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OBJECTIVES@#Multiple myeloma (MM) is a plasma cell malignancy occurring in middle and old age. MM is still an incurable disease due to its frequent recurrence and drug resistance. However, its pathogenesis is still unclear. Abnormal amino acid metabolism is one of the important characteristics of MM, and the important metabolic pathway of amino acids participates in protein synthesis as basic raw materials. Aminoacyl transfer ribonucleic acid synthetase (ARS) gene is a key regulatory gene in protein synthesis. This study aims to explore the molecular mechanism for ARS, a key factor of amino acid metabolism, in regulating amino acid metabolism in MM and affecting MM growth.@*METHODS@#The corresponding gene number was combined with the gene expression profile GSE5900 dataset and GSE2658 dataset in Gene Expression Omnibus (GEO) database to standardize the gene expression data of ARS. GSEA_4.2.0 software was used to analyze the difference of gene enrichment between healthy donors (HD) and MM patients in GEO database. GraphPad Prism 7 was used to draw heat maps and perform data analysis. Kaplan-Meier and Cox regression model were used to analyze the expression of ARS gene and the prognosis of MM patients, respectively. Bone marrow samples from 7 newly diagnosed MM patients were collected, CD138+ and CD138- cells were obtained by using CD138 antibody magnetic beads, and the expression of ARS in MM clinical samples was analyzed by real-time RT-PCR. Human B lymphocyte GM12878 cells and human MM cell lines ARP1, NCI-H929, OCI-MY5, U266, RPMI 8266, OPM-2, JJN-3, KMS11, MM1.s cells were selected as the study objects. The expression of ARS in MM cell lines was analyzed by real-time RT-PCR and Western blotting. Short hairpin RNA (shRNA) lentiviruses were used to construct gene knock-out plasmids (VARS-sh group). No-load plasmids (scramble group) and gene knock-out plasmids (VARS-sh group) were transfected into HEK 293T cells with for virus packaging, respectively. Stable expression cell lines were established by infecting ARP1 and OCI-MY5 cells, and the effects of knockout valyl-tRNA synthetase (VARS) gene on proliferation and apoptosis of MM cells were detected by cell counting and flow cytometry, respectively. GEO data were divided into a high expression group and a low expression group according to the expression of VARS. Bioinformatics analysis was performed to explore the downstream pathways affected by VARS. Gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) and high performance liquid chromatography (HPLC) were used to detect the valine content in CD138+ cells and ARP1, OCI-MY5 cells and supernatant of knockdown VARS gene in bone marrow samples from patients, respectively.@*RESULTS@#Gene enrichment analysis showed that tRNA processing related genes were significantly enriched in MM compared with HD (P<0.0001). Further screening of tRNA processing-pathway related subsets revealed that cytoplasmic aminoacyl tRNA synthetase family genes were significantly enriched in MM (P<0.0001). The results of gene expression heat map showed that the ARS family genes except alanyl-tRNA synthetase (AARS), arginyl-tRNA synthetase (RARS), seryl-tRNA synthetase (SARS) in GEO data were highly expressed in MM (all P<0.01). With the development of monoclonal gammopathy of undetermined significance (MGUS) to MM, the gene expression level was increased gradually. Kaplan-Meier univariate analysis of survival results showed that there were significant differences in the prognosis of MM patients in methionyl-tRNA synthetase (MARS), asparaginyl-tRNA synthetase (NARS) and VARS between the high expression group and the low expression group (all P<0.05). Cox regression model multivariate analysis showed that the high expression of VARS was associated with abnormal overall survival time of MM (HR=1.83, 95% CI 1.10 to 3.06, P=0.021). The high expression of NARS (HR=0.90, 95% CI 0.34 to 2.38) and MARS (HR=1.59, 95% CI 0.73 to 3.50) had no effect on the overall survival time of MM patients (both P>0.05). Real-time RT-PCR and Western blotting showed that VARS, MARS and NARS were highly expressed in CD138+ MM cells and MM cell lines of clinical patients (all P<0.05). Cell counting and flow cytometry results showed that the proliferation of MM cells by knockout VARS was significantly inhibited (P<0.01), the proportion of apoptosis was significantly increased (P<0.05). Bioinformatics analysis showed that in addition to several pathways including the cell cycle regulated by VARS, the valine, leucine and isoleucine catabolic pathways were upregulated. Non-targeted metabolomics data showed reduced valine content in CD138+ tumor cells in MM patients compared to HD (P<0.05). HPLC results showed that compared with the scramble group, the intracellular and medium supernatant content of ARP1 cells and the medium supernatant of OCI-MY5 in the VARS-shRNA group was increased (all P<0.05).@*CONCLUSIONS@#MM patients with abnormal high expression of VARS have a poor prognosis. VARS promotes the malignant growth of MM cells by affecting the regulation of valine metabolism.
