| BackgroundMultiple myeloma(MM)is a malignant disease in which terminal differentiated plasma cells accumulate in bone marrow.MM is a complex hereditary hematological malignancy.It is characterized by abnormal infiltration of clonal plasma cells in bone marrow and is the second most common hematological malignancy after non-Hodgkin’s lymphoma.Compared with normal bone marrow plasma cells,there is obvious molecular heterogeneity in gene changes and gene expression changes.These changes in expression can be driven either directly by the above gene changes or indirectly by changes in signals,such as changes in external stimuli mediated by changing microenvironments.Both of them can directly act on gene expression or its mediators.The prominent example of the latter is microRNA(microRNA).MicroRNA is a non-protein-coded RNA that regulates the stability and translation of RNA.MicroRNAs inhibit the expression of target genes after transcription,and under specific conditions,such as in different cell types,specific transcripts are found to up-regulate gene expression in eukaryotes.A single microRNA is usually involved in the regulation of hundreds of RNA.MicroRNAs are small single-stranded non-coding RNAs that play a role in post-transcriptional regulation of RNA silencing and gene expression.More and more studies have shown that microRNAs play an important role in tumorigenesis.Understanding the regulatory mechanism of microRNAs in this gene regulatory network will help to elucidate the complex biological processes in malignant tumors.MicroRNAs regulate about 30% of human genes and affect cell growth,differentiation,proliferation,metabolism and death.Regarding the treatment of MM,although there are many treatments,MM is still incurable and the 5-year survival rate is 40%.Therefore,there is an urgent need to find new treatments to improve the prognosis of MM.Changes in gene copy number,chromosome translocation,mutation,transcription and epigenetics during the development of MM lead to serious disorders in the transcription of microRNAs.This,in turn,leads to abnormalities in the translation of RNA and signal transduction in cells.With regard to multiple myeloma,a number of global microRNA genomic studies have been published that affect gene expression,biological correlation and survival,suggesting that there may be a link between the pathogenesis of myeloma and molecular subentities in high-risk populations based on specific chromosomal aberrations or gene expression.After microRNAs are secreted from cells to plasma or serum,they have high stability and specificity.At present,it has been found that the expression of some microRNAs in plasma or serum of different cancer patients increases or decreases,which is consistent with the expression of microRNAs in cells or bone marrow.Because of the advantages of convenient sampling,less invasiveness and less trauma to patients,there are some plasma or serum M.IRNA has been used as a molecular marker in the diagnosis,treatment or prognosis of tumors.At present,there is a certain understanding of the role of microRNAs in MM.The microRNA-17-92 gene clusters include microRNAs-17,microRNAs-18,microRNAs-19 a,microRNAs-19b-1,microRNAs-20,microRNAs-32 and microRNAs-92-1.These genes are important microRNAs activated by Myc,which are closely related to the prognosis of MM and are highly expressed in high-risk groups of MM.Relevant studies suggest that the expression of microRNA-17-3p is high in MM,and it can promote the proliferation of MM cells,inhibit apoptosis and play an oncogene role.However,it is not clear how microRNA-17-3p can promote cell proliferation and inhibit apoptosis,and there are few studies on its other roles and mechanisms in MM cells.The study of plasma microRNA-17-3p in patients with MM is rare.In order to explore the role of plasma microRNAs in MM and its mechanism,we conducted the following experiments: 1)In this study,we collected bone marrow samples of MM patients and made microarray of microRNAs to understand the difference in the expression of microRNAs,and found that the expression of microRNAs-17-3p in the bone marrow of MM patients was higher than that of normal people,the difference was statistically significant;2)We detected the expression of microRNAs-17-3p in the plasma of MM patients and its diagnosis with MM.The relationship between interruption,treatment and prognosis;3)To detect the expression of microRNA-17-3p in M M cell lines and its biological function;4)To verify the role of microRNA-17-3p in vivo experiments by xenotransplantation model in nude mice.Part I To study the expression profile of microRNAs in bone marrow samples of multiple myelomaResearch