Wnt/β-Catenin and Hippo Pathway Deregulation in Mammary Tumors of Humans, Dogs, and Cats

Mammary cancer is a common neoplasm in women, dogs, and cats that still represents a therapeutic challenge. Wnt/β-catenin and Hippo pathways are involved in tumor progression, cell differentiation, and metastasis. The aim of this study was to evaluate mRNA and protein expression of molecules involved in these pathways in human (HBC), canine (CMT), and feline mammary tumors (FMT). Real-time quantitative polymerase chain reaction (qPCR) for β-catenin, CCND1, YAP, TAZ, CTGF, and ANKRD1, western blotting for YAP, TAZ, and β-catenin, and immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), ERBB2, β-catenin, and YAP/TAZ were performed on mammary tumor tissues. The protein expression of active β-catenin was higher in tumors than in healthy tissues in all 3 species. The mRNA expression of the downstream gene CCND1 was increased in HBC ER+ and CMTs compared to healthy tissues. Membranous and cytoplasmic protein expression of β-catenin were strongly negatively correlated in all 3 species. Tumors showed an increased protein expression of YAP/TAZ when compared to healthy tissues. Notably, YAP/TAZ expression was higher in triple negative breast cancers when compared to HBC ER+ and in FMTs when compared to CMTs. The mRNA expression of β-catenin, YAP, TAZ, CTGF, and ANKRD1 was not different between tumors and healthy mammary gland in the 3 species. This study demonstrates deregulation of Wnt/β-catenin and Hippo pathways in mammary tumors, which was more evident at the protein rather than the mRNA level. Wnt/β-catenin and Hippo pathways seem to be involved in mammary carcinogenesis and therefore represent interesting therapeutic targets that should be further investigated.

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Survivin Regulates Bad Gene Expression by Binding to Its Promoter and Modulates Cell Cycle and Apoptosis in Esophageal Carcinoma Cell

Journal of Oncology ◽

10.1155/2021/1384289 ◽

2021 ◽

Vol 2021 ◽

pp. 1-11

Author(s):

Yan Chen ◽

Shayahati Bieerkehazhi ◽

Xiumei Li ◽

Lili Ma ◽

Waresijiang Yibulayin ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Protein Expression ◽

Esophageal Carcinoma ◽

Mrna Expression ◽

Carcinoma Cell ◽

Positive Expression ◽

Tumor Tissues ◽

Mrna And Protein Expression ◽

Expression Rate

Esophageal cancer (EC) is the eighth most prevalent cancer and the sixth leading cause of cancer-related mortality worldwide. As an antiapoptotic and a proapoptotic protein, respectively, survivin and Bad play an important role in carcinogenesis of the most human cancers including EC. However, the regulatory relationships between them remain unclear. We sought to investigate the effects of survivin knockdown and overexpression on the expression of Bad gene, cell cycle progression, and apoptosis of esophageal carcinoma cell. The mRNA expression levels of survivin and Bad were determined in EC tissue samples. The knockdown and overexpression experiments were performed in ECA109 and KYSE450 cells via transfection with survivin overexpression and shRNA plasmids. A Bad overexpression experiment was conducted to confirm the biological effect on knockdown of survivin via modulating Bad expression. RT-qPCR and Western blot analysis were used to detect mRNA and protein expression, respectively. Cell cycle and apoptosis were analyzed by flow cytometry. The chromatin immunoprecipitation (ChIP) was conducted to determine the binding sites of survivin on the promoter of Bad gene. By analyzing the mRNA expression of survivin and Bad in 40 ESCC patient specimens, we found that the positive expression rate of survivin in tumor tissues (88%, 35/40) was remarkably high, compared with the distal nontumor tissues (48%, 19/40, p < 0.01). On the other hand, the positive expression rate of Bad in tumor tissues (70%, 28/40) was remarkably low, compared with the distal nontumor tissues (95%, 38/40, p < 0.01). Overexpression of survivin decreases Bad mRNA and protein expression and promotes transformation of cell cycle to S phase. Conversely, knockdown of survivin increases Bad mRNA and protein expression and induces cell cycle arrest and apoptosis. Bad overexpression inducing apoptosis of esophageal carcinoma cell shows the similar apoptotic effect with survivin knockdown. ChIP assays indicate that survivin directly binds to the Bad promoter region, diminishing the transcriptional activity of Bad. In conclusion, the result suggested that survivin regulates Bad gene expression by binding to its promoter and modulates cell cycle and apoptosis in esophageal carcinoma cell.

