Quantification of Pathogenicity Genes Expression in Pectobacterium carotovorum by qRT-PCR

Abstract Gastric cancer is concerned as the second leading cause of tumor-associated death worldwide after lung tumors and is specified as one of the most common malignant cancers. Given the increasing importance of HOX genes in gastric cancer research, this study was aimed at exploring the expression profile of HOTAIR, HOXC13, HOXC10, HOXC13-AS, and HOXC‑AS3 in gastric cancer. To achieve this goal, 30 pairs of tumor and normal margin samples were assessed for these genes expression analyses via qRT-PCR. result we found that, the HOTAIR, HOXC13, and HOXC10 expression was significantly increased in cancer tissue samples in comparison with adjacent normal tissue samples, P<0.01. Moreover, there were significant positive correlations between the expressions of genes studied: HOXC‑AS3 and HOXC10 (r=0.52, P<0.003), HOXC13-AS and HOXC13 (r=0.57, P<0.001), HOTAIR and HOXC‑AS3 (r=0.39, P<0.03), HOTAIR and HOXC13-AS (r=0.36, P<0.05). The HOXC13 and HOXC10 expression exhibited a significant relationship with both distant metastasis and perineural invasion (metastasis: P<0.014, r=0.443 and P<0.0041, r=0.51 perineural invasion: P<0.025, r=0.41 and P<0.0017, r=0.55). The HOXC13-AS displayed a significant relationship with the sex factor so that in females the expression of this gene was higher than males (P<0.030, r=0.4). Our findings suggest that the expression of HOXC genes and HOTAIR lncRNA increased through gastric tumor progression and thus they possibly participate in malignant transformation and gastric carcinoma metastasis.

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mTORC1/NF-κB axis controls amino acid catabolism by regulating the expression of the key enzymes in human hepatocytes

10.21203/rs.2.24282/v1 ◽

2020 ◽

Author(s):

Meng Zhang ◽

Yuting Fu ◽

Yuhao Chen ◽

Yuze Ma ◽

Zhixin Guo ◽

...

Keyword(s):

Amino Acid ◽

Mrna Level ◽

Building Blocks ◽

Human Hepatocytes ◽

Acid Decarboxylase ◽

Amino Acid Catabolism ◽

Qrt Pcr ◽

Mechanistic Target Of Rapamycin ◽

Intracellular Content ◽

Genes Expression

Abstract Background In addition to serving as building blocks for protein synthesis, amino acids also provide energy and precursors that are used by cells through catabolism. Mechanistic target of rapamycin complex 1 (mTORC1) is a central coordinator of cellular metabolism. However, little is known regarding the function of mTORC1 in amino acid catabolism. The aims of this study were to explore the mechanism by which mTORC1 controls the conversion of glutamate to α-ketoglutarate and ornithine to putrescine, and mTORC1 regulates the expression amino acid catabolism-related genes in hepatocyte. Methods HL-7702 hepatocytes were treated with glutamate, ornithine, rapamycin or SC75741, alone or in combination; the plasmids pRNAT-U6.1/Neo-shRaptor and pIRES2-EGFP-Rheb were transfected into HL-7702 cells to silencing Raptor or overexpressing Rheb . The intracellular content of glutamate, oxaloacetate, α-ketoglutaric acid, and aspartic acid, and the intracellular level of aspartate aminotransferase (AST), ornithine decarboxylase (ODC), glutamate dehydrogenase (GDH), and glutamic acid decarboxylase (GAD) were measured by ELISA. The concentrations of intracellular ornithine and putrescine were measured by HPLC. The mRNA level of amino acid catabolism-related genes was detected by qRT-PCR, and the protein level of mTORC1 and NF-κB was investigated by western blot. Results Our data showed that rapamycin inhibits the utilization of glutamate and ornithine in HL-7702 hepatocytes. mTORC1 regulates the expression of AST and ODC through the transcription factor NF-κB in response to glutamate or ornithine. Further, inactivated mTORC1 by Raptor silencing downregulated the expression of AST , ODC , GDH and GAD , while enhanced mTORC1 by Rheb overexpression upregulated NF-κB activation and the indicated genes expression in hepatocytes. Inhibited NF-κB by inhibitor SC75741 decreased the AST , ODC , GDH , and GAD expression. Conclusions Our results demonstrate that mTORC1 regulates amino acid catabolism by inducing the expression of AST , ODC , GDH , and GAD , which is mediated by NF-κB. This finding constitutes a novel mechanism by which amino acid catabolism is regulated in hepatocytes.

