| Background: Multiple myeloma(MM)is a hematological tumor characterized by malignant proliferation of clonal plasma cells.The current annual incidence rate is 4-6/100000,which is the second highest incidence of hematological malignancies[1-5].Its clinical manifestations are anemia,renal dysfunction,hypercalcemia,bone destruction,etc.With the extensive development of autologous hematopoietic stem cell transplantation and the emergence of new drugs such as proteasome inhibitors,immunomodulators and targeted drugs,multiple The complete remission rate and long-term survival rate of myeloma [6-7]have been significantly improved.However,more than half of patients with multiple myeloma are prone to osteolytic destruction,also known as Myeloma bone disease(MBD),which is characterized by severe bone pain,fractures,osteoporosis and hypercalcemia.[8-9].Bone destruction in myeloma bone disease is usually attributed to an imbalance between an increase in osteoclast activity and a decrease in osteoblast function.Osteogenesis is mediated by differentiation of bone marrow mesenchymal stem cells(BMMSCs),which have the potential for osteogenic differentiation [10].Previous studies have shown that osteogenic differentiation of BMMSCs in multiple myeloma patients is impaired [11].Therefore,improving the osteogenic differentiation ability of MM-MSC is a key issue that can promote bone regeneration and repair.Micro RNAs(miRNAs or miRs)are highly conserved short non-coding RNA molecules that have been previously thought to be involved in many biological processes such as inflammation,embryonic development,hematopoiesis,immunity,etc.,and are regulators of cell proliferation,differentiation,organ development and metabolism [12].Many studies have shown that dysregulation of miRNA expression is a possiblepathogenesis of multiple myeloma [13].In addition,miRNA plays a key role in MSC self-renewal and differentiation [14-15].In the process of osteogenic differentiation,many studies have reported that miRNAs including miR-133 [16],miR-141[17]and miR-34 a [18] are involved in the process of osteogenic differentiation and promote the regulation of key transcription factors.Although there have been different studies on the role of microRNAs and lnc RNAs in MSC osteogenic differentiation,previous studies have been limited to single-type or single RNA,or a single type of multiple RNAs to study the role of genes,failing to systematically understanding and interpreting gene regulation related to MSC development.Therefore,this study aimed to screen the difference of circRNA,LncRNA,mRNA and microRNA expression between mesenchymal stem cells of mesenchymal stem cells and normal control mesenchymal stem cells by second-generation sequencing technology,and to draw a network diagram and to select some of the differentially expressed RNAs for preliminary validation,Clarifying the mechanisms and pathways that regulate osteogenic differentiation,laying the foundation for further comprehensive study of the mechanism of RNA regulation of mesenchymal stem cells into osteogenic differentiation.Objectives: By analyzing the differential expression of circRNA,LncRNA,mRNA and microRNA in osteogenic differentiation of myeloma patients with bone marrow disease and normal control group.Bioinformatics analysis was performed through differentially expressed microRNAs to find the most likely miRNAs and Its target gene and related validation to clarify the role of microRNA in osteogenic differentiation of myeloma bone disease MSC.Methods: This study is divided into three parts.Phase I:Differences in MSC and myeloma cell content and antigen expression in different severity of MBD patients(1)Confirmation of MBD condition: Confirming the cases of multiple myeloma patients with myeloma bone disease,excluding other underlying metabolic diseases,eliminating infectious diseases,and ruling out bone destruction caused by other diseases.(2)Specimen collection: bone marrow and peripheral blood samples of patients with multiple myeloma and myeloma bone disease were selected as experimental group,normal human bone marrow and peripheral blood samples were selected as control group,and experimental group and control group were examined by flow cytometry.And identification of myeloma cells and mesenchymal stem cells,and identification of myeloma bone disease,immunophenotyping and isolation of myeloma cells and mesenchymal stem cells,and enrichment and purification of mesenchymal stem cells.Phase 2:Circ RNA,LncRNA,mRNA,microRNA differentially screening.Extracting RNA from the experimental group and the control group separately,and pre-processing the RNA sample to perform second-generation sequencing of the RNA full transcript.According to the second-generation sequencing results,the differentially expressing circRNA,LncRNA,mRNA,and mirro RNA were analyzed.According to bioinformatics analysis,they were constructed with the target gene to adjust the network map;then,some differential expression was selected to verifying bone differentiation.Phase 3: Verification experiment(based on the results of the data filtered)(1)Alizarin red S staining and RT-q PCR was conducted to confirm the difference of osteoblast differentiation between MM-MSC and N-MSC.(2)The selected microRNA was determined,and the expression level of microRNA from MM-MSC and N-MSC was analyzed by RT-q PCR.(3)Predicting the target genes that the selected microRNAs may bind through a network database.(4)Luciferase assay to determine the target gene involved in osteogenic differentiation(5)Target gene was screened by lentiviral vector overexpression,and its protein expression level was analyzed by WB.(6)Analysis of the protein expression level of osteogenic differentiation pathway by Western Blot Verification experiment(based on the results of the data filtered)Results:1.Circ RNA,LncRNA,mRNA and microRNA were differentially expressed between MM-MSC and N-MSC.After bioinformatics analysis,miR-203a-3p.1 and miR-221-5p were selected for osteogenic differentiation assay.2.Compared with normal(N)-MSC,the osteogenic differentiation ability of MM-MSC is reduced.Further studies showed that miR-203a-3p.1 was down-regulated in N-MSCs after induction of osteoblasts,while no significant changes were observed in MM-MSCs.Down-regulation of miR-203a-3p.1 leads to increased osteogenic potential,manifested by an increase in mRNA expression levels of typical osteoblast differentiation markers,including alkaline phosphatase(ALP),osteopontin(OPN)and osteocalcin(OC).Bioinformatics and luciferase reporter assays indicate that Smad9 is a direct target of miR-203a-3p.1 in MM-MSCs.RT-q PCR and Western blot analysis showed a negative correlation between Smad9 and miR-203a-3p,and overexpression of smad9 significantly enhanced the effect of miR-203a-3p.1 inhibitor on osteoblast markers,miR-203 a The-3p.1 inhibitor regulates osteogenic differentiation of MM-MSCs by up-regulating Smad9.The Wnt3 a / ?-catenin signaling pathway was also found to be activated following m R-203a-3p.1 inhibition.3.The osteogenic differentiation ability of MBD-MSCs is reduced compared with normal(N)-MSCs.Further studies showed that miR-221-5p was down-regulated in N-MSCs after induction of osteoblasts,but no significant changes were observed in MBD-MSCs.Inhibition of miR-221-5p promotes osteogenic differentiation of MBD-MSCs.Bioinformatics,luciferase reporter gene,RT-q PCR and WB analysis indicated that smad family member 3(smad3)is a direct target of miR-221-5p in MBD-MSCs.A negative correlation was found between the expression levels of smad3 and miR-221-5p.Studies on molecular mechanisms have shown that miR-221-5p regulates osteogenic differentiation of MBD-MSCs by up-regulating smad3.Inhibition of miR-221-5p activates the PI3K/Akt/m TOR signaling pathway and increases the osteogenic differentiation capacity of MBD-MSCs.Conclusions: Circ RNA,LncRNA,mRNA,and microRNA,which are differentially expressed in MM-MSC and N-MSC.miR-203a-3p.1 plays an important role in the osteogenicdifferentiation of MM-MSC by regulating Smad9 expression,miR-221-5p Osteogenic differentiation of MBD-MSCs can be regulated by up-regulating smad3. |
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