Preliminary Investigaion Of The Regulation Of Survivin By BCR-ABL In Chronic Myeloid Leukemia

Chronic Myelogenous Leukemia (CML), a malignant myeloproliferative disorder in the hematopoietic stem cells and the most common type of chronic leukemia, has a high incidence of1/105.The clinical stage of CML can be divided into chronic phase (CP), accelerated phase (AP) and blast phase (BP), The BP stage of CML has a poor prognosis and would lead to death in a short term.95%of CML patients have Ph chromosome and expression of BCR-ABL fusion gene. The Ph chromosome is the result of translocation of the long arm of chromosome9and22, The fusion gene BCR-ABL was formed from the translocation of the long arm of chromosome9(9q34) on the proto-oncogene ABL and chromosome22(the22q11) in the BCR gene. In CML, the BCR-ABL fusion gene transcripts to the8.5kb mRNA and further translates to produce the fusion protein p210BCR/ABL with a molecular mass of a 210KD. P210bcr/ab1has effect on various downstream signaling pathways, including the RAS/MAPK pathway, the JAK/STAT pathway, the PI3kinase pathway and MYC pathway. BCR-ABL interferes with cytokine regulation, promotes proliferation and inhibits apoptosis, Moreover, it disrupts essential adhesion procedures that required for normal growth and differentiation of hematopoietic stem cells, leading to abundant bone marrow progenitor cells proliferation, apoptosis inhibition, reduction of bone marrow stromal cells adhesion. Eventually, a large number of immature myeloid cells are released into the peripheral blood, resulting in the occurrence of chronic myeloid leukemia.BCR-ABL is considered to be the molecular pathological basis and an effective indicator for diagnosis, observation and prognosis of CML Imatinib(IM),a tyrosine kinase inhibitor for BCR-ABL, has been a first-line therapy for CML. IM can strongly inhibit ABL tyrosine kinase activity under both ex vivo and in vivo conditions, Moreover, The expression of V-ABL and the proliferation of BCR-ABL positive cells are specifically inhibited. Imatinib inhibits the BCR-ABL fusion protein autophosphorylation and substrate phosphorylation by occupying the ATP binding site of the BCR-ABL protein, leading to BCR-ABL positive cells proliferation inhibition or apoptosis. However, with the clinical application of IM, resistance develops over time in many patients. Two major reasons are responsible for the occurrence of drug resistance in CML. Firstly, the leukemia stem cells (LSCs) in CML are insensitive to imatinib.Secondly, the point mutations of BCR-ABL. More than50%IM-resistance occur in CML patients because of BCR-ABL point mutations, resulting in interfering with imatinib for binding at the ATP-binding site.MiRNA is a hot frontier research topic in the regulation of post-transcriptional gene expression. It is involved in tumor signal transduction and influences the occurrence of tumor. Studies have confirmed the low expression of miRNA-196b in acute myeloid leukemia (AML). And found that miRNA-196b played an important role in the development of MLL. They both believe that the research on miRNA-196b and its target genes are essential for normal hematopoiesis. In the previous study, our group had validated the target gene of miRNA-196b is BCR-ABL.miRNA-196b overexpression leaded to BCR-ABL down-regulation, miRNA-196b suppression enhanced up-regulation of BCR-ABL.Small interfering RNA (siRNA) is involved in RNA interference (RNAi) and regulates genes expression at transcriptional and post-transcriptional levels.Nowadays,the experimental techniques of chemical synthesis of siRNA interfering on the target gene are very mature. Some scholars have studied the siRNA of BCR-ABL fusion gene.The occurrence and progression of CML is highly dependent on BCR-ABL activity.Imatinib inhibits the expression of BCR-ABL fusion protein directly,miRNA-196b and siRNA-ABL regulate BCR-ABL expression by acting on the BCR-ABL fusion gene at mRNA level.Three methods all affect the BCR-ABL downstream signal transduction pathways finally. Therefore, we researched some of genes in the downstream of BCR-ABL signaling pathways.On the basis of the preliminary study, we found Survivin is a downstream gene of BCR-ABL signal pathways.Various studies had showed that Survivin was closely correlated with Cox-2expression in gastric cancer, lung cancer and elderly patients with acute leukemia.Survivin and COX-2might have synergistic effect or the expression level of Survivin might be dependent on Cox-2. Since Survivin is a downstream gene of BCR-ABL fusion gene and regulated by BCR-ABL, Cox-2might be a downstream gene of the BCR-ABL gene and regulated by the BCR-ABL.We choose three methods, including miRNA-196b, IM and siRNA-ABL, were used to treat K562cells respectively. The expression relationship between Survivin and Cox-2was observed and the effect of different methods on the expression of Survivin and Cox-2were analyzed.PI3K/Akt pathway is one of the BCR-ABL relevant signaling pathways. Its main function is cell growth promotion and apoptosis inhibition. PTEN gene can inhibit PI3K/Akt pathway and down-regulate HIF-1α downstream genes by modulating the expression of HIF-1α. Various downstream genes of HIF-1α have been confirmed, including VEGF, Survivin, Bcl-2, Bax, etc. The Survivin transcripts are elevated by HIF-la. We hypothesized the expression of Surviving PTEN and HIF-1α have certain relations in CML. Therefore, in this study we detected the expression of Survivin, Cox-2, PTEN and HIF-1α, and further analysis of the downstream signal transduction pathways of BCR-ABL.This thesis includes the following two aspects:Part I:Three ways to inhibit the expression of BCR-ABL, including miRNA-196b, IM and siRNA. K562-196b cells were constructed by infecting normal K562cells with the miRNA-196b overexpression lentivirus.The results showed that the green fluorescent protein was clearly observed under the fluorescence microscope, and the mRNA level of miRNA-196b was up-regulated in K562-196b group comparing to K562group cells, suggesting that K562-196b cell line was constructed successfully. The expressions of BCR-ABL were inhibited effectively in all three treated groups, suggesting that the three group cells could be used for subsequent experiments. The exoression of miRNA-196b in IM gropu was significantly higher than K562group (P<0.05), the result show that there was a regulatory loop between miRNA-196b and BCR-ABL. The results of proliferation and apoptosis showed that cell proliferation inhibition and apoptosis promotion were observed in all three treated groups comparing to the control groupPart Ⅱ:The mRNA expression level of Survivin, Cox-2HIF-1α, and PTEN in four groups was detected by real-time quantitative PCR, and analysis the relationship between these gene expressions.The results showed that the expression of Survivin mRNA decreased in K562-196b group (P=0.007) and K562-siRNA-ABL group (P=0.002). Survivin mRNA expression was slightly decreased in K562-IM comparing to K562group, but the difference was no statistically significant (P=0.715). The expression of Cox-2mRNA in three treatment groups as was K562group (P>0.05). Compared with K562group, HIF-1α mRNA was lower in K562-196b group (P=0.007) and K562-siRNA-ABL (P=0.049). The expression of HIF-1α mRNA in K562-IM group was consistent with the K562group (P=0.943). The expression of PTEN in three groups were increased (P<0.05).These results suggest that the Survivin is a downstream gene of BCR-ABL pathway in K562cells, while the expression of Cox-2was not regulated by BCR-ABL. In the three treated groups, HIF-la expression decreased, PTEN increased.The interaction between BCR-ABL fusion gene and Survivin contributes to our understanding of the pathogenesis of CML and the mechanism of drug resistance, providing more possibilities for combined use of drugs and a more solid molecular basis.