Diagnostic impact of promoter methylation and E-cadherin gene and protein expression levels in laryngeal carcinoma

Abstract Both somatostatin (SST) and somatostatin receptors (SSTRs) are proteins with important functions in both physiological tissue and in tumors, particularly in neuroendocrine tumors (NETs). NETs are frequently characterized by high SSTRs expression levels. SST analogues (SSAs) that bind and activate SSTR have anti-proliferative and anti-secretory activity, thereby reducing both the growth as well as the hormonal symptoms of NETs. Moreover, the high expression levels of SSTR type-2 (SSTR2) in NETs is a powerful target for therapy with radiolabeled SSAs. Due to the important role of both SST and SSTRs, it is of great importance to elucidate the mechanisms involved in regulating their expression in NETs, as well as in other types of tumors. The field of epigenetics recently gained interest in NET research, highlighting the importance of this process in regulating the expression of gene and protein expression. In this review we will discuss the role of the epigenetic machinery in controlling the expression of both SSTRs and the neuropeptide SST. Particular attention will be given to the epigenetic regulation of these proteins in NETs, whereas the involvement of the epigenetic machinery in other types of cancer will be discussed as well. In addition, we will discuss the possibility to target enzymes involved in the epigenetic machinery to modify the expression of the SST-system, thereby possibly improving therapeutic options.

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Proinflammatory and Anti-Inflammatory Cytokines Mediated by NF-κB Factor as Prognostic Markers in Mammary Tumors

Mediators of Inflammation ◽

10.1155/2016/9512743 ◽

2016 ◽

Vol 2016 ◽

pp. 1-10 ◽

Cited By ~ 15

Author(s):

Gustavo Rodrigues Martins ◽

Gabriela Bottaro Gelaleti ◽

Marina Gobbe Moschetta ◽

Larissa Bazela Maschio-Signorini ◽

Debora Ap. Pires de Campos Zuccari

Keyword(s):

Protein Expression ◽

Survival Time ◽

Inflammatory Cytokines ◽

Prognostic Markers ◽

Tumor Development ◽

Mammary Tumors ◽

Clinicopathological Characteristics ◽

Expression Levels ◽

Gene And Protein Expression ◽

High Gene

Inflammation results in the production of cytokines, such as interleukin- (IL-) 4 and IL-10 with immunosuppressive properties or IL-6 and TNF-αwith procarcinogenic activity. Furthermore, NF-κB is the major link between inflammation and tumorigenesis. This study verified the interaction between active inflammatory cytokines in the tumor microenvironment and serum of female dogs with mammary tumors and their correlation with the clinicopathological characteristics and overall survival. Measurement of gene expression was performed by qPCR and protein levels by ELISA/Luminex. High gene and protein expression levels of NF-κB, IL-6, and TNF-αwere found in association with characteristics that reflect worse prognosis and a negative correlation between TNF-αprotein expression and survival time was observed (p<0.05). In contrast, high gene and protein expression levels of IL-4 and IL-10 were associated with characteristics of better prognosis and an increased level of IL-4 and a longer survival time of animals were obtained (p<0.05). In addition, there was a positive correlation between TNF-αand IL-6 expression in association with NF-κB. The results show a significant correlation of these cytokines with tumor development, associated with NF-κB expression and cytokines promodulation, showing that these biological factors could be used as predictive and prognostic markers in breast cancer.

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

Blood ◽

10.1182/blood.v126.23.2161.2161 ◽

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.

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Differences in Expression Patterns of DNMTs and TSG Proteins in Lymphoid Tissue Section Play an Important Role in Their Association

Blood ◽

10.1182/blood.v126.23.2657.2657 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2657-2657

Author(s):

Lobna Alkebsi ◽

Hiroshi Handa ◽

Kenichi Tahara ◽

Hiroaki Shimizu ◽

Takuma Ishizaki ◽

...

