scholarly journals Determination of CYP450 Expression Levels in the Human Small Intestine by Mass Spectrometry-Based Targeted Proteomics

2021 ◽  
Vol 22 (23) ◽  
pp. 12791
Author(s):  
Alexia Grangeon ◽  
Valérie Clermont ◽  
Azemi Barama ◽  
Fleur Gaudette ◽  
Jacques Turgeon ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Small Intestine ◽  
Protein Expression ◽  
Mrna Levels ◽  
Targeted Proteomics ◽  
Tissue Samples ◽  
Human Organ ◽  
Expression Levels ◽  
Protein Levels ◽  
Human Small Intestine

The human small intestine can be involved in the first-pass metabolism of drugs. Under this condition, members of the CYP450 superfamily are expected to contribute to drug presystemic biotransformation. The aim of this study was to quantify protein expression levels of 16 major CYP450 isoforms in tissue obtained from nine human organ donors in seven subsections of the small intestine, i.e., duodenum (one section, N = 7 tissue samples), jejunum (three subsections (proximal, mid and distal), N = 9 tissue samples) and ileum (three subsections, (proximal, mid and distal), N = 9 tissue samples), using liquid chromatography tandem mass spectrometry (LC-MS/MS) based targeted proteomics. CYP450 absolute protein expression levels were compared to mRNA levels and enzyme activities by using established probe drugs. Proteins corresponding to seven of sixteen potential CYP450 isoforms were detected and quantified in various sections of the small intestine: CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, CYP3A5 and CYP4F2. Wide inter-subject variability was observed, especially for CYP2D6. CYP2C9 (p = 0.004) and CYP2C19 (p = 0.005) expression levels decreased along the small intestine. From the duodenum to the ileum, CYP2J2 (p = 0.001) increased, and a trend was observed for CYP3A5 (p = 0.13). CYP3A4 expression was higher in the jejunum than in the ileum (p = 0.03), while CYP4F2 expression was lower in the duodenum compared to the jejunum and the ileum (p = 0.005). CYP450 protein levels were better correlated with specific isoform activities than with mRNA levels. This study provides new data on absolute CYP450 quantification in human small intestine that could improve physiologically based pharmacokinetic models. These data could better inform drug absorption profiles while considering the regional expression of CYP450 isoforms.

scholarly journals Correlation between Protein and mRNA Abundance in Yeast

1999 ◽  
Vol 19 (3) ◽  
pp. 1720-1730 ◽  
Author(s):  
Steven P. Gygi ◽  
Yvan Rochon ◽  
B. Robert Franza ◽  
Ruedi Aebersold
Keyword(s):  
Protein Expression ◽  
Capillary Liquid Chromatography ◽  
Metabolic Labeling ◽  
Mrna Levels ◽  
Mrna Transcript ◽  
Expression Levels ◽  
Protein Levels ◽  
Liquid Chromatography Tandem Mass ◽  
Steady State Levels ◽  
2D Gel

ABSTRACT We have determined the relationship between mRNA and protein expression levels for selected genes expressed in the yeastSaccharomyces cerevisiae growing at mid-log phase. The proteins contained in total yeast cell lysate were separated by high-resolution two-dimensional (2D) gel electrophoresis. Over 150 protein spots were excised and identified by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein spots were quantified by metabolic labeling and scintillation counting. Corresponding mRNA levels were calculated from serial analysis of gene expression (SAGE) frequency tables (V. E. Velculescu, L. Zhang, W. Zhou, J. Vogelstein, M. A. Basrai, D. E. Bassett, Jr., P. Hieter, B. Vogelstein, and K. W. Kinzler, Cell 88:243–251, 1997). We found that the correlation between mRNA and protein levels was insufficient to predict protein expression levels from quantitative mRNA data. Indeed, for some genes, while the mRNA levels were of the same value the protein levels varied by more than 20-fold. Conversely, invariant steady-state levels of certain proteins were observed with respective mRNA transcript levels that varied by as much as 30-fold. Another interesting observation is that codon bias is not a predictor of either protein or mRNA levels. Our results clearly delineate the technical boundaries of current approaches for quantitative analysis of protein expression and reveal that simple deduction from mRNA transcript analysis is insufficient.