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Inactivation of parkin by promoter methylation correlated with lymph node metastasis and genomic instability in nasopharyngeal carcinoma

Tumor Biology ◽

10.1177/1010428317695025 ◽

2017 ◽

Vol 39 (3) ◽

pp. 101042831769502 ◽

Cited By ~ 6

Author(s):

Haifeng Ni ◽

Zhen Zhou ◽

Bo Jiang ◽

Xiaoyang Yuan ◽

Xiaolin Cao ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Nasopharyngeal Carcinoma ◽

Protein Expression ◽

Mrna Expression ◽

Genomic Instability ◽

Promoter Methylation ◽

Chain Reaction ◽

Node Metastasis ◽

Mrna And Protein Expression ◽

Polymerase Chain

This study aimed to investigate the inactivation of the parkin gene by promoter methylation and its relationship with genome instability in nasopharyngeal carcinoma. Parkin was considered as a tumor suppressor gene in various types of cancers. However, its role in nasopharyngeal carcinoma is unexplored. Genomic instabilities were detected in nasopharyngeal carcinoma tissues by the random amplified polymorphic DNA. The methylation-specific polymerase chain reaction, semi-quantitative reverse transcription polymerase chain reaction, and immunohistochemical analysis were used to detect methylation and mRNA and protein expression of parkin in 54 cases of nasopharyngeal carcinoma tissues and 16 cases of normal nasopharyngeal epithelia tissues, and in 5 nasopharyngeal carcinoma cell lines (CNE1, CNE2, TWO3, C666, and HONE1) and 1 normal nasopharyngeal epithelia cell line (NP69). mRNA expression of parkin in CNE1 and CNE2 was analyzed before and after methyltransferase inhibitor 5-aza-2-deoxycytidine treatment. The relationship between promoter methylation and mRNA expression, demethylation and mRNA expression, and mRNA and protein expression of the gene and clinical factors and genomic instabilities were analyzed. The mRNA and protein expression levels were significantly reduced in 54 cases of human nasopharyngeal carcinoma compared with 16 cases of normal nasopharyngeal epithelia. Parkin-methylated cases showed significantly lower mRNA and protein expression levels compared with unmethylated cases. After 5-aza-2-deoxycytidine treatment, parkin mRNA expression was restored in CNE1 and CNE2; 92.59% (50/54) of nasopharyngeal carcinoma demonstrated genomic instability. Parkin is frequently inactivated by promoter methylation, and its mRNA and protein expression correlate with lymph node metastasis and genomic instability. Parkin deficiency probably promotes tumorigenesis in nasopharyngeal carcinoma.

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Survivin regulates Bad gene expression by binding to its promoter and modulates cell cycle and apoptosis in esophageal carcinoma cell

10.21203/rs.2.20789/v1 ◽

2020 ◽

Author(s):

YAN CHEN ◽

SHAYAHATI BIEERKEHAZHI ◽

XIUMEI LI ◽

LILI MA ◽

WARESIJIANG YIBULAYIN ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Protein Expression ◽

