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Experimental Study of MicroRNA Gene Sequence Variations in Multiple Myeloma

Author: ZhangNingYu
Tutor: DuXin
School: Southern Medical University,
Course: Clinical medicine internal medicine hematology
Keywords: Small RNA Sequence mutations Multiple myeloma Polymerase chain reaction ( PCR ) Single strand conformation polymorphism
CLC: R733.3
Type: Master's thesis
Year: 2010
Downloads: 176
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Abstract


Multiple myeloma (multiple myeloma, MM) is a B-cell malignancy, a malignant clonal proliferation of plasma cells in the bone marrow, serum or urine M protein monoclonal immunoglobulin or its ingredients, normal immunoglobulin by suppression and extensive osteolytic lesions and / or osteoporosis is characterized. Multiple myeloma incidence of about one hundred thousandth ten ten thousandths, second only to acute leukemia, the highest incidence of hematological malignancies, and there is a growing trend. Diagnosis of 97% -99% of patients over the age of 40 years old, and gradually increase the incidence increased with age, especially, mainly in the 60-year-old or older. Despite some progress in recent years, drug treatment, most patients with multiple myeloma or due to recurrence and disease progression can not be cured. As China has entered the aging society, this age-malignant disease are increasingly common, serious impact on the survival time and quality of life of the patients. Pathogenesis of multiple myeloma is unclear, the the existing findings show mainly with two aspects of the role of genetic alterations and cytokine, but so far there is no clear answer. Research progress is rather slow due to the occurrence of the disease involved in the development of more complex molecular mechanism. Small RNA (micro RNA, miRNA) discovery of a new idea for the research in this area, miRNA is widely present in the cytoplasm of eukaryotic cells, a class of small molecules of single-stranded RNA derived from endogenous hairpin like transcription product, about 18-25nt, does not have the open reading window (ORF), and do not encode proteins, and as a sequence-specific, post-transcriptional inhibition of gene expression regulatory factors, complete or incomplete pairing with the complementary mRNA, the degradation of target translated mRNA or repressor of its transcription. miRNA hundreds of target genes, regulation and control more than half of the human protein-coding genes. Target genes through regulation of miRNA involved in a series of important processes in the life course, including ontogeny and hematopoietic organ formation, apoptosis, cell proliferation and differentiation, and fat metabolism. More than 50% of the miRNA and tumor-related region of the map of the human genome, suggesting that miRNA play a very important role in tumorigenesis. It has been found that miRNA expression patterns and genetics abnormalities of miR-1 and miR-133a has the same chromosomal locus, overexpression exception of cases of the t (14; 16). Selective upregulation of some miRNA in multiple myeloma and significantly associated with some tumor genes, such as miR-181a and miR-181b is highly expressed in MM, miR-17 and miR-32 selective upregulation in MM cell lines and raised in the normal group. miR-19a and miR-19b can down-regulate the expression of the tumor suppressor gene SOCS-1 (SOCS-1 in MM is often silent, play an important role as an IL-6 inhibitor). MM, miR-335 and miR-342-3p, miR-561, and three host gene expression was significantly associated, may play an important role in cell homing and interaction with the bone marrow microenvironment. Through the literature review, 12 kinds of abnormal miRNA are summarized as follows: miR-17, miR-32, miR-19a, miR-19b-1, miR-19b-2 of miR-181a-1, miR expression in multiple myeloma -181a-2, miR-181b-1, miR-181b-2, miR-335, miR-342, miR-561. In view of the above findings, the close relationship of the miRNA and MM-depth study of these miRNA is particularly important. the miRNA generation of after the following process: gene transcription produces about 300-1000bp primary transcripts - the original tiny RNA (pri-miRNAs), and then processed into about 70 tiny precursor complexes ('Microprocessor'-complex) -90nt hairpin-like precursor RNA (pre-miRNA); Pre-miRNA in the role of exportin-5 transport into the cytoplasm, another double-stranded RNA-specific RNase Ⅲ-Dicer recognition, further cut into about 18-25 nt RNA, small molecules that mature miRNA. Due to the the miRNA expression conservative, its gene sequence changes may have a significant impact on the downstream cleavage modification, and thus play a role in the pathogenesis. Including original small RNA (pri-miRNAs), the precursor of small RNA (pre-miRNAs) and mature small RNA (mature miRNA) sequences to change the will of miRNA processing, the selection process have an impact. Overseas research shows that exist in the pri-miRNA sequence mutations will affect the maturation of pre-miRNA, mature miRNA mutations will directly affect the expression of miRNA maturation process, and with the combination of the target area. In addition, miRNAs mutation sequence of a target region will change the activity of the miRNA, and (or) to affect their target gene expression. MiRNA gene mutation tumor confined to acute myeloid leukemia, lymphoma, liver cancer, studies have not been reported in multiple myeloma. The experimental multiple myeloma study in 12 abnormal expression of miRNA