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Genome-wide Identification and Transcriptional Profiling of New Cobra-like Genes in Bread Wheat (Triticum Aestivum)

10.21203/rs.3.rs-796690/v1 ◽

2021 ◽

Author(s):

Muhammad Zaheer ◽

Shoaib Ur Rehman ◽

Sultan Habibullah Khan ◽

Shahmeer Shahid ◽

Awais Rasheed ◽

...

Keyword(s):

Drought Stress ◽

Transcriptional Profiling ◽

Database Search ◽

Qrt Pcr ◽

Genome Wide ◽

Genes Expression ◽

Karyotype Structure ◽

Functional Investigation ◽

Gene Ontologies ◽

Membrane Cell

Abstract Background: The COBL gene encodes a plant-specific glycosylphosphatidylinositol (GPI)-anchored protein. Recently identified COBRA genes supposed as a key regulator of the orientation of cell expansion in the root indicating that Cobra gene family members are likely to be important new players at the plasma membrane-cell wall interface. Methods and Results: By performing a database search and domain prediction, we identified five genes named as TaCOBL 1, TaCOBL 2, TaCOBL 3, TaCOBL 4 and TaCOBL 5, and selected for further analysis. Chromosomal locations of each gene were drawn on karyotype. Structure of genes, promoter analysis and phylogenetic analysis were done using bioinformatics tools and databases. Set of novel SNPs were also predicted. Gene ontologies were analyzed, and pathways involving cobra genes were predicted. Whole genes of 3kb to 4kb were successfully amplified. Five set of primers were designed targeting TaCOBL 1, TaCOBL 2, TaCOBL 3 TaCOBL 4 and TaCOBL 5 genes. Expression of COBL genes were checked in root and shoot by using qPCR. The qRT-PCR revealed that expression TaCOBL genes can be regulated under abiotic stress such drought stress. Conclusion: The comprehensive annotation and expression profiling of COBL genes performed in this study enhanced our understanding and these genes were found to play a significant role in drought stress. Our findings lay the groundwork for further functional investigation of COBL genes mechanism.

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The Effect of 5-Aza-2′-Deoxycytidine in Combination to and in Comparison with Vorinostat on DNA Methyltransferases, Histone Deacetylase 1, Glutathione S-Transferase 1 and Suppressor of Cytokine Signaling 1 Genes Expression, Cell Growth Inhibition and Apop

International Journal of Hematology-Oncology and Stem Cell Research ◽

10.18502/ijhoscr.v14i1.2360 ◽

2020 ◽

Author(s):

Masumeh Sanaei ◽

Fraidoon Kavoosi ◽

Zahra Esmi

Keyword(s):

