Gene Mutation and Overexpression of Newly Diagnosed Multiple Myeloma Patients / 中国实验血液学杂志

OBJECTIVE@#To analyze the characteristics of gene mutation and overexpression in newly diagnosed multiple myeloma (NDMM) patients.@*METHODS@#Bone marrow cells from 208 NDMM patients were collected and analyzed. The gene mutation of 28 genes and overexpression of 6 genes was detected by DNA sequencing. Chromosome structure abnormalities were detected by fluorescence in situ hybridization (FISH).@*RESULTS@#Gene mutations were detected in 61 (29.33%) NDMM patients. Some mutations occurred in 5 or more cases, such as NRAS, PRDM1, FAM46C, MYC, CCND1, LTB, DIS3, KRAS, and CRBN. Overexpression of six genes (CCND1, CCND3, BCL-2, CCND2, FGFR3, and MYC) were detected in 83 (39.9%) patients, and cell cycle regulation gene was the most common. Single nucleotide polymorphisms (SNP) changes were detected in 169 (81.25%) patients, the TP53 P72R gene SNP (70.17%) was the most common. Abnormality in chromosome structure was correlated to gene overexpression. Compared to the patients with normal chromosome structure, patients with 14q32 deletion showed higher proportion of CCND1 overexpression. Similarly, patients with 13q14 deletion showed higher proportion of FGFR3 overexpression, whereas patients with 1q21 amplification showed higher proportion of CCND2, BCL-2 and FGFR3 overexpression.@*CONCLUSION@#There are multiple gene mutations and overexpression in NDMM. However, there is no dominated single mutation or overexpression of genes. The most common gene mutations are those in the RAS/MAPK pathway and the genes of cyclin family CCND are overexpression.

The role of licorice DXS knockout and overexpression in glycyrrhizic acid biosynthesis / 药学学报

1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is a rate-limiting enzyme involved in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for terpenoid precursor biosynthesis. DXS plays an essential role in glycyrrhizic acid (GA) biosynthesis. Based on our previous transcriptome study, there was a negative correlation between DXS expression and GA content. Therefore, we explored the regulatory role of DXS in GA biosynthesis using both gene overexpression and gene knockout in a hairy root culture system. DXS was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MN158121). A plant binary expression vector pCA-DXS was constructed by a gene fusion method. The sgRNA sequence was designed based on the first exon of DXS to construct the gene editing vector pHSE-DXS. Hairy roots overexpressing or knocking out DXS were generated through an Agrobacterium-mediated method with licorice hypocotyls as explants. Wild-type hairy roots and negative control hairy roots containing empty plasmids were also evaluated. UPLC was used to determine the GA content in each licorice hairy root line. Results showed that the content of GA in the hairy root group knocking out DXS was significantly higher than that in the wild-type and negative control groups, while in the hairy root group overexpressing DXS was significantly lower, suggesting that DXS plays a negative role in GA biosynthesis. This study provides a foundation for determining the function of DXS in terpenoid metabolism and for further establishment of a molecular regulatory network of GA biosynthesis.

Ferulate 5-hydroxylase gene (F5H) regulation of glycyrrhizic acid biosynthesis determined by gene overexpression and knockout / 药学学报

Ferulate 5-hydroxylase (F5H) is a key enzyme involved in the phenylpropane metabolism pathway. Based on our previous transcriptome sequencing study, F5H played a negative regulatory role in glycyrrhizic acid (GA) biosynthesis. Therefore, in this study we cloned the F5H gene and investigated its regulatory effect on GA accumulation through gene overexpression and knockout. F5H was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MK882511). A plant binary expression vector pCA-F5H was constructed by inserting F5H into pCAMBIA1305.1 at Spe I and Bgl II sites. The sgRNA sequences were designed based on the first exon of F5H. The CRISPR/Cas9 gene editing vector pHSE-F5H was constructed by inserting F5H sgRNA into pHSE401 at two Bsa Ⅰ sites. PCA-F5H and pHSE-F5H were transfected into Agrobacterium tumefaciens ATCC15834, which was used to induce hairy root overexpressing or knocking out F5H with licorice hypocotyl as explants. At the same time, wild type and negative control hairy roots were also generated. UPLC was used to assay the GA content in different hairy root lines, and results showed that the GA content in hairy root lines knocking out F5H was significantly higher, whereas in hairy root lines overexpressing F5H GA content was lower than that in the wild-type and negative control. In this work, through a reverse genetics strategy, the negative regulatory effect of F5H on GA biosynthesis was confirmed through gene overexpression and knockout. This work will lay a foundation for further elucidation of the molecular regulatory network of GA biosynthesis.