gene sequence mutations, using polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) silver staining four human multiple myeloma tumor cell lines (KM-3, ARH-77, U266, RPMI8226) in bone marrow samples of 20 patients with multiple myeloma and 20 patients with haematological indices return to normal after treatment, non-blood cancer patients above 12 miRNA gene mutations in experimental data by conventional statistical methods test significant differences, and analysis of its pathophysiology and clinical significance of the test results and the theoretical basis for the diagnosis of multiple myeloma molecular biology and molecular targeted therapy. Objective To understand the multiple myeloma (multiple myeloma, MM) patients with small RNA (MicroRNA, miRNA) gene mutation exists, whether the relationship with the tumor, what clinical significance. The method of collecting bone marrow samples of 20 cases of multiple myeloma patients and haematological indices return to normal after treatment of 20 cases of non-blood cancer patients, the group of cases of bone marrow cells were CD138 positive immunomagnetic bead separation and purification of plasma cells, flow cytometry purity; controls extraction of mononuclear cells in bone marrow specimens; 4 kinds of human multiple myeloma cell lines cultured and passaged. The case group and the control group and cell line samples were extracted genomic DNA using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) silver staining in the specimen 12 kinds of miRNAs (miR-17, miR-32, miR -19a, miR-19b-1, miR-19b-2, miR-181a-1, miR-181a-2, miR-181b-1, miR-181b-2, miR-335, miR-342, miR-561 ) gene mutations, the case group and the control group, the mutation rate by the Fisher exact test, P lt; 0.05 was considered statistically significant. The experimental results with clinical analysis. Result of human multiple myeloma cell line KM-3, RPMI8226 miRNA-335 gene mutations detected; 20 cases of multiple myeloma patients were detected mutations, miR-19a, miR-19b and miRNA-335 gene mutations each In one case, the patient group total mutation rate was 15%; mutation were not detected in the normal control group. The statistical results show that P gt; 0.05, two groups mutation rate differences not statistically significant, is not yet the mutation rate of the patient group than in the control group. The mutation of the case group 1 patients had died, two cases to detect mutations in end-stage disease. Discussion through the experimental study, we have mastered the cell culture, mononuclear cell extraction and separation of CD138 cells, polymerase chain reaction - single strand conformation polymorphism analysis and other experimental techniques. For the first time in patients with multiple myeloma and multiple myeloma cell line KM-3 RPMI8226 detect miRNA genes miR-19a, miR-19b and miRNA-335 sequence mutation: multiple myeloma cell lines KM 3 and the control group RPMI8226 miRNA-335 gene mutations detected; 3 cases of 20 patients with MM mutation, miR-19a, miR-19b and miRNA-335 gene mutation example, the case group total mutation rate of 15%; 20 cases were not detected mutation. The statistics showed that the case group and the control group, no significant difference in mutation rate, still can not believe that the mutation rate of the patient group than the control group. These results suggest that miRNA genes miR-19a, miR-19b and miRNA-335 in the presence of mutations in MM specific mutations need further validation. miRNA gene sequence mutations may be associated with multiple myeloma pathogenesis play an important role in the pathogenesis of multiple myeloma, in view of the miRNA target gene SOCS-1 and MEST sequence mutation seems to contain an important mechanism for downstream dysregulation . Combined with the clinical outcome of the case group experimental group were analyzed: the mutation of the case group 1 cases had died two cases to detect mutations in end-stage disease. Clinical features occurred miR-19a, miR-19b and miRNA-335 gene mutations in cases with poor prognosis in detection of mutations, miR-19a, miR-19b and miRNA-335 mutation may be related to the prognosis of disease progression, mutations detection may contribute to the analysis of MM progression and prognosis analysis. Conclusion miRNA gene sequences in the presence of the mutation rate in patients with multiple myeloma and multiple myeloma cell lines, mutations in three kinds of miRNA genes miR-19a, miR-19b and miRNA-335 in hematology normal in the control group have been found in the above-mentioned mutations. Whether these mutations have a specific need to expand the amount of samples tested further validation. miR-19a, miR-19b and miRNA-335 gene sequence mutations may be associated with multiple myeloma pathogenesis sequence mutations may be its target gene SOCS-1 and abnormal regulation of MEST one of the reasons why the specific mechanisms need further study . Clinical features suggestive occur miR-19a and miR-19b and miRNA-335 gene mutations in patients with the disease and poor prognosis, the detection of mutations may contribute to the progression and prognosis analysis, multiple myeloma is expected to become the new signs of disease detection and prognosis speculated that the indicators have important theoretical and clinical significance.

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CLC: > Medicine, health > Oncology > Hematopoietic and lymphoid neoplasms > Bone marrow tumor
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