scholarly journals Yiqi Huoxue Recipe Improves Liver Regeneration in Rats after Partial Hepatectomy via JNK Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Wen-cong Li ◽  
Su-xian Zhao ◽  
Wei-guang Ren ◽  
Hui-juan Du ◽  
Yu-guo Zhang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Liver Regeneration ◽  
Partial Hepatectomy ◽  
Underlying Mechanism ◽  
Mrna Levels ◽  
Protective Effects ◽  
Jnk Pathway ◽  
Expression Levels ◽  
Protein Levels ◽  
Mrna And Protein Expression

The liver is the only visceral organ that exhibits a remarkable capability of regenerating in response to partial hepatectomy (PH) or chemical injury. Improving liver regeneration (LR) ability is the basis for the favourable treatment outcome of patients after PH, which can serve as a potential indicator for postoperative survival. The present study aimed to investigate the protective effects of Yiqi Huoxue recipe (YQHX) on LR after PH in rats and further elucidate its underlying mechanism. A two-thirds PH rat model was used in this study. Wistar rats were randomly divided into four groups: sham-operated, PH, YQHX + PH, and Fuzheng Huayu decoction (FZHY) + PH groups. All rats were sacrificed under anesthesia at 24 and 72 h after surgery. The rates of LR were calculated, and the expression levels of cyclin D1 and c-jun were determined by immunohistochemical staining. The protein levels of p-JNK1/2, JNK1/2, p-c-jun, c-jun, Bax, and Bcl-2 were detected by Western blotting, while the mRNA levels of JNK1, JNK2, c-jun, Bax, and Bcl-2 were examined by real-time polymerase chain reaction (RT-PCR). At the corresponding time points, YQHX and FZHY administration dramatically induced the protein levels of p-JNK1/2 compared to the PH group p<0.05, while FZHY + PH group showed prominently increase in p-JNK1/2 protein levels compared to the YQHX + PH group p<0.05. A similar trend was observed for the expression levels of p-c-jun. Compared to the PH group, YQHX and FZHY markedly reduced the mRNA and protein expression levels of Bax at 24 h after PH, while those in the FZHY + PH group decreased more obviously p<0.05. Besides, in comparison with the PH group, YQHX and FZHY administration predominantly upregulated the mRNA and protein expression levels of Bcl-2 at 24 and 72 h after PH p<0.05. In conclusion, YQHX improves LR in rats after PH by inhibiting hepatocyte apoptosis via the JNK signaling pathway.

scholarly journals Bovine gonadotrophs express anti-Müllerian hormone (AMH): comparison of AMH mRNA and protein expression levels between old Holsteins and young and old Japanese Black females

2019 ◽  
Vol 31 (4) ◽  
pp. 810 ◽  
Author(s):  
Onalenna Kereilwe ◽  
Hiroya Kadokawa
Keyword(s):  
Protein Expression ◽  
Mrna Levels ◽  
Expression Levels ◽  
Black Females ◽  
Protein Levels ◽  
Gonadotrophin Secretion ◽  
Old Japanese ◽  
Mrna And Protein Expression ◽  
Gonadotrophin Releasing Hormone ◽  
Polymerase Chain

Anti-Müllerian hormone (AMH) is secreted from ovaries and stimulates gonadotrophin secretion from bovine gonadotroph cells. Other important hormones for endocrinological gonadotroph regulation (e.g. gonadotrophin-releasing hormone, inhibin and activin) have paracrine and autocrine roles. Therefore, in this study, AMH expression in bovine gonadotroph cells and the relationships between AMH expression in the bovine anterior pituitary (AP) and oestrous stage, age and breed were evaluated. AMH mRNA expression was detected in APs of postpubertal heifers (26 months old) by reverse transcription-polymerase chain reaction. Based on western blotting using an antibody to mature C-terminal AMH, AMH protein expression was detected in APs. Immunofluorescence microscopy utilising the same antibody indicated that AMH is expressed in gonadotrophs. The expression of AMH mRNA and protein in APs did not differ between oestrous phases (P&gt;0.1). We compared expression levels between old Holsteins (79.2±10.3 months old) and young (25.9±0.6 months old) and old Japanese Black females (89.7±20.3 months old). The APs of old Holsteins exhibited lower AMH mRNA levels (P&lt;0.05) but higher AMH protein levels than those of young Japanese Black females (P&lt;0.05). In conclusion, bovine gonadotrophs express AMH and this AMH expression may be breed-dependent.

scholarly journals The mRNA of TCTP functions as a sponge to maintain homeostasis of TCTP protein levels in hepatocellular carcinoma

2020 ◽  
Vol 11 (11) ◽  
Author(s):  
Wei Liu ◽  
Qi Liu ◽  
Beilei Zhang ◽  
Zhibin Lin ◽  
Xia Li ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Protein Expression ◽  
Combined Treatment ◽  
Migration And Invasion ◽  
Protein Accumulation ◽  
Mrna Levels ◽  
Expression Levels ◽  
Protein Levels ◽  
Hcc Cells ◽  
In Cells

AbstractTranslationally controlled tumor protein (TCTP) is a highly conserved protein that accumulated in the tumorigenesis of various malignancies. Despite the important role of TCTP protein in tumor progression, the precise function and underlying mechanistic regulation of TCTP mRNA in hepatocellular carcinoma (HCC) remain unclear. In this study, we found that TCTP protein was overexpressed in HCC patients but TCTP mRNA expression levels were reversed. TCTP knockout HCC cells exhibited attenuated abilities of proliferation, migration, and invasion. The knockdown of TCTP by siRNA effectively reduced TCTP mRNA levels but not protein levels in HCC cells. Moreover, although the constitutive knockdown of TCTP inhibited almost 80% of TCTP protein expression levels in tumors of wildtype transgenic mice (TCTP KD/WT), partial restoration of TCTP protein expression was observed in the tumors of heterozygous TCTP mice (TCTP KD/TCTP±). The blockage of mRNA synthesis with ActD stimulated TCTP protein expression in HCC cells. In contrast, combined treatment with ActD and CHX or MG132 treatment alone did not lead to the TCTP protein accumulation in cells. Furthermore, following the introduction of exogenous TCTP in cells and orthotopic HCC tumor models, the endogenous TCTP protein did not change with the recombinational TCTP expression and kept a rather stable level. Dual-luciferase assays revealed that the coding sequence of TCTP mRNA functions as a sponge to regulate the TCTP protein expression. Collectively, our results indicated that the TCTP mRNA and protein formed a closed regulatory circuit and works as a buffering system to keep the homeostasis of TCTP protein levels in HCC.

scholarly journals Potential prognostic value of PD-L1 and NKG2A expression in Indonesian patients with skin nodular melanoma

2021 ◽  
Author(s):  
Ridwan Dwi Saputro ◽  
Hanggoro Tri Rinonce ◽  
Yayuk Iramawasita ◽  
Muhammad Rasyid Ridho ◽  
Maria Fransiska Pudjohartono ◽  
...  
Keyword(s):  
Target Genes ◽  
Therapy Response ◽  
Mrna Levels ◽  
Independent Predictive Factor ◽  
Clinicopathologic Characteristics ◽  
Tissue Samples ◽  
Nodular Melanoma ◽  
Expression Levels ◽  
Melanoma Tissue

Abstract Objective Biomarker mRNA levels have been suggested to be predictors of patient survival and therapy response in melanoma cases. This study aimed to investigate the correlations between the mRNA expression levels of PD-L1 and NKG2A in melanoma tissue and clinicopathologic characteristics and survival in Indonesian patients with primary nodular melanoma. Results Thirty-two tissue samples were analyzed. Upregulated PD-L1 was associated with shorter overall survival (hazard ratio: 2.930; 95% confidence interval: 1.011–8.489, p = 0.048) compared with patients with normoregulated PD-L1. A significant positive correlation was found between the expression levels of PD-L1 and NKG2A (rs: 0.768, p < 0.001). However, no clinicopathologic associations with PD-L1 and NKG2A mRNA levels were statistically proven. Comparison with other studies suggested that the choice of adjuvant therapy and the presence of TILs affect the prognostic role of PD-L1 expression. NKG2A was not proven to be an independent predictive factor but may become an adjunct target for therapy. The strong correlation between PD-L1 and NKG2A suggests that anti-PD-1 and anti-NKG2A agents could be effective in patients with PD-L1 upregulation. The combination of the mRNA levels of these two target genes may provide a novel prognostic and therapeutic direction for immunotherapy.