Esophageal Carcinoma ◽

Mrna Expression ◽

Carcinoma Cell ◽

Positive Expression ◽

Tumor Tissues ◽

Mrna And Protein Expression ◽

Expression Rate

Abstract Esophageal cancer (EC) is the eighth most prevalent cancer and the sixth leading cause of cancer-related mortality worldwide. As an anti-apoptotic and a pro-apoptotic protein respectively, survivin and Bad play important role in carcinogenesis of the most human cancers including EC. However the regulatory relationships between them remain unclear. We sought to investigate the effects of survivin knockdown and overexpression on the expression of Bad gene, cell cycle progression and apoptosis of esophageal carcinoma cell. The mRNA expression levels of survivin and Bad were determined in EC tissue samples. The knockdown and overexpression experiments were performed in ECA109 and KYSE450 cells via transfection with survivin overexpression and shRNA plasmids. RT-qPCR and Western blot analysis were used to detect mRNA and protein expression respectively. Cell cycle and apoptosis were analyzed by flow cytometry. The chromatin immunoprecipitation (ChIP) was conducted to determine the binding sites of survivin on the promoter of Bad gene. By analyzing the mRNA expression of survivin and Bad in 40 ESCC patient specimens, we found that the positive expression rate of survivin in tumor tissues(88%, 35/40) was remarkably high, compared with the distal non-tumor tissues (48%, 19/40, p<0.01). On the other hand, the positive expression rate of Bad in tumor tissues (70%, 28/40) was remarkably low, compared with the distal non-tumor tissues (95%, 38/40, p<0.01). Overxpression of survivin decreases Bad mRNA and protein expression, and promotes transformation of cell cycle to S phase. Conversely, knockdown of survivin increases Bad mRNA and protein expression, and induces cell cycle arrest and apoptosis. ChIP assays indicate that survivin directly binds to the Bad promoter region, diminishing the transcriptional activity of Bad. In conclusion, the result suggested that survivin regulates Bad gene expression by binding to its promoter and modulates cell cycle and apoptosis in esophageal carcinoma cell.

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Novel regulation of renal gluconeogenesis by Atp6ap2 in response to high fat diet via PGC1-α/AKT-1 pathway

Scientific Reports ◽

10.1038/s41598-021-90952-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Safia Akhtar ◽

Silas A. Culver ◽

Helmy M. Siragy

Keyword(s):

Protein Expression ◽

High Fat Diet ◽

Normal Diet ◽

Receptor Expression ◽

Wild Type ◽

High Fat ◽

Renal Gluconeogenesis ◽

Mrna And Protein Expression ◽

Polymerase Chain ◽

Renal Expression

AbstractRecent studies suggested that renal gluconeogenesis is substantially stimulated in the kidney in presence of obesity. However, the mechanisms responsible for such stimulation are not well understood. Recently, our laboratory demonstrated that mice fed high fat diet (HFD) exhibited increase in renal Atp6ap2 [also known as (Pro)renin receptor] expression. We hypothesized that HFD upregulates renal gluconeogenesis via Atp6ap2-PGC-1α and AKT pathway. Using real-time polymerase chain reaction, western blot analysis and immunostaining, we evaluated renal expression of the Atp6ap2 and renal gluconeogenic enzymes, PEPCK and G6Pase, in wild type and inducible nephron specific Atp6ap2 knockout mice fed normal diet (ND, 12 kcal% fat) or a high-fat diet (HFD, 45 kcal% fat) for 8 weeks. Compared with ND, HFD mice had significantly higher body weight (23%) (P < 0.05), renal mRNA and protein expression of Atp6ap2 (39 and 35%), PEPCK (44 and 125%) and G6Pase (39 and 44%) respectively. In addition, compared to ND, HFD mice had increased renal protein expression of PGC-1α by 32% (P < 0.05) and downregulated AKT by 33% (P < 0.05) respectively in renal cortex. Atp6ap2-KO abrogated these changes in the mice fed HFD. In conclusion, we identified novel regulation of renal gluconeogenesis by Atp6ap2 in response to high fat diet via PGC1-α/AKT-1 pathway.

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Expression Characteristics and Significant Prognostic Values of PGK1 in Breast Cancer

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.695420 ◽

2021 ◽

Vol 8 ◽

Author(s):

Yanping Li ◽

Shanshan Wang ◽

Xiaoyuan Zhang ◽

Rui Yang ◽

Xiaonan Wei ◽

...

Keyword(s):

Breast Cancer ◽

Protein Expression ◽

Mrna Level ◽

Vital Role ◽

Migration And Invasion ◽

Breast Cancer Patients ