| BackgroundsMicro RNAs(mi RNAs) are a kind of length 20 to 22 nucleotides of single non-coding small RNA molecules which widely exists in eukaryotic cells, and regulates the target gene expression through binding to its target gene m RNA to induced degradation or deter its translation process. Mi RNAs plays an important role in the process of cell growth and development, involved in cell proliferation, differentiation, apoptosis and so on. Mi RNA plays key roles in tumor formation through regulating different pathways, and its abnormal expression is closely related to the occurrence of human tumor development. In recent years, studies have shown that mi RNA not only takes significant part in the process of regulating hematopoietic stem/progenitor cell proliferation, differentiation and apoptosis, but also involves in the occurrence and development of leukemia.Acute lymphoblastic leukemia(ALL) is originated from the lymphoid cell line(precursor B, T, or mature B lymphocytes) with the malignant clon, it is also a common malignant tumor in hematological disorders. ALL has a bimodal age distribution, having a high incidence in ages 2–5 and another peak in incidence above 50 years old. There is an increased incidence in people after age 60 years old, the long-term disease-free survival rate was about 35% ~ 40% under the age of 60, and below 10% after 60 years old. 85 percent of ALL children and 75 percent of ALL adults are Acute Blymphoblastic leukemia(B-ALL).B-ALL with t(9;22)(q34;q11) is a type of chromosomal abnormalities in B-ALL patients, named B-ALL with BCR/ABL positive. Despite improvements in early diagnosis and treatment responses by emerging technologies and newly developed chemo/targeted therapies, the high mortality has not been markedly changed in this type of B-ALL. The overall 5-year survival for these patients is still low(<10%) and the recurrence rate is too high, even in early-stage groups. Although attempts have been made to identify important genes and pathways in contribution to tumorigenesis of B-ALL with BCR/ABL positive, most of the studies are focused on the molecular network of protein-coding genes. However, knowledge of genomic aberrations associated with non-coding genes such as micro RNA and their contributions to B-ALL with BCR/ABL positive is relatively limited.ObjectivesIn view of this, this study is designed to analyze the expressions of mi RNAs in the bone marrow of B-ALL with BCR/ABL positive patients and B-ALL with BCR/ABL positive cell lines, and to investigate its function in B-ALL with BCR/ABL positive derived cell line. Furthermore, the potential new targets of mi RNAs as well as their biological function will be further studied by employing bioinformatics-associated technologies. By elucidating the expression, function and mechanisms of disease related mi RNAs in B-ALL with BCR/ABL positive patients, this study maybe provide new clues and basic rationales for the diagnosis, treatment and prognosis of B-ALL with BCR/ABL positive.Materials and MethodsA total of 139 cases with bone marrow were involved in the study, newly diagnosed with B-ALL(68 cases), newly diagnosed with CML(7 cases) and APL(11 cases) as disease control group, healthy control group(53 cases). Through multiple nested PCR combined with cytology, immunology and cytogenetics technology confirmed BCR/ABL negative or positive B-ALL and other leukemia; BCR/ABL positive B-ALL leukemia cells were sorted by Flow cytometry; total RNA was extracted from the sorted leukemia cells; the differentially expressed mi RNAs were sequenced and screened by next generation sequencing and further verified by fluorescence quantitative RT-PCR in large clinical samples to find the BCR/ABL positive B-ALL relevant mi RNAs; BCR/ABL positive B-ALL relevant mi RNAs were selected; their analogs were electric transfected into BCR/ABL positive B-ALL leukemia cell lines; the effects of these mi RNAs related to BCR/ABL positive B-ALL leukemia cell growth was detected through CCK-8; mi RNAs associated with target genes, the analysis and discussion on mi RNAs and BCR/ABL fusion gene regulation effect and binding sites were studied through bioinformatics analysis.Results1. There existed the differencial expression of mi RNAs in BCR/ABL positive B-ALL, the expression level of these mi RNAs were different from the normal control group and CN-B-ALL. 2. The differencial expression of mi RNAs in BCR/ABL positive B-ALL, also had differencial levels in different types of leukemia. 3. The differencial expression of mi RNAs were validated in BCR/ABL positive B-ALL cell line. 4. Enforcement of over expression of BCR/ABL positive B-ALL related mi RNAs(mi R-183、4485、5008、5582), could inhibite cell proliferation in BCR/ABL positive B-ALL cell lines. 5. BCR/ABL positive B-ALL related mi RNAs might have direct target in BCR or ABL gene or/and BCR/ABL fusion gene, which directly or indirectly involved in the occurred or development of B-ALL with BCR/ABL fusion gene.ConclusionsThere exists the related or specific expression of mi RNAs including mi R-183、4485、5008、5582 and so on in BCR/ABL positive B-ALL, these low level of mi RNAs may be involved in the occurrence and development of the disease process. The results of our study can be useful for the diagnosis, prognosis, and designing to provide theoretical and experimental basis of targeted drugs for the BCR/ABL positive B-ALL patients. |
PDF Full Text Request