Protein Expression Patterns of Candidate Genes Involved in Apoptosis and Cell Cycle Control in Genetic Subgroups of Chronic Lymphocytic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2796-2796
Author(s):  
Christof Schneider ◽  
Dirk Winkler ◽  
Meike Loddenkemper ◽  
Alexander Krober ◽  
Peter Lichter ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Expression ◽  
Cyclin D1 ◽  
Candidate Genes ◽  
Cell Lines ◽  
Normal Karyotype ◽  
Lymphocytic Leukemia ◽  
Expression Levels ◽  
Protein Levels ◽  
Atm Protein

Abstract Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a highly variable clinical course. Genomic aberrations (such as 13q−, 11q−, +12q, 17p−) can be found in about 80% of CLL cases and define pathogenic as well as clinical subgroups. Similarly, the mutational status of the variable region of the immunoglobulin heavy-chain gene (VH) identifies subgroups with different maturation stage and clinical outcome. In this study protein expression levels of candidate genes involved in cell cycle and apoptosis control (p53, ATM, Akt1, PI3-K, p21, p27, cdk4, Cyclin-D1, D2, D3, Bax, Bcl-2, Apaf-1, Smac, XIAP, cIAP2, survivin) were examined by Western Blotting. A total of 87 CLL cases derived from the subgroups with 11q- (n=22), 17p-/p53 mutation (n=18), +12q (n=24), 13q- (n=8) or a normal karyotype (n=15) were studied and compared to the cell lines EHEB and JVM-2. VH-mutation status was available for 65 cases (unmutated n=48, mutated n=17). Due to limitations in sample availability not all proteins could be examined in all cases. A highly homogenous expression pattern for all the proteins studied was observed in the CLL subgroup with a normal karyotype. This pattern was independent of the VH-status. CLL samples with normal karyotype, +12q and 13q deletion showed equal levels of ATM as compared to EHEB and JVM-2. As compared to cases with a normal karyotype the ATM level within the 11q- subgroup was reduced in 5 cases and absent in 1 case among 11 evaluable 11q- cases. The 17p- subgroup was comprised of 3 cases with concomitant 17p- and 11q- and 15 cases with 17p- but no 11q-. The latter group showed ATM protein levels comparable to the levels of the normal karyotype group. In the group with 17p- and 11q- there was an ATM expression level similar to the groups with 17p- and normal karyotype in two cases while one case had a reduced ATM protein level comparable to the 11q- subgroup. All cases with 17p- exhibited a stronger expression of p53 as compared to the cell lines and all other cases, except for one case with normal karyotype and one with an 11q-. No p53 mutations could be detected in exons 5–9 by sequencing in these two cases. High levels of survivin protein were found in all cases with 17p- and/or 11q-, 13q-, +12q while the subgroup with a normal karyotype showed lower levels. High levels of cdk4 protein were expressed in cases with 17p-, 11q- and 13q- while cdk4 protein levels were low in the subgroup with +12q and normal karyotype. Regarding p21, p27, Bcl2, Bax, Smac, Apaf-1, Cyclin D1–D3, cIAP2, XIAP, Akt1 and PI3K no variation in the expression levels were observed across the genetic CLL subgroups. Comparing the CLL cases to the cell lines the differences in expression levels were found for the cell cycle regulators Cyclin D1, D2, D3, p21 and p27. While the cell lines showed strong protein levels for Cyclin D1, D2, D3 and p21, they were nearly absent in the CLL cases. Expression of p27 was higher in all CLL cases as compared to JVM-2 and EHEB. In conclusion, the 17q- subgroup was the only group with a high level of p53 protein expression indicating that p53 is the affected gene in this subgroup. In contrast, the ATM protein levels are reduced only in a part of the 11q- cases indicating a possible role of additional candidate genes. Cases with +12q and normal karyotype showed weak expression of cdk4 pointing out a possible function in these subgroups.