Histone Deacetylase ◽

Dna Methyltransferases ◽

Cytokine Signaling ◽

Cell Growth Inhibition ◽

Glutathione S Transferase ◽

Suppressor Of Cytokine Signaling ◽

Histone Deacetylase 1 ◽

Qrt Pcr ◽

Genes Expression ◽

Apoptotic Effect

Background: Aberrant methylation and histone deacetylation of tumor suppressor genes (TSGs) are the most epigenetic alterations involving in tumorigenesis. Overexpression of DNA methyltransferases (DNMTs) and histone deacetylase 1 (HDAC1) have been reported in several cancers. The reversion of hypermethylation and deacetylation by epi-drugs such as 5-aza-2′-deoxycytidine (5-AZA-CdR) and vorinostat (SAHA) can restore normal expression of TSGs. Previously, we reported that 5-AZA-CdR and valproic acid (VPA) can inhibit DNMT1 in hepatocellular carcinoma (HCC). The aim of this study was to investigate the effect of 5-AZA-CdR in combination to and in comparison with SAHA on DNMT1, DNMT3a, DNMT3b, histone deacetylase 1 (HDAC1), glutathione S-transferase 1 (GSTP1) and suppressor of cytokine signaling 1 (SOCS1) genes expression, cell growth inhibition and apoptotic induction in hepatocellular LCL-PI 11 cell line. Materials and Methods: The cells were treated with 5-AZA-CdR and SAHA and then MTT assay, cell apoptosis assay and Real-time quantitative RT-PCR (qRT-PCR) were done. Results: Both agents indicated significant inhibitory and apoptotic effect (P< 0.001). The apoptotic effect of SAHA was more than that of 5-Aza-CdR. The result of qRT-PCR indicated that 5-Aza-CdR decreased DNMT1, DNMT3a, DNMT3b and increased GSTP1and SOCS1 genes expression and SAHA decreased HDAC1 and increased GSTP1 and SOCS1 genes expression significantly. Maximal apoptosis and genes expression were seen with combined treatment. Conclusion: 5-AZA-CdR and SAHA down-regulated DNMT1, DNMT3a, DNMT3b, and HDAC1 and up-regulated GSTP1 and SOCS1 gene expression by which inhibited cell viability and induced apoptosis, suggesting that they could be used in the treatment of HCC.

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Quantification of NMDAR Subunit Genes Expression by qRT-PCR

Methods in Molecular Biology - NMDA Receptors ◽

10.1007/978-1-4939-7321-7_2 ◽

2017 ◽

pp. 83-92

Author(s):

Emilie Pallesi-Pocachard

Keyword(s):

Qrt Pcr ◽

Genes Expression ◽

Nmdar Subunit

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Targeted Therapy of AML1/ETO-Positive AML Cells in Experimental Model.

Blood ◽

10.1182/blood.v112.11.3358.3358 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3358-3358

Author(s):