objectives: The purpose of this study is to detect the expression profiles of microRNAs in CD138 + cells of multiple myeloma patients and to select microRNAs for further study in abnormally expressed microRNAs.Research methods: Total RNA was extracted from bone marrow and quantitatively analyzed.Poly(A)polymerase of RNA was tailed,linked and biotin labeled.Then microarray hybridization,washing and scanning were performed.Finally,abnormal expression of microRNAs was detected by image acquisition and data analysis.Research results: The results showed that the expression of microRNAs in CD138 + cells of bone marrow of 7 normal persons and 21 newly diagnosed and untreated patients with multiple myeloma was analyzed by SAM.A total of 6631 microRNAs were detected and 336 abnormal microRNAs were detected.Among them,61 microRNAs were lower in newly diagnosed and untreated MM than in normal persons,and 275 microRNAs were higher in newly diagnosed and untreated MM than in normal persons.The top 10 differentially expressed microRNAs in MM patients and normal controls were up-regulated in MM.They were: has-mi-130b-3p,hsa-mi-664b-3p,hsa-mi-193b-3p,hsa-mi-6501-3p,hsa-mi-1972,hsa-mi-17-3p,hsa-mi-2467-3p,hsa-mi-4461,hsa-mi-29c-5p,hsa-mi-4299.Because some scholars have found that microRNA-17-3p is highly expressed in MM cells,which is consistent with the results of this study,we chose microRNA-17-3p as the research object to further understand its role in MM.Research conclusions: There are miRNAs with differential expression between multiple myeloma patients and normal people,and miR-17-3p is of great research value.Part II Detection of the expression of plasma microRNA-17-3p in patients with MM and its relationship with the diagnosis and prognosis of MMResearch objectives: The purpose of this study is to detect the levels of plasma microRNA-17-3p in patients with multiple myeloma and normal controls,compare the differences between them,analyze the differences of plasma microRNA-17-3p expression between newly diagnosed and untreated MM,complete remission of MM,relapsed and refractory MM and normal controls,and the relationship between the levels of plasma microRNA-17-3p in newly diagnosed and untreated MM and clinical diagnostic indicators,and the expression of plasma microRNA-17-3p.Whether it is related to the prognosis of patients,etc.Research methods: Real-time PCR was used to detect the difference in the expression of plasma microRNA-17-3p in different groups;correlation analysis was used to analyze the correlation between the expression of plasma microRNA-17-3p and the proportion of plasma cells and the content of serum M protein in newly diagnosed and untreated patients with multiple myeloma;and the accuracy of plasma microRNA-17-3p as a molecular marker for the diagnosis of multiple myeloma was evaluated by the receiver working curve(ROC curve).Research results: The results showed that the content of plasma microRNA-17-3p in newly diagnosed and untreated MM patients was higher than that in normal persons,and the results were statistically significant;the content of plasma microRNA-17-3p in patients with complete remission of MM was not significantly different from that in normal persons;the content of plasma microRNA-17-3p in relapsed and refractory MM patients was higher than that in normal persons,and the results were statistically different.Moreover,plasma microRNA-17-3p was positively correlated with plasma cells and serum M protein in newly diagnosed and untreated MM patients,and the results were statistically significant.The results of ROC curve suggested that plasma microRNA-17-3p might be used as a diagnostic index for MM.Research conclutions: It can be concluded that plasma microRNA-17-3p in MM patients is related to the diagnosis and prognosis of MM patients,and may be a potential biomarker of MM.Part III Detection of the expression of microRNA-17-3p in MM cell lines and its biological functionResearch objectives: The purpose of this study is to study the expression characteristics of microRNA-17-3p in MM cells and to determine its function in MM cell lines,so as to predict and verify the target gene of microRNA-17-3p,and to verify the role of the target gene in MM cell lines.Research methods: Real-time PCR was used to detect the relative expression of microRNA-17-3p in multiple myeloma(MM)cell lines,CCK-8 was used to detect the effect of microRNA-17-3p on the proliferation of MM cell lines,soft agar was used to detect the effect of microRNA-17-3p on the cloning ability of MM cell lines,Western blot was used to detect the expression of protein,and double luciferase reporter gene was used to verify the