Upregulation of the Anti-Apoptotic Protein, Survivin, in Hematopoietic Cells in Sickle Cell Anemia and Effects of Hydroxyurea Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1532-1532
Author(s):  
Carolina Lanaro ◽  
Carla Fernanda Franco-Penteado ◽  
Mariana R. B. Mello ◽  
Kleber Yotsumoto Fertrin ◽  
Marcos André C Bezerra ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Sickle Cell Anemia ◽  
Survivin Expression ◽  
Erythroid Differentiation ◽  
Mrna Levels ◽  
Gene Expressions ◽  
Protein Levels ◽  
Survivin Protein ◽  
Inflammatory State

Abstract Abstract 1532 Poster Board I-555 Survivin (BIRC5) is a member of the inhibitors of apoptosis family implicated in both prevention of cell death and control of mitosis. Although the actions of survivin in control of cancer cell division and apoptosis have been studied, its role in nonneoplastic diseases is not elucidated. Chronic inflammation is associated with STAT-3 upregulation, which can induce survivin production. Sickle cell anemia (SCA) has been characterized as a chronic inflammatory state and growing evidence indicates that inflammatory stress within the microvasculature may play a significant role in the vasoocclusion that is characteristic of SCA. Long-term treatment with hydroxyurea (HU) has been shown to reduce the production of inflammatory cytokines in SCA patients and leukocyte number. Since enhanced survivin expression has been reported in leukocytes under inflammatory conditions, and during hematopoietic cell survival and proliferation, the aim of this study was to investigate changes in survivin levels during erythroid differentiation, and determine expression in neutrophils (NS), mononuclear cells (MC) and red blood cell (RBC) in steady-state SCA patients (n≥10), SCA patients on HU therapy (n≥16), and healthy controls (HC, n≥5). Survivin and STAT-3 gene expression were determined by qRT-PCR analysis in primary human erythroblasts cultures for 7, 10 and 13 days and leukocytes separated from peripheral blood samples. Survivin protein expression was determined by flow cytometry with survivin-specific antibodies. Survivin gene expression was significantly increased during erythroid differentiation, but survivin mRNA levels showed similar patterns between SCA and HC (7d: 0.8±0.1 × 0.7±0.08; 10d: 1.7±0.3 × 1.6±0.2; 13d: 2.2± 0.27 × 1.8±0.19,U.A.,P>0.05,respectively). However, protein levels of survivin in mature RBC (glicophorin A +) was significantly higher in SCA patients compared to HC (41.90± 2.9 × 25.76±1.9, P=0.0006, respectively). BIRC-5 gene expression in MC was significantly higher in SCA patients compared to HC (0.9±0.1 × 0.5±0.2, P=0.04, respectively). Survivin protein levels in MC from SCA was significantly increased to compared to HC (51.7±3.2 × 39.7±1.7, MFI, P=0.01,respectively). Survivin protein levels are elevated in NS of SCA patients compared to HC (28.4±1.6 × 21.9±1.5, MFI, P=0.02,respectively). No significant alterations in the mRNA levels of the gene encoding STAT-3 were found during erythroid differentiation (7d: 1.1±0.04 × 1.1±0.08; 10d: 0.6±0.07 × 0.8±0.08; 13d: 0.6±0.07 × 0.9±0.1, P>0.05,respectively) or MC cells (1.2±0.1 × 1.1± 0.1, P>0.05,respectively) in SCA patients compared to HC. Patients on HU therapy demonstrated lower survivin MC gene expressions and protein levels compared to non-treated patients (0.6±0.3 × 0.9±0.1; 37.9±1.5 × 51.7±3.3, P=0.02; P<0.0001,respectively), but no difference was shown in STAT-3 gene expressions (1.1±0.04 × 1.2 ±0.1, respectively). Survivin protein levels were not significantly different in NS and RBC in patients on HU therapy compared to SCA (27.1±1.8 × 28.4± 1.6; 45.9± 3.2× 41.9± 2.9, MFI, P>0.05, respectively). Our data showed that survivin gene and protein expression are upregulated in MC in SCA patients, independently of STAT-3 expression. In addition, a high protein expression was observed in NS and RBC in these patients. HU therapy was associated with lower survivin expression in MC, but not NS and RBC, indicating that the beneficial effect that HU has on the inflammatory state, may participate in the reduced levels of survivin. In conclusion, the exact importance of survivin in SCA vasooclusion is not clear, but data indicates a high expression of this protein in leukocytes and RBC of SCA patients and may imply a role for this protein in leukocytosis and RBC proliferation in SCA. Disclosures No relevant conflicts of interest to declare.