Michal Zapotocky ◽

Julia Starkova ◽

Ester Mejstrikova ◽

Karel Smetana ◽

Jan Trka

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Cell Line ◽

Target Genes ◽

Myeloid Cells ◽

Surface Markers ◽

Qrt Pcr ◽

Hla Dr ◽

Genes Expression ◽

Leukaemic Cells

Abstract In t(8;21) acute myeloid leukaemia (AML), the leukaemogenesis is supposed to be promoted by interference with expression of AML1 target genes. Repressor complex associated with AML1/ETO fusion protein recruits class I histone deacetylases (HDAC). Valproic acid (VPA) was found to have an extensive effect on AML blasts, via inhibition of class I HDAC. It was shown previously that VPA treatment disrupts the AML1/ETO-HDAC1 complex from AML1 promoter thus leading to apoptosis at different cell lines. However, there is still lack of in-depth morphological and immunophenotypical proof of the hypothesized restoration of differentiation after treatment with VPA. Although very little is known about AML1 target genes, it was shown that AML1 protein binds to promotor region of IL-3 and PU.1 and regulates their expression. There are also reports demonstrating possible candidate target genes of AML1 transcription factor (BPI, IGFBP7). We aimed to characterize the differentiation effect of VPA on AML1/ ETO-positive leukaemic cells and to determine the expression pattern of selected genes. Kasumi-1 (AML1/ETO-positive) cell line and MV4-11 (MLL/AF4-positive) cells were treated with VPA (0,5 mM and 1,0 mM concentrations) and examined by flow cytometry, morphological evaluation and qRT-PCR. Paediatric patients’ bone marrow samples (AML1/ ETO-positive) from the time of diagnosis were taken for in vitro experiment. We optimised the method of patients’ samples cultivation using conditioned medium with cytokines (IL6, FLT3 ligand, TPO, SCF) and we treated leukaemic cells with VPA. We examined immunophenotype and cell cycle of these samples after 24 and 48 hours of cultivation. We show that treatment of AML1/ETO-positive myeloid cells with HDACi VPA resulted in decreased expression of early myeloid progenitor antigens (CD33/34/117) and increased expression of antigens typical for differentiated myeloid cells (CD11a/11b). Cell morphology, nucleolar morphology and cytochemistry evaluation indicated the maturation process and decreased proliferation activity. All these phenomenons were not observed in control MLL/AF4-positive myeloid cells. We quantified the level of expression of selected genes (PU.1, C/EBPalpha, BPI, IGFBP7) and we observed the increase of genes expression after VPA treatment in AML1/ETO-positive cells. In VPA treated AML1/ETO-positive cells PU.1 increased expression 6.2 times (p<0.001), C/EBPalpha 3 times (p<0.001), BPI 2.6 times (p<0.001) and IGFBP7 7 times (p<0.001). Different situation occurred in MLL/AF4-positive cell line, where PU.1 conversely decreased expression 2.5 times (p=0.01), IGFBP7 decreased 2.4 times (p=0.01) and the expression of C/EBPalpha and BPI remained unchanged (p=0.32 and p=0.75) after VPA treatement. Samples recovered from patientś bone marrow reacted differently to VPA therapy. The first patient increased the expression of HLA-DR (p=0.04) and CD11b (p=0.02) and at the same time decreased the expression of CD38 (p=0.006) and CD117 (p=0.03) surface markers, which represents the signs of differentiation. The second patient’s sample presented with expression of HLA-DR (p=0.1) unchanged; expression of CD11b (p=0.05), CD38 (p=0.002), CD117 (p=0.04) and CD34 (p=0.02) decreased. The concentration of VPA used in our experiment seems to have strong cytotoxic effect on the other patient’s leukaemic cells, as they passed into the apoptosis, the amount of 10% of persistent blast cells was not sufficient for the analysis. Cell cycle examination confirmed the results of the experiment with cell lines; patientś samples treated with VPA decreased the proliferation and the number of cells undergoing apoptosis increased. Taken together, we provide a valid evidence of differentiation of AML1/ETO-positive cell line, demonstrated by flow cytometry and confirmed morphologically. This process goes hand in hand with the increase of the repressed genes expression as measured by qRT-PCR in contrast with non-CBF leukemic cells. Patients’ data are not completely in line with those experimental findings, as the flow cytometry analysis showed uneven changes in surface markers expression pattern. VPA induces differentiation and apoptosis; therefore it seems to be a promising drug in treatment of AML/ETO-positive paediatric AML. Supported by Grant Agency of Charles University 71/2006.

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Cell cycle genes expression in human corneal endothelium: study by microarray and qRT-PCR

Acta Ophthalmologica ◽

10.1111/j.1755-3768.2008.414.x ◽

2008 ◽

Vol 86 ◽

pp. 0-0

Author(s):

A PIPPARELLI ◽

G THURET ◽

S PISELLI ◽

M PEOC'H ◽

S ACQUART ◽

...

Keyword(s):

Cell Cycle ◽

Corneal Endothelium ◽

Cell Cycle Genes ◽

Human Corneal Endothelium ◽

Qrt Pcr ◽

Genes Expression

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Association of High Cardiovascular Fitness and the Rate of Adaptation to Heat Stress

BioMed Research International ◽

10.1155/2018/1685368 ◽

2018 ◽

Vol 2018 ◽

pp. 1-6

Author(s):

Małgorzata Żychowska ◽

Alicja Nowak-Zaleska ◽

Grzegorz Chruściński ◽

Ryszard Zaleski ◽

Jan Mieszkowski ◽

...

Keyword(s):

Heat Stress ◽

Cardiorespiratory Fitness ◽

Adaptive Response ◽

Interleukin 10 ◽

Cardiovascular Fitness ◽

Blood Samples ◽

Qrt Pcr ◽

Genes Expression ◽

Before And After ◽

Adaptation To Heat

This study aimed to compare changes in genes expression associated with inflammation and apoptosis in response to heat stress caused by sauna between people with varying cardiorespiratory fitness levels. We hypothesis that high cardiorespiratory level caused higher positive changes after four weeks of sauna bathing. Blood samples were taken at rest before and after the first and last sauna sessions and 48 hours after the last sauna session and used to assay HSP70 (HSPA1A), HSP27 (HSPB1), interleukin 6 (IL6), and interleukin 10 (IL10) genes expression in blood with quantitative real-time qRT-PCR. Overall, small decreases in rest values of HSPA1A and IL6 mRNA, increase in HSPB1 mRNA, and a significant increase in IL10 mRNA were observed after four weeks of exposure to heat stress. Our findings suggest that an adaptive response to heat stress (an anti-inflammatory response) occurs faster in people with higher cardiorespiratory fitness.

