The Role Of AIMP1 Gene In The Development Of Multiple Myeloma And Its Mechanism

Background and Objective:Multiple Myeloma(MM)is a common malignant tumor of the blood system.The main pathological feature is the malignant proliferation of plasma cells in the bone marrow with bone damage,which is manifested by extensive osteolytic lesions and unexplained pathological fractures.As one of the protagonists in the bone marrow microenvironment,osteoclasts are activated abnormally due to the interaction between a large number of myeloma cells and the bone marrow microenvironment,which triggers a series of symptoms related to bone damage.One important potential oncogene AIMP(amino acid-tRNA synthetase-interacting multifunctional protein)was screened out to be associated with poor prognosis of patients with multiple myeloma(MM)using chip dataset.This study aims to investigate the relationship between AIMP gene expression on the proliferation of multiple tumor cells and osteoclast differentiation in vitro;explore the mechanism of proliferation of MM cells and osteoclast differentiation induced by AIMP;meanwhile to search for a traditional Chinese medicine that can inhibit the proliferation of multiple myeloma cells during MM progression.Methods:1.Firstly,a large number of bone marrow samples of MM patients after initial diagnosis and high-dose chemotherapy were collected and analyzed using the Affymetrix U Plus.gene chip to obtain a genome-wide expression profile.The expression of one gene AIMP and the survival rate of MM patients were analyzed.The relationship between the prognosis and the expression of AIMP in the serum of normal and MM patients was measured using enzyme-linked immunosorbent assay(ELISA).2.The AIMP1-targeted cDNA or shRNA virus solution was used to infect myeloma wild-type cells H929 and OCI,and puromycin was used to screen for stable expressing cell lines.Western blot(WB)was used to verify the AIMP1 expression in stable transfer cells,and the proliferation of transfected cells and control cells was detected by MTT colorimetry.3.The recombinant plasmid pET32a-AIMP1 was transformed into Escherichia coli BL for amplification,and expression was induced by isopropyl-?-D-thiogalactopyranoside(IPTG).The target protein AIMP1 was purified using a nickel column.4.A mouse-derived AIMP1 overexpression plasmid was transiently transfected into the mouse-derived macrophage Raw264.7.Western blot(WB)was used to verify the expression of AIMP1 in the cells after transient transduction.Tartrate-resistant acid phosphatase(TRAP)staining method was used to identify the differentiation of osteoclasts before and after transient induction,and the expression of regulatory protein NFATc1,which affects osteoclast differentiation,was detected by WB.Purified protein AIMP1 at different concentrations was used to induce the differentiation of mouse-derived macrophages Raw264.7 into osteoclasts.Tartrate-resistant acid phosphatase(TRAP)staining was used to identify the differentiation of osteoclasts before and after induction.The expression of regulatory protein NFATc1,which affects osteoclast differentiation,was detected by WB.5.Homo sapiens Long non-coding RNA data sequencing analysis technology service was provided by company.After extraction total RNA from samples,removing rRNA,and constructing a chain-specific library,the Illumina X Ten/NovaTM platform was used for Pair-End sequencing.A total of 89.87 Gb Raw Data was collected.The quality control program processes the raw data and obtains Clean Data for subsequent data analysis.6.MTT colorimetric method was used to detect the effect of purified protein AIMP1 at different concentrations on the proliferation rate of ARP1,CAG,H929,and OCI in MM wild-type cells.WB was used to detect the expression of MAPK pathway-related proteins in MM wild-type cells H929 and OCI.A model of xenograft tumor mice was constructed and the purified protein AIMP1 was administrated to intervene.The tumor diameter was measured with a vernier caliper.When the tumor diameter reached 20 mm,the mice were killed,the tumor was peeled off,weighed,and frozen after taking pictures.7.Taking advantages of the high-throughput protein-chip platform to find a single protein or protein family that interacts with the target protein AIMP1,the protein ANP32A that can interact with AIMP1 was selected,and the co-immunoprecipitation(Co-IP)experiment was used to verify the interaction between AIMP1 and ANP32A in the cells.8.MTT colorimetric method was used to detect the effect of bufalin at different concentrations on the proliferation of MM cells.Results:1.Gene chip results showed that the expression of AIMP1 gene was significantly higher in MM patients than in monoclonal gammopathy of undetermined significance(MGUS)and normal plasma(NP)(P<0.0001.).In the results of the Assessment of Proteasome Inhibition for Extending Remission(APEX)clinical trial,the OS of patients with high expression of AIMP1 was significantly lower than that of patients with low expression.Kaplan Meier survival analysis results showed that in patients treated with Total Therapy 2(TT2),patients with high AIMP1 expression have lower overall survival(OS)and event-free survival(EFS)than patients with lower expression.The level of AIMP1 in the serum of MM patients was higher than that of normal people by ELISA test.2.The WB experiments verified that,AIMP1 shRNA and AIMP1 cDNA were transfected into MM cells and cell models of AIMP1 stable knockdown and overexpression was constructed successfully.The results of MTT detection of cell proliferation showed that compared with control cells,the proliferation of MM cells knocked down by AIMP1 was significantly inhibited,while the proliferation of MM cells overexpressed by AIMP1 was significantly promoted.3.SDS-PAGE test results showed that the protein sample obtained after purification and elution using a nickel column had a molecular weight which was basically the same as the molecular weight of AIMP1 without obvious banding.According to the gray value,the purity of AIMP1 was about 95.9%.4.The WB experiments confirmed that,mouse AIMP1 overexpression plasmid was transiently transfected into mouse macrophage Raw.cells,and AIMP1 was overexpressed at the protein level.Raw264.7 cells were stimulated with a receptor activator of nuclear factor ?B ligand(RANKL)50 ng/?l and a macrophage colony-stimulating factor(M-CSF)10 ng/?l.After co-stimulation,Raw264.7 cells overexpressing AIMP1 had stronger differentiation ability than the control group.The number and size of osteoclasts were significantly higher than those of the control group.WB results showed that NFATc1 was significantly higher in Raw.cells overexpressing AIMP1 than in control.The results of tartrate-resistant acid phosphatase(TRAP)staining showed that the purified protein AIMP1 could induce the differentiation of mouse macrophage Raw264.7 into osteoclasts.With the increase of protein concentration,the number and size of differentiated osteoclasts also increased as well as the expression of NFATc1 in macrophages.5.According to the results of transcriptome sequencing analysis of differentially expressed genes,GO enrichment and KEGG enrichment analysis,it can be found that in cell lines that overexpress and knock down AIMP1 and add exogenous AIMP1,MAPK,NF-?B and other signaling pathways are significantly enriched.6.MTT colorimetric detection showed that the purified protein AIMP1 at different concentrations could promote the proliferation of MM wild-type cells ARP1,CAG,H929,and OCI in vitro in a dose-dependent manner;WB detection showed that the purified protein AIMP1 at different concentrations could promote the expression of ERK1/2 and its phosphorylated proteins in the MAPK pathway of MM wild-type cells H929 and OCI.7.Taking advantage of the high-throughput protein-chip platform,we found the protein ANP32A that interacts with the target protein AIMP1,and confirmed it by the Co-IP results.8.MTT colorimetric detection showed that bufalin at different concentrations could inhibit the proliferation of MM wild-type and AIMP1-overexpressed cells.Conclusions:The high expression of AIMP1 is associated with the prognosis of relapse in patients with MM;In vitro,AIMP1 overexpression can promote MM cell proliferation and promote RANKL-mediated osteoclast differentiation;interaction between AIMP1 and ANP32A may be related to the proliferation of MM cells.