Erythroid-Expression of HMGA2 Increases Fetal Hemoglobin in Human Adult Erythroblasts

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2161-2161
Author(s):  
Jaira F. de Vasconcellos ◽  
Y. Terry Lee ◽  
Colleen Byrnes ◽  
Laxminath Tumburu ◽  
Antoinette Rabel ◽  
...  
Keyword(s):  
Protein Expression ◽  
Fetal Hemoglobin ◽  
Cell Counting ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Globin Mrna ◽  
Expression Levels ◽  
Gamma Globin ◽  
Empty Vector ◽  
Gene And Protein Expression

Abstract HMGA2 is a member of the high-mobility group A family and plays a role in the regulation of gene transcription and chromatin structure. HMGA2 is a validated target of the let-7 family of miRNAs. Let-7 miRNAs are highly regulated in erythroid cells during the fetal-to-adult developmental transition (1). Recent studies demonstrated that the LIN28 -let-7 axis mediated up-regulation of fetal hemoglobin (HbF) expression to >30% of the total globin levels in cultured erythroblasts from adult humans (2) and the amelioration of hypoxia-related sickling of cultured mature erythrocytes from pediatric patients with sickle cell disease (3). Interestingly, increased expression of endogenous HbF in a patient receiving gene therapy was also associated with truncated HMGA2 protein expression after lentiviral integration and disruption of let-7 targeting at the HMGA2 gene locus (4). Therefore, we hypothesized that HMGA2 may be involved in fetal hemoglobin regulation as a downstream target of the let-7 miRNAs. To study the effects of HMGA2 upon erythropoiesis and globin expression, lentiviral constructs were designed for let-7 resistant expression of HMGA2 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2 -SPTA1-OE), with a matched empty vector control. Transductions were performed in CD34+ cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Overexpression of HMGA2 was confirmedby Q-RT-PCR (control: below detection limits; HMGA2 -SPTA1-OE: 2.51E+04 ± 3.44E+04 copies/ng) and Western blot analyses at culture day 14. Cell counting revealed no significant changes between HMGA2 -SPTA1-OE and control (empty vector) transductions at culture day 14. Terminal maturation with loss of CD71 from the erythroblast cell surface and enucleation assessed by thiazole orange staining were analyzed in the control and HMGA2 -SPTA1 -OE samples at the end of the culture period. Globin genes expression levels were evaluated for HMGA2 -SPTA1-OE by Q-RT-PCR. HMGA2 -SPTA1-OE caused a significant increase in gamma-globin mRNA expression levels compared to controls (control: 5.02E+05 ± 8.62E+04 copies/ng; HMGA2 -SPTA1-OE: 1.45E+06 ± 7.31E+05 copies/ng; p=0.037). Consistent with the increase in gamma-globin mRNA levels, HPLC analyses at culture day 21 demonstrated modest but significant increases in HbF levels in HMGA2 -SPTA1-OE compared to controls (HbF control: 5.41 ± 2.15%; HMGA2 -SPTA1-OE: 16.53 ± 4.43%; p=0.006). Possible effect(s) and downstream mechanism(s) triggered by HMGA2 -SPTA1-OE were investigated. Q-RT-PCR analyses demonstrated no significant changes in the let-7 family of miRNAs in HMGA2 -SPTA1-OE compared to controls. Expression patterns of several transcription factors such as BCL11A, KLF1, SOX6 and GATA1 were investigated by Q-RT-PCR and no significant changes were detected in HMGA2 -SPTA1-OE compared to controls. While BCL11A mRNA levels were decreased by HMGA2 -SPTA1 -OE, the differences did not reach statistical significance (control: 4.26E+02 ± 8.18E+01 copies/ng; HMGA2 -SPTA1 -OE: 2.84E+02 ± 1.48E+02 copies/ng; p=0.104). However, nuclear BCL11A protein levels assessed by Western analysis were suppressed in HMGA2 -SPTA1 -OE. In summary, these results demonstrate that HMGA2, a validated target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in human erythroblasts, and reduces levels of BCL11A protein. These data thus support the notion that suppression of let-7 miRNAs increases fetal hemoglobin, in part, by the targeting of erythroblast HMGA2 mRNA. (1) Noh SJ et al. J Transl Med. 7:98 (2009). (2) Lee YT et al. Blood. 122:1034-41 (2013). (3) Vasconcellos JF et al. PLoS One. 9:e106924 (2014). (4) Cavazzana-Calvo M et al. Nature. 467:318-22 (2010). Disclosures No relevant conflicts of interest to declare.

