The Clinical Value Of MiRNA-192-5p In Multiple Myeloma And Its Role In Pathogenesis

Multiple myeloma is a complicated malignant hematologic disease,which is characterized by abnormal clonal proliferation of plasma cells in bone marrow,and a large number of secreting monoclonal immunoglobulin and its fragments,so that the majority of MM patients have different degrees of bone,kidney and other organs damage.The incidence of MM is only inferior to that of non-Hodgkin’s lymphoma,accounting for 13% of all hematological tumors.MM mainly occurred in the elderly,mostly in the age between 50-75 years old.In addition,the ratio of male to female was 3:2.Diagnostic criteria of the International Myeloma Working Group on the formulation of multiple myeloma: the proportion of clone plasma cell in bone marrow was more than 10% and(or)biopsy confirmed the presence of a plasma cell tumor;monoclonal immunoglobulin was present in blood and urine;There were anemia,hypercalcemia,bone destruction,renal damage and immunodeficient myeloma related end organ and tissue injury.The pathogenesis of multiple myeloma is complex and mainly related to the regulation mechanism of bone marrow microenvironment,genetic mutation,DNA methylation and histone modification.In clinical treatment,although the immune regulating factor(lenalidomide,pomalidomide)or proteasome inhibitor(bortezomib,carfilzomib)and other emerging therapies can significantly improve the treatment response rate and overall survival rate,but MM still can not be cured.With the development of molecular biology technology,more and more studies have shown that non-coding RNA,especially micro RNA(miRNA),plays an important role in the pathogenesis of multiple myeloma.miRNA is a kind of short endogenous single stranded non-coding regulatory RNA molecules,which is about as large as 18 to 25 nucleotides.It can regulate the expression of target genes at the post transcriptional level by binding to the 3’ non-coding region(3’-untranslated region,3’-UTR)of specific messenger RNAs.In recent years,some studies have found that miRNA molecules were found to have abnormal expression in a variety of different tumor types.These miRNA molecules were as tumor suppressor or promoter to regulate tumor cell proliferation,migration,cell cycle and differentiation.Circulating miRNA molecules have potential as novel biomarkers.In solid tumors,such as breast cancer,the expression of circulating plasma miR-195 was increased,and the abnormal expression of miR-195 was different from that of other cancer patients and healthy people.In addition,the expression levels of circulating plasma miR-195 and let-7a in breast cancer patients were significantly lower than those in healthy controls.In hematological malignancies,circulating serum miR-128 expression was significantly higher in patients with acute lymphoblastic leukemia,but in between acute myeloid leukemia and the normal control group showed no significant difference;the expression of let-7b and miRNA-233 in serum circulating AML and ALL patients were higher than those in control group;the above results showed that circulating serum miR-128,let-7b and miRNA-233 have some reference value for the diagnosis and classification of leukemia.Based on the above,we raise the following question: whether circulating miRNA molecules have abnormal expression in the peripheral blood of patients with MM? Is abnormal expression of circulating miRNA associated with the diagnosis and prognosis of MM? What do they play role in the pathogenesis of MM? In order to solve these problems,my subject by using gene chip,fluorescence quantitative PCR,a number of statistical data analysis,flow cytometry technology is discussed in the following three parts: 1)Screening and identification of abnormal expression miRNA of multiple myeloma in serum;2)Circular miRNA molecules were screened out: the correlation analysis between the expression level of miRNA-192-5p in multiple myeloma serum in different stages of disease and clinical parameters;3)The role of miRNA-192-5p in the pathogenesis of multiple myeloma.The first part: abnormal expression of miRNA molecules in multiple myelomaSome studies have found that some of the abnormal expression of miRNA molecules in the majority of patients with cancer can be detected in the peripheral serum,and said that these miRNA molecules are circulating miRNA molecules.Circulating miRNA not only has significant tumor correlation and tissue specificity,but also has good stability.In addition,the detection of circulating miRNA molecules also has the advantages of small trauma,easy access and so on.Therefore,circulating miRNA molecule is expected to be a new non-invasive biomarker in tumor diagnosis.The main purpose of this study is to screen out the abnormal expression of miRNA molecules in peripheral serum of MM patients.First,we randomly selected three primary MM patients and three healthy controls.Then,We used a highly specific Taq Man micro RNA microarray to screen the abnormal expression of miRNA molecules in the serum.It was found that there were 6 low expression of miRNA and 25 high expression of miRNA,in which the expression of miRNA-192-5p in serum of patients with MM was obvious lower than that of miRNA molecule.We verified the chip screening results by using the quantitative PCR technique in 20 cases of primary MM patients and 20 healthy subjects and found that the expression level of miRNA-192-5p in serum of patients with MM was significantly lower than that in normal controls(p<0.01).At the same time,the expression levels of miRNA-192-5p were detected in MM LP-1 cells and normal human bone marrow CD138+ cells by using quantitative PCR technology.The results showed that the expression level of miR-192-5p in MM cell line LP-1 was significantly lower than that in CD138+ cell line,which was similar to that of MM.It has been shown that the abnormal expression of miRNA molecules in circulating serum is associated with the risk stratification and survival in patients with MM.For example,the expression of circulating miRNA-15 a,miRNA-16-1,and miRNA-17-92 has been shown to be associated with the prognosis of patients with MM.The expression level of circulating miR-15 a of stage II and III patients was significantly lower than stage I patients in ISS staging of MM;On the contrary,the expression level of miR-181 a and miR-181 b of serum was significantly increased in patients with ISS II and III;and expression levels of miR-866-5p,miR-17 and miR-18 a were higher in overall short survival of patients.Therefore,the prognosis of MM patients were stratified according to their expression level to improve the sensitivity of MM risk stratification based on ISS and fluorescence in situ hybridization(fluorescence in situ hybridization,FISH).The second part: the correlation analysis between the expression levels of miRNA-192-5p in serum of patients with multiple myeloma and clinical parameters of MMIn this study,we investigated the expression levels of miRNA-192-5p in peripheral blood of patients with MM at different stages of disease,and the correlation between miRNA-192-5p and clinical parameters.The main purpose of the experiment was to evaluate the application value of abnormal expression of miRNA-192-5p in the diagnosis of MM,the progression of disease and prognosis.First of all,the expression of miRNA-192-5p was detected in peripheral serum of 50 cases of primary MM patients,15 cases with complete remission,15 cases of clinical recurrence patients and 80 healthy subjects by using fluorescence quantitative PCR technology;Clinical parameters of blood beta 2-macroglobulin(β2-microglobulin,β2-MG),serum calcium,albumin,hemoglobin,serum creatinine,lactate dehydrogenase,M protein,blood and urine light chain and FISH were collected from MM patients in each group and the independent samples T-test and Pearson correlation were used to analyze the correlation between the expression leves of miRNA-192-5p and all above clinical parameters.Findings:(1)The expression leves of miRNA-192-5p was significantly lower in the serum of patients with primary and recurrent MM;(2)ROC curve was used to evaluate the efficacy of miRNA-192-5p in the diagnosis of MM.It was found that the area under the ROC curve was 0.65,p=0.025,and sensitivity and specificity were 77% and 56%,respectively.(3)The expression level of serum miRNA-192-5p in primary MM patients with high levels of serum 2-MG and serum creatinine was lower than that of low levels of serum 2-MG and serum creatinine.(4)The expression level of serum miRNA-192-5p in primary MM patients with low hemoglobin and albumin content was lower than that of high hemoglobin and albumin.(5)The expression level of miRNA-192-5p in serum of patients with primary MM was negatively correlated with the levels of serum Ig G,Ig A,kappa and lambda.(6)The expression level of miRNA-192-5p in serum of primary MM patients with t(4;14)and 13q14 mutation was significantly reduced.(7)Further clinical analysis of 50 patients with primary MM patients showed that the expression level of miRNA-192-5p in serum of patients with non-remission group was significantly lower than that in MM remission group.These results show that although the miRNA-192-5p is an auxiliary diagnostic standard in the diagnosis of multiple myeloma,it may be a good marker for monitoring the progression of multiple myeloma.The third part: the role of miRNA-192-5p in the pathogenesis of multiple myelomaStudies have shown that miRNA molecules play an important role in the occurrence and development of MM.For example,miR-21 can regulate MAPK/ERK signaling through SPRY2,which can mediate the growth and drug-fast of myeloma cells.Mi R-148 a can regulate cell proliferation and apoptosis by targeting CDKN1 B and PTEN.In the first two parts of the study,we found that the expression level of miRNA-192-5p was low in peripheral serum of MM patients.Moreover,the low expression level of miR-192-5p was associated with severe disease and poorer prognosis.However,the role of this molecule in the pathogenesis of MM needs further study.Therefore,this part of the experiment used the multiple myeloma cell line LP-1 cells as a model to transfect miR-192-5p mimic and over-express miR-192-5p.The biological effects of miR-192-5p on MM cells were investigated by flow cytometry.The results showed that over expression of miR-192-5p could not only induce the apoptosis of MM cells,but also significantly enhance effect of bortezomib(Bortezomib,BTZ)on apoptosis of MM cells,which is the main chemotherapeutic agent of MM.In addition,overexpression of miRNA-192-5p can inhibit the proliferation of MM cells,and the longer the time was,the more inhibition effect was significant.These results indicate that miRNA-192-5p can inhibit the proliferation of MM cells,induce the apoptosis of MM cells,and enhance therapeutic sensitivity of MM to the mainstream chemotherapeutic drug BTZ.miRNA-192-5p may act as a tumor suppressor gene in MM,and may be a potential therapeutic target for the treatment of MM.In summary,this study confirmed that the expression level of miRNA-192-5p was low in the peripheral serum of MM patients by using chip screening,fluorescence quantitative PCR Technology,and the abnormal expression of miRNA-192-5p could be used as the auxiliary diagnostic criteria of MM and the marker of disease progression.Overexpression of miR-192-5p could induce the apoptosis and inhibit the proliferation of MM cells.BTZ cells,which could enhance the apoptosis of cells.Moreover,when overexpression of miR-192-5p combined with BTZ,it can enhance the effect of BTZ on apoptosis of MM cells.