Keyword(s):

B Cells ◽

Protein Expression ◽

Mrna Expression ◽

Lymphoid Tissue ◽

Methylation Status ◽

Lymphoid Tissues ◽

Expression Levels ◽

Protein Expressions ◽

Mrna Expression Levels ◽

E Cadherin

Abstract In situ, patterns of expression of DNMTs (DNA methytransferases) in normal reactive tonsillar tissue have been examined. Difference in pattering of expression of DNMTs and TSG (Tumor suppressor genes) proteins in lymphoid tissue section is an important question in relation to their association with each other as well as relationship to mRNA gene expression level. In order to examine this issue, we examined DNMTs and TSG proteins expression by immunohistochemistry in sections of paraffin-embedded specimens obtained from 33 subjects of lymphoma and 16 subjects of Non-malignant tissues after receiving written informed consent. The specimens were stained with anti-DNMTs (DNMT1, DNMT3A and DNMT3B) and anti-TSG (E-cadherin, H-cadherin and ADAMTS18) antibodies. In addition, using fresh-frozen optimal cutting temperature (OCT) compound-embedded tissue specimens before any treatment, we examined mRNA expression levels and promoter methylation status of E-cadherin (CDH1), H-cadherin (CDH13) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS18) using quantitative real-time PCR (qRT-PCR) and methylation-specific PCR (MSP), respectively. The expression of nuclear DNMTs proteins (DNMT1 and 3A) in lymphoma section was observed in [17/33 (51%); 27/33 (81%)], whereas in non-malignant tissues was [14/16 (87.5%); 13/16 (81%)], respectively. The DNMT3B protein expression was not detected in our tissue samples, which might be explained by the fact that DNMT3B characterized by alternative splicing as shown previously. Membrane proteins (E-cadherin, H-cadherin and ADAMTS18) showed low expression [12/33 (36%); 10/33 (30%); 6/33 (18%), respectively], when compared to non-malignant tissue sections [12/16 (75%); 7/16 (43%); 8/16 (50%), respectively]. The expression levels of CDH1, CDH13 and ADAMTS18 mRNAs were non-significantly reduced in their corresponding protein negative expression compared to the levels in cases with positive protein expression (p =0.112, p =0.378, p =0.077, respectively). We could not find any correlation between mRNA/protein expression levels of DNMTs and the methylation status of CDH1, CDH13 and ADAMTS18. Importantly, by immunostaining especially in non-malignant lymphoid tissues, we found that DNMT1 was highly detected in germinal center B cells (GC B cells) with gradual decrease or no expression in the mantle, marginal, interfollicular and T cells zones. Whereas DNMT3A was preferentially and scattered like expressed in the cells of the surrounding zones out of the germinal centers. Furthermore, E-cadherin, H-cadherin and ADAMTS18 proteins expression were detected on the cell surface membrane of the cells outside the GC but at rates somehow more than those cells inside the GC (Fig. 1). This is supported by the significant association observed between the frequency of DNMT3A with both E-cadherin and ADAMTS18, protein expressions (Chi square: p <0.05), while no association with H-cadherin protein expression. In addition, DNMT1 protein expression did not show significant association with the protein expressions of E-cadherin, H-cadherin and ADAMTS18. Moreover, the mRNA expression levels of DNMT3A and 3B showed high significant levels (p <0.05) in cases with negative protein expressions of both E-cadherin and ADAMTS18 when compared to cases with positive protein expressions (Fig. 2A and C). The DNMT1 mRNA expression level did not show any significant difference between the negative and positive protein expressions of E-cadherin, H-cadherin and ADAMTS18 (Fig. 2B). Furthermore, there was no significant association between the mRNA levels of DNMTs and H-cadherin protein expression. Expression of H-cadherin protein was frequently observed in the endothelial venules and trabeculae of the lymphoid tissues (Fig. 1) which might cause of its lack of association with both DNMT1 and DNMT3A. In conclusion, these results indicate that as a result of differences in pattering of DNMTs and TSG protein expressions detected in lymphoid tissues by immunohistochemistry staining, it might be one of the reasons of the association with each other and their mRNA expression levels across the spectrum of lymphomas and non-malignant lymphoid tissues. Disclosures No relevant conflicts of interest to declare.

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Nitrergic neuromuscular transmission in the mouse internal anal sphincter is accomplished by multiple pathways and postjunctional effector cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00331.2014 ◽

2014 ◽

Vol 307 (11) ◽

pp. G1057-G1072 ◽

Cited By ~ 17

Author(s):

C. A. Cobine ◽

A. G. Sotherton ◽

L. E. Peri ◽

K. M. Sanders ◽

S. M. Ward ◽

...