Relationship of ERCC1 genotype variant with mRNA expression and ERCC1 protein levels in advanced urothelial carcinoma (UC).

2013 ◽  
Vol 31 (6_suppl) ◽  
pp. 260-260
Author(s):  
Elizabeth A. Guancial ◽  
Lillian Werner ◽  
Joaquim Bellmunt ◽  
Nikitas Nikitas ◽  
Edward C. Stack ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Excision Repair ◽  
Predictive Biomarker ◽  
Mrna Levels ◽  
Nuclear Staining ◽  
Protein Levels ◽  
Tissue Arrays ◽  
Platinum Based Chemotherapy ◽  
The Relationship

260 Background: DNA repair factors may be predictive for response to chemotherapies that produce DNA damage. While low ERCC1 protein and mRNA levels have been reported as associated with improved outcomes in metastatic UC patients treated with platinum-based chemotherapy, the relationship between genotype, mRNA expression, and protein level is unknown. The ERCC1 germline 19007C>T single-nucleotide polymorphism (SNP) is functionally associated with reduced translation of ERCC1 mRNA. We investigated the relationship between ERCC1 germline SNP, ERCC1 tumor mRNA and protein expression, in a cohort of patients with advanced UC who received first-line, platinum-based chemotherapy. Methods: A cohort of clinically annotated, uniformly-treated advanced UC patients with FFPE primary tumor tissue available was identified through the Hellenic cooperative Oncology Group (HECOG) (N=93). Genomic DNA extraction, nested PCR, and restriction fragment length polymorphism techniques for the 19007C>T SNP were performed to identify C/C, C/T and T/T genotypes. ERCC1 mRNA expression was interrogated using Nanostring nCounter profiling. IHC analysis was performed on tissue arrays using an ERCC1 antibody. Percent of positive nuclear staining was categorized as quartiles using previously identified cut-points. Results: ERCC1 C/T genotype was identified in 30/61 samples (49%) and T/T in 14/61 samples (23%). In 54 patients with both SNP and mRNA data available, T/T genotype was associated with the highest level of mRNA expression, followed by the C/T genotype (p=0.04). Neither ERCC1 genotype (N=44) nor ERCC1 mRNA expression (N=54) was associated with ERCC1 protein expression as measured by IHC (p=0.52 and p=0.13, respectively). Conclusions: ERCC1 19007C>T is associated with increased ERCC1 mRNA expression. However, neither genotype nor mRNA are surrogates for ERCC1 protein detected by IHC in advanced UC tumors. This suggests that while genotype influences mRNA expression of ERCC1, the use of the nucleotide excision repair pathway as a predictive biomarker of platinum-sensitivity may be more complex than previously appreciated and require the integrative use of proteomics, genomics and epigenomics.

Sign in / Sign up

Export Citation Format

Share Document