target gene of microRNA-17-3p.Research results: The results showed that the expression of microRNA-17-3p was high in M M cell lines,which was consistent with that in bone marrow and plasma.Through CCK-8,soft-agar and flow cytometry,we found that microRNA-17-3p promoted MM cell proliferation,inhibited apoptosis and had the ability of clone formation.The software predicted the target genes of microRNA-17-3p,and screened out the 10 most probable genes with high scores.After overexpressing or knocking down the expression of microRNA-17-3p,the expression of these 10 genes was detected by qRT-PCR.The results indicated that the expression of LMLN was contrary to that of microRNA-17-3p,and might be the target gene of microRNA-17-3p.Western blot analysis showed that the expression of LMLN protein was contrary to that of microRNA-17-3p,suggesting that microRNA-17-3p negatively regulated the expression of LMLN protein.By double luciferase reporter gene experiment,it was found that microRNA-17-3p binds to the 3’UTR end target of LMLN,and the two do not bind after mutation,suggesting that LMLN is the target gene of microRNA-17-3p.After overexpression or knockdown of LMLN,the effect of LMLN on MM cell lines was observed.Experiments such as CCK-8 and soft-agar showed that LMLN inhibited the proliferation of MM cells,contrary to the effect of microRNA-17-3p.LMLN was confirmed to be the target gene of microRNA-17-3p,and LMLN played the role of tumor suppressor gene in MM cells.Research conclutions: It can be concluded that microRNA-17-3p plays an oncogene role in M M cells,LMLN is the target gene of microRNA-17-3p,and LMLN plays an anti-oncogene role in M M cells.Mir-17-3p may be a potential therapeutic target for MM.Part IV Nude mouse xenotransplantation model to verify the role of microRNA-17-3p in vivo experimentsResearch objectives: The purpose of this part is to further study the role of microRNA-17-3p in nude mice xenotransplantation model and to verify the role of microRNA-17-3p in vivo.Research methods: Lentiviruses were packaged with over-expression and low-expression of microRNA-17-3p and control plasmids,and then transfected into MM cell lines to interfere with the expression of microRNA-17-3p.Real-time PCR was used to detect the expression of microRNA-17-3p in MM cell lines,and CCK-8 was used to detect the effect of microRNA-17-3p on the proliferation of MM cell lines.Then a large number of infected MM cell lines were amplified and injected into nude mice subcutaneously.When the tumors grow to a certain extent,the mice are executed and weighed.The tumors are stripped out and weighed.The differences of tumors volume,tumors weight and mice weight among groups are compared.Research results: The results indicated that the over-expression of microRNA-17-3p and the low-expression of microRNA-17-3p and the control plasmid-encapsulated virus were transfected into MM cell line NCI-H929,then the transfected NCI-H929 cells were subcutaneously transfected into nude mice to observe and record the volume growth of subcutaneous transplanted tumors.Finally,the tumors were weighed and measured to compare the differences between the groups.The results showed that the tumors in the overexpression group were generally larger and heavier than those in the control group,while the tumors in the knockdown group were smaller and lighter than those in the control group,with statistical significance.Research conclutions: It can be concluded that mir-17-3p plays an oncogene role in vivo and can promote the growth of tumors.This study preliminarily explored the role of microRNA-17-3p in MM,which provides a possibility for finding biomarkers for diagnosis and prognosis of MM,and is expected to become a therapeutic target for MM.Conclutions1.The relative expression of microRNA-17-3p in bone marrow and plasma of patients with multiple myeloma is higher than that of normal persons,which may be a potential plasma molecular marker for the diagnosis of multiple myeloma.2.The expression of microRNA-17-3p in multiple myeloma cell lines is higher than that in normal human bone marrow mononuclear cells,and it plays an oncogene role in multiple myeloma cell lines.3.LMLN is the target gene of microRNA-17-3p,and microRNA-17-3p negatively regulates the expression of LMLN at both the level of mRNA and protein.LMLN plays an anti-oncogene role in multiple myeloma cell lines,contrary to microRNA-17-3p,which further proves that LMLN is the target gene of microRNA-17-3p.4.Animal experiments showed that microRNA-17-3p acted as an oncogene in mice,which was consistent with the experimental results of cell biology. |
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