Effect of neurogenin 3 overexpression on the differentiation of human umbilical cord mesenchymal stem cells into insulin-producing cells / 解剖学报

Objective To investigate whether the overexpression of neurogenin 3 ( Ngn3 ) can promote the induced differentiation of human umbilical cord mesenchymal stem cells ( HUCMSCs) into insulin-producing cells (IPGs). Methods HUCMSCs were isolated and cultured, identified by flow cytometry, and the differentiation potential was identified by adipogenesis and osteogenesis induction. HUCMSCs were induced into IPCs in different stages, including a low glucose induction stage and a high glucose induction stage. The experiment was divided into two groups, the control group was induced by the above scheme, while the experimental group was additionally infected the lentivirus overexpression vector carrying the target gene Ngn3 on the 6th day of the same induction process. After induction, the changes of cell structure were observed by electron microscope ; mRNA and protein was collected and Real-time PCR and Western blotting were used to compare the expressions of insulin and musculoaponeurotic fibrosarcoma oncogene homolog A Mafa in the two groups ; medium supernatant was collected and C-peptide content was determined by ELISA. Results HUCMSCs were successfully isolated with positive expression of CD 105 and CD90 and negative expression of CD34 and CD45, which had adipogenic and osteogenic differentiation ability. Then, HUCMSCs were induced into IPCs by stage induction, and the cells expressed Mafa and insulin positively. Ngn3 was overexpressed in the experimental group during the induction. After induction, electron microscopy showed that the cell structure was more mature in the experimental group. The expression levels of insulin and Mafa in the experimental group were significantly higher than those in the control group. During the induction process, the amount of C-peptide secreted by the experimental group was higher than that of the control group. Conclusion Lentivirus-mediated Ngn3 overexpression improves the differentiation efficiency of HUCMSCs into IPCs.

Effects of 3β-HSD gene silence or overexpression on DEHP induced oxidative damage in MCF-7 cells / 中华劳动卫生职业病杂志

Objective@#To study the oxidative damage of di- (2-ethylhexyl) phthalate (DEHP) on MCF-7 cells, and to investigate the effects of 3β-hydroxysteroid dehydrogenase (3β-HSD) gene silence or overexpression on DEHP-induced oxidative damage.@*Methods@#MCF-7 cells, 3β-HSD gene silencing cells and 3β-HSD gene overexpression cells were treated with different doses of DEHP (0,0.05,0.1,0.2,0.4,0.8 mmol/L) for 24h, then intracellular oxidative damage index such as MDA, SOD, GSH, GSH-PX were detected, DNA repair gene hOGG1, hMTH1 mRNA expression were tested by Q-PCR, hOGG1, hMTH1 protein expression were detected by western blot.@*Results@#After MCF-7 cells were treated by DEHP, MDA levels increased; SOD activity, GSH content, GSH-PX activity decreased, hOGG1 and hMTH1 mRNA expression levels increased, hOGG1 and hMTH1 protein expression levels increased, the differences were statistically significant when compared with control (P<0.05 or P<0.01) . In 3β-HSD gene silencing cells which were treated by DEHP, when compared with the same dose group of MCF-7 cells, MDA content increased, SOD activity, GSH content, GSH-PX activity decreased, hOGG1 and hMTH1 mRNA expression levels decreased, hOGG1 and hMTH1 protein expression levels decreased, the difference were statistically significant (P<0.05 or P<0.01) . In 3β-HSD gene overexpression cells which were treated by DEHP, when compared with the same dose group of MCF-7 cells, MDA content decreased; SOD activity, GSH content, GSH-PX activity increased, of hOGG1 and hMTH1 mRNA expression levels increased, hOGG1 and hMTH1 protein expression levels increased, the difference were statistically significant (P<0.05 or P<0.01) .@*Conclusion@#DEHP could cause oxidative damage in MCF-7 cells, induce the changes of related genes and proteins, 3β-HSD plays an antioxidant role in the process of DEHP ox-idative damage.

