| Multiple Myeloma(MM)is the second most common hematologic tumor,accounting for 13% of hematologic tumors,and is characterized by the accumulation of malignant plasma cells infiltrating the bone marrow.Malignant plasma cells proliferate and produce secretory proteins that implicate multiple parts of the body,resulting in typical clinical features such as anemia,hypercalcemia,renal insufficiency,and osteolysis.These injuries can lead to pain,infection and an increased risk of fractures,so quality of life is limited.Early diagnosis and treatment of MM is therefore expected to improve patient survival,prognosis and quality of life.Genomic instability is a major genetic event which deal the progression from normal plasma cells to MM.This leads to the complex and variable genotype of MM tumor with high heterogeneity.Researchers make progress in treatment as more research into MM pathogenesis is done: from initial supportive therapies to current high-dose therapies including the clinical application of new drugs such as chemotherapy drugs and proteasome inhibitors and immunosuppressants.While,MM remains an incurable disease due to its genetic heterogeneity and development of resistance in the microenvironment,despite advances in treatment.As a result,the concept of precision medicine is increasingly being proposed for better analysis and targeted treatment of patients.This concept,based on gene sequencing,has been increasingly used to investigate the pathogenesis and prognostic role of differential gene molecules in MM.MiRNAs are non-coding RNAs that influence a range of biological activities,such as cell cycle,differentiation,apoptosis,migration and the like,by regulating the translation and stability of messenger RNAs(m RNAs).Functional studies confirm that miRNAs dysregulation leads to the development of many cancers in which miRNAs may act as tumor suppressors or oncogenes,and specific miRNA and miRNA-targeted molecules are increasingly used in the diagnosis and treatment of clinical cancers.miRNAs have been found to play a key role in the development of MM.In this study,we used bioinformatics to screen for differentially expressed miRNAs in MM and further investigate their clinical value as biomarkers for the diagnosis of MM patients and their pathogenesis.Part I:Objective:The primary purpose of this part was to identify miRNAs differentially expressed in MM patients’ plasma and evaluate their potentials as biomarkers for MM diagnosis.Methods:Serum from the GEO database was first downloaded and a series of datasets from healthy and MM patient plasma were selected for genome-wide sequencing as samples.Data was then normalized and analyzed using R language to obtain a list of miRNAs for differential expression of MM patients compared to healthy controls group,and the relative expression of miRNAs from MM patients compared to healthy controls was measured by quantitative real-time polymerase chain reaction(q RT-PCR).Statistical analysis of the data was performed using SPSS software,and P<0.05 was considered statistically significant.Then the feasibility of differential miRNAs as potential diagnostic biomarker was assessed by mapping ROC curves and correlating them with clinically significant indicators.Results:1.Through high-throughput screening,we identified six differentially expressed miRNAs as candidates: hsa-miR-18a-5p,hsa-miR-30e-5p,hsa-miR-142-3p,hsa-miR-3198,hsa-miR-3679-5p,and hsa-miR-3937.2.Using fluorescence quantitative PCR,we validated the relative expression levels of hsa-miR-142-3p,hsa-miR-3198,hsa-miR-3679-5p,and hsa-miR-3937 in plasma.This miRNAs in MM patient plasma were higher than healthy controls,and the results were consistent with high-throughput data predictions,with statistically significant differences.Finally,we selected hsa-miR-142-3p with stable and significant differences in expression as a signature molecule for further study.3.By drawing the ROC curve and further analyzing the correlation,it was shown that hsa-mir-142-3p,an indicator of MM diagnosis,had a sensitivity of 80% and specificity of 70% at 2.883 and was positively correlated with the proportion of abnormal plasma cells in bone marrow.Conclusion:The relative expression of hsa-miR-142-3p in MM patient plasma was higher than that of the normal controls,the miRNA was positively associated with abnormal plasma cells in bone marrow from clinical diagnostic criteria.ROC curve analysis also indicated that hsa-miR-142-3p may be a potential novel biomarker for MM with high sensitivity and specificity.Part IIObjective: To investigate the expression level of hsa-miR-142-3p in MM cell lines and its biological role.Predict its upstream and downstream genes and explore mechanism of action at the cellular level.Methods:First,mononuclear cells in healthy human bone marrow were used as a control group,and MM cell lines U266 and RPMI-8226 were used as experimental groups.Expressions of hsa-miR-142-3p in two groups of cells were verified by q RT-PCR.Hsa-miR-142-3p was transfected into cells by lentivirus,hsa-miR-142-3p stable cell lines were constructed,and their effects on cell viability was determined by cck-8.The miRNA target genes were then predicted from the database,then the ce RNA networks were constructed,and core downstream target genes and their pathways were enriched and analyzed.Results1.Empirically,hsa-miR-142-3p was characterized by high expression in U266 cells compared to controls,but not in PRMI-8226 cells.2.Hsa-miR-142-3p stable cell line constructed by lentivirus showed higher proliferation than normal cell group and negative control group.3.Bioinformatics analysis revealed that the central target protein of hsa-miR-142-3p was PI3K-Akt,which is the pathway most gene regulated,and Competing endogenouse RNA(ce RNA)revealed a network of interactions between hsa-miR-142-3p and its upstream and downstream genes.Conclusion:Hsa-miR-142-3p is relatively highly expressed in U266 cells and promotes cell proliferation,affecting cell viability.Its core proteins,ROCK2 and TGFBR1,may influence cell proliferation through the PI3K-Akt pathway. |
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