Subject(s)
Humans , Valine-tRNA Ligase , Multiple Myeloma/genetics , Metabolomics , Amino Acids , RNA, TransferABSTRACT
OBJECTIVES@#Liver cancer is the sixth most common malignant tumor in the world. Hepatocellular carcinoma (HCC) accounts for 85%-90% of all patients with liver cancer. It possesses the characteristics of insidious onset, rapid progression, early recurrence, easy drug resistance, and poor prognosis. NIMA related kinase 2 (NEK2) is a cell cycle regulating kinases, which regulates cell cycle in mitosis. Cellular senescence is a complex heterogeneous process, and is a stable form of cell cycle arrest that limits the proliferative potential of cells. This study aims to investigate the relationship between the expression level of NEK2 and the senescence in hepatoma cells, and to explore the effect of NEK2 expression on hepatoma cell senescence and the underlying molecular mechanism.@*METHODS@#A total of 581 senescence-relevant genes were obtained from the GenAge website. The gene expression data of tumor tissues of 370 HCC patients were downloaded from the Cancer Genome Atlas database. The co-expression of NEK2 and aging-related genes was analyzed by R-package. KEGG was used to analyze the significant gene enrichment pathway of differentially expressed genes in NEK2 overexpression HEK293. The stable transfected cell lines with overexpression and knockdown of NEK2 were constructed in hepatoma cell line SMMC-7721 and HepG2, and senescence-associated β-galactosidase (SA-β-gal) staining was used to detect senescence, the cell proliferation was detected by CCK-8 method and clone formation experiment, the cell cycle was analyzed by flow cytometry, and the expression of proteins related to p53/p21, p16/Rb, and phosphatase and tensin homolog deleted on chromosome ten (PTEN)/Akt signal transduction pathway was detected by Western blotting.@*RESULTS@#There were 320 senescence related genes co-expressed with NEK2. KEGG analysis showed that the senescence signaling pathway was significantly enriched in HEK293 cells with overexpression of NEK2.Compared with SMMC-7721 or HepG2 without knockdown of NEK2, the senescent cells of SMMC-7721 and HepG2 with knockdown of NEK2 were increased, cell proliferation and clone formation were decreased significantly, the percentage of cells in G0/G1 phase was increased, the expression levels of phospho-Akt (p-Akt) and phospho-Rb (p-Rb) protein were decreased significantly, and the expression level of p16 protein was increased significantly (all P<0.05). Compared with SMMC-7721 or HepG2 transfected with blank plasmid, the senescent cells of SMMC-7721 and HepG2 overexpressing NEK2 were decreased, the cell proliferation and clone formation were increased significantly, the percentage of cells in G0/G1 phase were decreased, the expression levels of p-Akt and p-Rb protein were increased significantly, and the expression level of p16 protein was decreased significantly (all P<0.05).@*CONCLUSIONS@#NEK2 may mediate the anti-aging effect of hepatoma cells through p16/Rb and PTEN/Akt signal transduction pathways, which provides a new theoretical basis for NEK2 to promote the progress of liver cancer and a new idea for the targeting treatment for liver cancer.
Subject(s)
Humans , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Proliferation/physiology , Cellular Senescence/genetics , HEK293 Cells , Liver Neoplasms/pathology , NIMA-Related Kinases/genetics , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
Objective:To investigate the effects of silencing HOXA13 gene on the malignant phenotypes of hepatocellular carcinoma cell lines HepG2and QGY-7703,and to provide a new molecular target for the diagnosis and treatment of hepatocellular carcinoma.Methods:The interference recombinant plasmid of plent-U6-GFP/si-HOXA13 was constructed,then it was stably transfected into the HepG2and QGY-7703 cells.The interfering effects of HOXA13 gene were determined by RT-PCR and Western blotting methods.The HepG2and QGY-7703 cells transfected with HOXA13 knockdown were used as experimental group,and the HepG2and QGY-7703 cells transfected with empty plasmid were used as control group.Then the growth speed was examined by MTT assay, the cell doubling time was examined by double time assay,the colony formation ability was examined by colony formation assay,and the cell cycle was examined by flow cytometry.Results:The MTT assay results showed that compared with control group,the growth speeds of HepG2and GY-7703 cells in experimental group were significantly decreased and the cell cycles were changed;the number of HepG2and QGY-7703 cells in G1phase was increased and the number of cells in S phase was decreased.The doubling time of HepG2and QGY-7703 cells in control group and experimental group were(4.59±0.27),(4.93±0.17),(6.02±0.86),and(6.43±0.66)h, and the differences between control group and experimental group were significant(P<0.05).The colony number of HepG2and QGY-7703 cells in control group and experimental group were 264.00 ± 12.62,269.00 ± 4.55, 165.00± 10.61,and 215.00 ± 4.43,and the differences between control group and experimental group were significant(P<0.01).Conclusion:HOXA13 can increase the proliferation,enhance the clone formation,decrease the number of cells at G1phase and increase the number of cells at S phase in the hepatocellular carcinoma HepG2 and QGY-7703 cells;and it may used as a new molecular target for diagnosis and treatment of hepatocellular carcinoma.
