Experimental Study Of MicroRNA Gene Sequence Variations In Multiple Myeloma

Multiple myeloma (MM) is a B-cell neoplasm with accumulation of clonal malignant plasma cells in the bone marrow, which is characterized by pathologic plasma cells (PCs) in marrow, monoclonal immunoglobulin (M protein) in blood or urine and widespread bone destruction. Now in China, the morbidity rate of MM is ranged between 1/100,000 and 2/100,000, second to acute leukemia as the most common hematological malignancy.97%-99% patients are over 40 years when they are first diagnosed with multiple myeloma. And, the risk increases with age, especially for people over 60.Accronding to its infection and bleeding tend, MM is the severe cause harming the health of human beings. Despite recent advances in drug development, multiple myeloma remains incurable for the majority of patients due to relapse and disease progression. As Chinese population entered the aging society, multiple myeloma becomes more and more common. It is necessary for researchers to study on pathogenesis of multiple myeloma, although progress in understanding the biology of multiple myeloma has been slow. Previous studies show that the incidence of multiple myeloma is also associated with genetic abnormalities and changes in a variety of cytokines. But to date there is no exact result. The discovery of microRNAs (miRNAs), a class of small non-coding RNA targeting multiple mRNAs, has revealed a further level of complexity to normal and cancer cell biology. MiRNAs are 18-25nt, single-stranded non-coding RNA molecules widespread in eucaryotic cells that are generated from endogenous hairpin-shaped transcripts. They regulate the expression of target genes at post-transcriptional level by either suppressing the mRNA translation or inducing the mRNA cleavage. MiRNAs regulate various key biological processes, such as cell proliferation and differentiation, apoptosis and insulin secretion via hundreds target genes. More than 50% of these miRNAs map to locations in the human genome that are associated with cancer, suggesting that loss or alteration of miRNA expression might be a common occurrence in cancer cells. Hematopoiesis is one of the most important highly regulated multistage processes, which includes orderly turn-on and turn-off of many genes; any wrong modulation may result in blood diseases. Several miRNAs have been found to be involved in hematopoiesis and hematopoietic tumor genesis. Specific miRNA signatures have been associated with different cytogenetic subtypes in acute leukemia. These findings prompted researchers to investigate potential associations between genetic abnormalities in MM and singular miRNA expression profiles. For example, miR-1 and miR-133a clustered on the same chromosomal loci, were specifically over expressed in the cases with t(14;16),which illustrates that miRNA expression pattern in MM is associated with genetic abnormalities. Studies also demonstrate that multiple new miRNAs are commonly up-regulated and down-regulated in multiple myeloma, including 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. Selective up-regulation of miR-32 and miR-17 was identified in MM subjects and cell lines, miR-19a and 19b, were shown to down regulate expression of SOCS-1, a gene frequently silenced in MM that plays a critical role as inhibitor of IL-6 growth signaling. MiR-181a and b over expressed in MM and MGUS samples. Three miRNAs (miR-335, miR-342-3p, and miR-561) that were differentially expressed along with their corresponding host genes, may play a role in plasma cell homing and/or interactions with the bone marrow microenvironment. MiRNAs are initially transcribed as 300-1000bp long primary transcripts (pri-miRNAs), which are then processed into 70-90nt hairpin-shaped precursor miRNAs (pre-miRNA) by the 'Microprocessor'-complex. After processing by'Microprocessor'-complex, pre-miRNAs are subsequently exported to cytoplasm by exportin-5 and cleaved by another RNaseâ…¢, Dicer, to generate 18-25nt mature miRNAs, which are then incorporated into RNA-induced silencing complexes to regulate gene expression. MiRNAs are members of highly conserved gene families. Sequence variations in miRNA genes, including pri-miRNAs, pre-miRNAs and mature miRNAs, could potentially influence the processing and/or target selection of miRNAs. Some studies have shown that variants located within the mature miRNA may directly affect miRNA expression, the maturation process and binding to target sites. Variants occurring in other regions of primary miRNA (pri-miRNA) may affect precursor miRNA (pre-miRNA) maturation. In addition, variants in the miRNA target sites could alter miRNA activity and/or affect the expression of their target genes. Whether sequence variation in microRNA genes are also associated with MM still unknown and thus require extensive investigations. In the present study, genomic sequences coding for the precursors of 12miRNA genes were were detected by the methods of polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) and silverstaining technique in CD 138+ bone marrow PCs from subjects with MM (n = 20), MM-derived cell lines (n= 4) and controls (n= 20). To our knowledge, this is the first evidence of miRNA mutation occurred in MM, providing insights that may contribute to the understanding of their biological roles in MM, lead to the identification of new biomarkers and altered molecular pathways of the disease. Objectives To investigate sequence variations of 12miRNA genes in multiple myeloma (MM) and to explore the clinical significance of such sequence variations in the tumorigenesis of MM.Methods Myeloma cells were obtained from patients with Multiple myeloma (n= 20) and were separated by CD138 Positive Selection Cocktail. The phenotypes of myeloma cells were identified by flow cytometry. Separate mononuclear cells from bone marrow of controls (n= 20). Culture and passage of MM-derived cell lines under appropriate conditions. Genomic DNA was extracted from all above-mentioned tissues. The 12 MicroRNA genes (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) were detected by the methods of polymeras chain reaction singles trand conformation polymorphism (PCR-SSCP) and silverstaining technique. Conventional statistics were used to test differences for significance. Both clinical features and laboratory results were analyzed simultaneously.Results The electrophoretic pattern showed a total of three variations in miR-19a, miR-19b and miRNA-335, which were observed in 3 MM cells(15%). We also found variations of miR-335 in MM-derived cell lines KM-3 and RPMI8226. However, no sequence alterations in the miRNA genes were observed in our set of controls. Statistical results showed no statistically significance of the difference between two groups. One of the three MM subjects was died, and two of them were detected mutations at the terminal stage of the disease.Discussions Through the above experimental study, we've grasped experimental skills, such as cell culture, abstraction and separation CD138+ cell. We primarily detected MicroRNA gene mutations in MM cell and MM derived cell lines. In total, we found variations of miR-335 in MM-derived cell lines KM-3 and RPMI8226 and three variations in three miRNAs, including miR-19a, miR-19b and miRNA-335, in 3 out of 20MM tissues(15%).No sequence variations were found in controls. It seems that sequence variations in miRNA genes are associated with the pathogenesis of multiple myeloma. Statistical results showed no statistically significance of the difference between two groups, we need to expand the sample size for further verification. Our experimental results suggest the possibility of a main mechanism underlying tumorigenesis. One of the three patients with such mutations was dead and two of them were in terminal stage of the disease when detected. It seems that miRNA sequence varieations are associated with progression and prognosis of MM.Conclusions A relative high frequence of miRNA gene mutation was found in MM and MM derived cell lines, which suggests possibility of a main mechanism underlying tumorigenesis. Whether sequence altered miRNA genes exist in health, we need to expand the sample size for further verification. And, mutation occurs in patients with poor prognosis; we speculate that detecting of miRNA gene mutations in MM might be benefit to evaluate the progression and prognosis of disease, which have very important oretical significance and clinic value.