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Cancer V-ATPase Expression Signatures: A Distinctive Balance of SubunitCIsoforms in Esophageal Carcinoma

10.1101/489856 ◽

2018 ◽

Author(s):

Juliana do Couto Vieira Carvalho dos Santos ◽

Pedro Nicolau Neto ◽

Evenilton Pessoa Costa ◽

Frederico Firme Figueira ◽

Tatiana de Almeida Simão ◽

...

Keyword(s):

Expression Patterns ◽

Ion Homeostasis ◽

Proton Pumps ◽

Molecular Signatures ◽

Gene Expression Microarray ◽

Expression Microarray ◽

Qrt Pcr ◽

Oncomine Database ◽

Gene Expression Microarray Data ◽

Genes Expression

ABSTRACTV-ATPases are hetero-oligomeric enzymes consisting of 14 subunits and playing key roles in ion homeostasis and signaling. Differential expressions of these proton pumps have been implicated in carcinogenesis and metastasis. To elucidate putative molecular signatures underlying these phenomena, we evaluated the V-ATPase genes expression in Esophageal Squamous Cell Carcinoma (ESCC) using gene expression microarray data and extended the analysis to other cancers the Oncomine database. Among all differentially expressed genes, those encoding the V-ATPase C isoforms exhibited striking expression patterns validated by qRT-PCR in paired ESCC samples and respective normal surrounding tissues. Structural modeling of C2a isoform uncovered motifs for oncogenic kinases in an additional peptide stretch, and an actin-biding domain downstream to this sequence. This study reveals multi-cancer molecular signatures in the V-ATPase structure and establishes that the expression ratios of its subunits/isoforms could form a conformational code that controls the pump regulation and interactions related to tumorigenic events.

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Restoration of mRNA Expression of Solute Carrier Proteins in Liver of Diet-Induced Obese Mice by Metformin

Frontiers in Endocrinology ◽

10.3389/fendo.2021.720784 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jiamei Le ◽

Yi Fu ◽

Qiuqin Han ◽

Xindong Wei ◽

Houlin Ji ◽

...

Keyword(s):

Insulin Sensitivity ◽

High Fat Diet ◽

Obese Mice ◽

Carrier Proteins ◽

High Fat ◽

Qrt Pcr ◽

Genes Expression ◽

Solute Carrier ◽

Insight Into

Metformin (MET), the most common medicine for type 2 diabetes (T2DM), improves insulin sensitivity by targeting the liver, intestine and other organs. Its impact on expression of the solute carrier (Slc) transporter genes have not been reported in the mechanism of insulin sensitization. In this study, we examined Slc gene expression in the liver and colon of diet-induced obese (DIO) mice treated with MET by transcriptomic analysis. There were 939 differentially expressed genes (DEGs) in the liver of DIO mice vs lean mice, which included 34 Slc genes. MET altered 489 DEGs in the liver of DIO mice, in which 23 were Slc genes. Expression of 20 MET-responsive Slc DEGs was confirmed by qRT-PCR, in which 15 Slc genes were altered in DIO mice and their expressions were restored by MET, including Slc2a10, Slc2a13, Slc5a9, Slc6a14, Slc7a9, Slc9a2, Slc9a3, Slc13a2, Slc15a2, Slc26a3, Slc34a2, Slc37a1, Slc44a4, Slc51b and Slc52a3. While, there were only 97 DEGs in the colon of DIO mice with 5 Slc genes, whose expression was not restored by MET. The data suggest that more genes were altered in the liver over the colon by the high fat diet (HFD). There were 20 Slc genes with alteration confirmed in the liver of DIO mice and 15 of them were restored by MET, which was associated with improvement of insulin sensitivity and obesity. The restoration may improve the uptake of glucose, amino acids, mannose, fructose, 1,5-anhydro-D-glucitol and bumetanide in hepatocytes of the liver of DIO mice. The study provides new insight into the mechanism of metformin action in insulin sensitization and obesity.

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