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Objective To study the distributions of three single nucleotide polymorphisms (SNPs) in β-arrestin2 (ARRB2) which including rs3786047,rs1045280 and rs2036657 and to elucidate the relationship between these SNPs and response to methadone maintenance treatment (MMT) among heroin-dependent patients of Han ethnicity population in Hunan.Methods Han MMT patients were recruited in four random-chosen MMT clinics from Hunan province.Demographics,history of drug-use and MMT were recorded.ARRB2 SNPs were genotyped to determine the association between SNPs and response to MMT.Results Distributions of the three SNPs were in Hardy-Weinberg equilibrium in both groups (responders vs.non-responders).There was no statistical significance in the distribution frequency of genotype on rs3786047 (x2=0.486 2,P=0.784),rs1045280 (x2=1.591 9,P=0.451) and rs2036657 (x2=1.061 5,P=0.588) in ARRB2 among the responders or the non-responders.Conclusion Associations between the ARRB2 genotypes,rs3786047,rs1045280 and rs2036657,and MMT response in Han MMT patients in Hunan province did not appear.
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Objective To study the distributions of three single nucleotide polymorphisms (SNPs) in β-arrestin2 (ARRB2) which including rs3786047,rs1045280 and rs2036657 and to elucidate the relationship between these SNPs and response to methadone maintenance treatment (MMT) among heroin-dependent patients of Han ethnicity population in Hunan.Methods Han MMT patients were recruited in four random-chosen MMT clinics from Hunan province.Demographics,history of drug-use and MMT were recorded.ARRB2 SNPs were genotyped to determine the association between SNPs and response to MMT.Results Distributions of the three SNPs were in Hardy-Weinberg equilibrium in both groups (responders vs.non-responders).There was no statistical significance in the distribution frequency of genotype on rs3786047 (x2=0.486 2,P=0.784),rs1045280 (x2=1.591 9,P=0.451) and rs2036657 (x2=1.061 5,P=0.588) in ARRB2 among the responders or the non-responders.Conclusion Associations between the ARRB2 genotypes,rs3786047,rs1045280 and rs2036657,and MMT response in Han MMT patients in Hunan province did not appear.
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AIM:To examine the latent membrane protein 1(LMP1)-DNA sequence in nasopharyngeal carcinoma(NPC) and detect mRNA expression of LMP1, EBNA1, EBNA2, and to explore the relationship between EBV infectious status, expression products and NPC carcinogenesis.METHODS: LMP1 DNA was detected in NPC by PCR. Direct sequence was applied to analyze the difference between NPC-LMP1-DNA and B95-8-LMP1-DNA. mRNA expressions of LMP1, EBNA1, EBNA2 in NPC were detected by nested RT-PCR.RESULTS: LMP1 DNA existed in all 47 NPC tissues. Several single nucleotide variations were found between NPC-LMP1-DNA and B95-8-LMP1-DNA. The notable variation was the lost of XhoⅠrestriction site in NPC. Direct sequence showed 30 bp deletion in NPC. The mRNA expressions of LMP1, EBNA1 and EBNA2 in NPC were 76.6%, 80.0% and 74.5% respectively by nested RT-PCR. The expression of EBNA1 in NPC was promoted by Q promoter while the expression of EBNA1 in B95-8 was promoted by C promoter.CONCLUSION: The way of EBV involved in NPC is complex. Latent genes such as LMP1, EBNA1 and EBNA2 as well as early lytic gene BARF1 may all play certain roles in NPC carcinogenesis.
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AIM: Our study focused on investigation of tissue specific regulatory activity of the newly cloned promoter: PLUNC-p with driving enhanced green fluorescent protein ( EGFP ). METHODS: Transgenic Xenopus Laevis system was applied.RESULTS: The green fluorescence protein directed under PLUNC-p was expressed strictly in branchial arches and epidermis of Xenopus Laevis embryos while CMV promoter showed ubiquitous regulation characteristic.CONCLUSION: PLUNC-p is able to direct epithelia specific expression of EGFP . This property of PLUNC-p might raise the possibility that lead target genes to express tissue-specifically.