The Expression Analysis Of MiRNA And Its Target Genes During The Period Of CML Patients Taking Imatinib

Chronic myeloid leukemia is also known as CML. It is a kind of malignant clonal proliferative disease, which originates from multifunctional hematopoietic stem cells of bone marrow. Ninety-five percent of CML patients have Ph chromosome in genetics, it is a fusion gene which fused by ABL gene on chromosome 9 and BCR gene on chromosome22. It is encoding P210 fusion protein and which constitutes the characteristic molecular biomarkers of chronic myeloid leukemia. BCR-ABL fusion gene can cause leukemia,largely because of P210 fusion protein can disorder the tyrosine kinase activity. It can lead to the change of biological characteristics, such as excessive proliferation of hematopoietic cells, the adhesion for bone marrow stromal has decreased and the loss of apoptosis. BCR-ABL fusion protein can activate multiple signaling pathways which has been associated with cell proliferation. And it can change the repair pathways for DNA damage, finally cause fatal hematological malignancy. Therefore, many existing therapeutic strategies for CML has been associated with the bcr-abl gene and its encoding fusion protein.The greatest advances in CML treatment is the application of tyrosine kinase inhibitors imatinib which has a favorable treatment effect on patients in chronic phase, it can also significantly prolong the survival time for patients with accelerated phase and blast crisis. It is one of the main drugs in the treatment of chronic myeloid leukemia and has been recommended by National Comprehensive Cancer Network(NCCN) as the preferred treatment. In our country, it has been applicated as clinical treatment for CML since 2000. The clinical observation shows that conventional doses of imatinib(300~400mg/d) can achieve satisfactory curative effect for most patients with chronic phase Ph+CML in recent years. However, with the clinical application of imatinib, the resistance phenomenon has also gradually appeared, and most occurs in chronic myeloid leukemia patients with accelerated phase(AP) or blast crisis(Bc). In recent years, many studies on imatinib resistant mechanism suggested that the mutation and amplification of bcr-abl gene and the secondary of other karyotypic change such as del(7q), de1(20q), tri8,mono7 mainly contributed to the mechanism of imatinib resistant. Now many studies have shown that micro RNA(mi RNA) can be used as tumor suppressor genes and(or)oncogenes, the abnormal expression of mi RNA can cause a series of drug resistance by promoting cancer cell self-renewal.mi RNA is a class of small non-coding RNA, its formation has experienced the original transcript, precursor and mature individual. The final mature individual is about19-24 base in size, it can regulate the expression of target gene in the translation and transcription level through specific binding to the target m RNA3’ untranslated region(UTR). The current study found that mi RNA is closely related to the development,differentiation, apoptosis and proliferation of biological cells.Therefore, this study aims to use Real-time quantitative PCR(RT-PCR) to detect the different expression of mi RNA of bone marrow mononuclear cells in chronic phase or progression phase of CML. Specific mi RNA molecules were screened, such as micro RNA-21(mi R-21), mi R-10 a, mi R-31, mi R-34 a, mi R-34 a, mi R-155, if there is significant difference in expression, we will further observe the mi RNA and its targetgenes in bone marrow mononuclear cells of patients who are sensitive and resistance to imatinib. To provide a new therapeutic target for CML.Experiment 1:Objective: Exploring the expression levels of mi RNA in chronic phase and progression phase of CML patientsMethods: Collected the bone marrow samples of CML patients in chronic phase and progression disease total of 35 cases. Prepared the bone marrow mononuclear cells and extracted total RNA, monitored the concentration and purity of RNA which had been extracted by UV spectrophotometer. Finally, we detected the relative expression levels of mi R-21, mi R-10 a, mi R-31, mi R-34 a, mi R-155 in the chronic group and progress group by the RT-PCR technology.Results: The expression of mi R-21 in bone marrow mononuclear cells in progress group was significantly higher than the chronic group after chronic myeloid leukemia patients taking imatinib. There were no significant difference in the expression levels of mi R-10 a, mi R-31, mi R-34 a and mi R-155 between the chronic group and progress group(P>0.05).Conclusion: The high expression levels of mi R-21 may be associated with the disease progression in CML patients who are treated with imatinib.Experiment 2:Objective: Exploring the expression levels of mi R-21 in sensitive group and drug resistance group of CML patients after taking imatinib.Methods: Collected of bone marrow samples of CML patients in sensitive group and drug resistance group after taking imatinib total of 37 cases. Prepared the bone marrow mononuclear cells and extracted total RNA, monitored the concentration and purity of RNA which had been extracted by UV spectrophotometer. Finally, we detected the relative expression levels of mi R-21 in the bone marrow mononuclear cells in the sensitive group and drug resistance group by the RT-PCR technology.Results: The expression levels of mi R-21 in bone marrow mononuclear cells in drugresistance group was significantly higher than the sensitive group, the difference was statistically significant(p<0.0001).Conclusion: mi R-21 may be closely associated with the drug resistance of imatinib after CML patients taking it.Experiment 3:Objective: Exploring the expression levels of the target genes of mi R-21 in sensitive group and resistance group of CML patients after they taking imatinib.Methods: A total of 37 cases were collected from bone marrow samples in sensitive group and drug resistance group of CML patients after they taking imatinib. Prepared of bone marrow mononuclear cells and extracted total RNA, monitored the concentration and purity of RNA which had been extracted by UV spectrophotometer. Then prepared c DNA.Finally, we detected the relative expression levels of PDCD4 and PTEN in the bone marrow mononuclear cells in the sensitive group and drug resistance group by the RT-PCR technology.Results: The relative expression levels of PDCD4 in bone marrow mononuclear cells of drug resistance group was significantly lower than the sensitive group after they taking imatinib, and the difference was statistically significant(p=0.0002). The relative expression levels of PTEN in bone marrow mononuclear cells of drug resistance group was significantly lower than the sensitive group after they taking imatinib, and the difference was statistically significant(p<0.0001).Conclusion: mi R-21 may be closely associated with drug resistance during the period of imatinib treatment in CML patients by regulating its target genes PDCD4 and PTEN.