1After treated with arsenic trioxide, the inhibition rate of cell proliferation is increased remarkably with time and concentration, compared with the negative control group the differences were statistically significant (P<0.05), the inhibition rate was time and concentration dependent.3.2After treated with Different concentrations of As2O3, BALL-1cells wre blocked in G0, G1in different degree, percentage of G0/G1phase cells increased, and percentage of S、 G2/M phase decreased (P<0.05).3.3HHIP-M amplification is positive, while the HHIP-U amplification results is negative, suggesting HHIP gene is highly methylated in BALL-1cells. As the As2O3concentration increased, HHIP-M bands gradually weakened, while the HHIP-U strip increases gradually, suggesting that HHIP gene methylation degree is reduced gradually, but the degree of unmethylation increased gradually. The above results indicate:As2O3can reverse HHIP gene promoter hypermethylation status, and the effect depended on the concentration of 3.4As2O3influence the expression of HHIP and Gli mRNADetected the expression of Gli and HHIP mRNA by RT-PCR before and after treated with As2O3, the results showed that HHIP mRNA expression was significantly upregulated after treated with5.0umol/L As2O3for72h. HH pathway target genes GlimRNA expression were reduced.3.5As2O3influence the expression of dnmt1、 dnmt3b mRNAAs2O3can downregulate DNA methyltransferase(dnmtl、 dnmt3b) mRNA. experimental groups with As2O3increasing the concentration of dnmt1、 dnmt3b gene expression gradually weakened(P<0.05). But the expression of mbd2mRNA did not change significantly after treated with As2O3(p>0.05).Conelution1The expression of Gli and PTCH were increased in some leukemic cell lines, which suggest that Hedgehog pathway is aberrant activate in leukemia. Hh pathway activation may be associated with the pathogenesis of leukemia.2Promoter methylation induced the silence of HHIP gene, and HHIP lost its function on the Hh pathway. It may be the aberrant activation mechanism of Hedgehog pathway.3As2O3can demethylation HHIP gene in BALL-1cells in a concentration dependent manner.4As2O3demethylation effect mechanism for the inhibition of S-methionine dependence of the methyltransferase, such as dnmtl、dnmt3b,thereby restoring gene expression.