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Anal Sphincter ◽

Internal Anal Sphincter ◽

Neuromuscular Transmission ◽

Second Messengers ◽

Cell Types ◽

Effector Cells ◽

Expression Levels ◽

Gene And Protein Expression

The effector cells and second messengers participating in nitrergic neuromuscular transmission (NMT) were investigated in the mouse internal anal sphincter (IAS). Protein expression of guanylate cyclase (GCα, GCβ) and cyclic GMP-dependent protein kinase I (cGKI) were examined in cryostat sections with dual-labeling immunohistochemical techniques in PDGFRα+ cells, interstitial cells of Cajal (ICC), and smooth muscle cells (SMC). Gene expression levels were determined with quantitative PCR of dispersed cells from Pdgfrα egfp/+, Kit copGFP/+, and smMHC Cre-egfp mice sorted with FACS. The relative gene and protein expression levels of GCα and GCβ were PDGFRα+ cells > ICC ≫ SMC. In contrast, cGKI gene expression sequence was SMC = ICC > PDGFRα+ cells whereas cGKI protein expression sequence was neurons > SMC ≫ ICC = PDGFRα+ cells. The functional role of cGKI was investigated in cGKI −/− mice. Relaxation with 8-bromo (8-Br)-cGMP was greatly reduced in cGKI −/− mice whereas responses to sodium nitroprusside (SNP) were partially reduced and forskolin responses were unchanged. A nitrergic relaxation occurred with nerve stimulation (NS, 5 Hz, 60 s) in cGKI +/+ and cGKI −/− mice although there was a small reduction in the cGKI −/− mouse. Nω-nitro-l-arginine (l-NNA) abolished responses during the first 20–30 s of NS in both animals. The GC inhibitor ODQ greatly reduced or abolished SNP and nitrergic NS responses in both animals. These data confirm an essential role for GC in NO-induced relaxation in the IAS. However, the expression of GC and cGKI by all three cell types suggests that each may participate in coordinating muscular responses to NO. The persistence of nitrergic NMT in the cGKI −/− mouse suggests the presence of a significant GC-dependent, cGKI-independent pathway.

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PDE4B Proposed as a High Myopia Susceptibility Gene in Chinese Population

Frontiers in Genetics ◽

10.3389/fgene.2021.775797 ◽

2022 ◽

Vol 12 ◽

Author(s):

Fuxin Zhao ◽

Wei Chen ◽

Hui Zhou ◽

Peter S. Reinach ◽

Yuhan Wang ◽

...

Keyword(s):

Protein Expression ◽

Normal Control ◽

High Myopia ◽

Susceptibility Gene ◽

A549 Cells ◽

Dna Pooling ◽

Myopia Progression ◽

Expression Levels ◽

Replication Stage ◽

Gene And Protein Expression

Myopia is the most common cause of refractive error worldwide. High myopia is a severe type of myopia, which usually accompanies pathological changes in the fundus. To identify high myopia susceptibility genes, DNA-pooling based genome-wide association analysis was used to search for a correlation between single nucleotide polymorphisms and high myopia in a Han Chinese cohort (cases vs. controls in discovery stage: 507 vs. 294; replication stage 1: 991 vs. 1,025; replication stage 2: 1,021 vs. 52,708). Three variants (rs10889602T/G, rs2193015T/C, rs9676191A/C) were identified as being significantly associated with high myopia in the discovery, and replication stage. rs10889602T/G is located at the third intron of phosphodiesterase 4B (PDE4B), whose functional assays were performed by comparing the effects of rs10889602T/T deletion of this risk allele on PDE4B and COL1A1 gene and protein expression levels in the rs10889602T/Tdel/del, rs10889602T/Tdel/wt, and normal control A549 cell lines. The declines in the PDE4B and COL1A1 gene expression levels were larger in the rs10889602T/T deleted A549 cells than in the normal control A549 cells (one-way ANOVA, p < 0.001). The knockdown of PDE4B by siRNA in human scleral fibroblasts led to downregulation of COL1A1. This correspondence between the declines in rs10889602 of the PDE4B gene, PDE4B knockdown, and COL1A1 protein expression levels suggest that PDE4B may be a novel high myopia susceptibility gene, which regulates myopia progression through controlling scleral collagen I expression levels. More studies are needed to determine if there is a correlation between PDE4B and high myopia in other larger sample sized cohorts.

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