Over-expression of ROBO4 reduces permeability of human renal glomerular endothelial cells in high glucose medium / 解放军医学杂志

Objective To investigate the effect of over-expression of ROBO4 on permeability of human renal glomerular endothelial cells (HRGECs) in high glucose medium.Methods HRGECs infected with recombinant lentiviral vector ROBO4 were cultured in high glucose or low glucose medium in vitro.The protein levels of ROBO4 and ARF6 in each group were detected by Western blotting.The endothelial permeability was measured by the effiux of fluorescein isothiocyanate-dextran (FITC-Dextran)permeated through the monolayer endothelial cells using Transwell cell model system.The cell viability after lentivirus transfection was measured by CCK8 assay.Results The transfection rate of lentiviruses in HRGECs reached 80％ 72h after,and obvious overexpression of ROBO4 protein was in transformed cells compared with the empty vector group (P＜0.05).The lentivirus-mediated ROBO4 transfection did not affect cell viability of HRGECs.Compared with the low glucose group,the expression of ROBO4 increased obviously after 12h,but declined after 24h (P＜0.05),and reached to minimun after 72h (P＜0.05).On the contrary,the expression of ARF6 increased after 12h,and the increase reached to the maximum after 72h (P＜0.05).Furthermore,the vascular permeability increased gradually after 24h,and reached to the maximum after 72h (P＜0.05) in high glucose group.Compared with the empty vector group,the over-expression of ROBO4 inhibited the expression of ARF6 significantly,and the FITC-Dextran permeability reduced obviously.Conclusion Over-expression of ROBO4 may significantly enhance the barrier functions of HRGEC in high glucose medium,and ROBO4 activation may be a potential therapeutic approach in diabetic nephropathy.

Report on recent insight into molecular genetic alterations and target therapy in T-cell acute lymphoblastic leukemia in the 54th ASH annual meeting / 白血病·淋巴瘤

T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematologic malignancy associated with poor prognosis.Increasing data regarding to alteration of gene expression signatures of oncogenes and tumor suppressors involved in the pathogenesis of T-ALL and the major mechanisms of T-cell transformation may contribute to define the biological markers for treatment response and prognosis,and has important clinical implications.In this review,advance knowledge concerning the characteristics of early T-cell precursor ALL,the alteration of TAL1 and NOTCH1 related genes and target inhibiton effects based on these alterations from 2012 the 54th ASH annual meeting ars summarized.

Expression of Nanog Gene with the Mediation of Ientiviral Vector in Mouse ES Cells / 中国生物工程杂志

In order to further study mouse embryonic stem cells(ES cells),lentiviral vector PLL-IRES-Nanog-Neo was constructed.Mouse ES cells overexpressed nanog by mediation of lentiviral were cultured on mouse fetal fibroblast feeders after 2 weeks under G418 media and examined according to gowth characteristics. Results were showed that 918 bp nanog fragments were expressed in mouse ES cells mediated by lentiviral vector PLL-IRES-Nanog-Neo,mouse nanog-ES cells were taken on mass-like image and positve with alkaline phosphatase staining and Oct4 and SSEA1 immunocytochemistry under no LIF condition in the media. It is concluded that mouse ES cells Elevated nanog gene expression by mediation of lentiviral were constucted and cultured.

From mouse to man,from gene to clinic:Progress in heart research using genetically modified mouse models / 西安交通大学学报(医学版)

Since the 1990s,models of genetically modified mice(gene knockout,mutation or transgene overexpression) have been increasingly used in heart research.The findings from this class of murine models have contributed significantly to a number of research breakthroughs achieved in the last 15 years.Currently,the number of genetically modified mouse strains has been increasing steadily.These models that targeted a family or a functionally relevant group of proteins have formed systemic tools for research use.The database of mouse cardiac phenotype has been established.Meanwhile,the research methodology and strategic approach exploring mouse cardiac phentypes at molecular to in vivo levels have been mature.It is expected that the use of this class of genetically targeted mouse models will become increasingly important in future research.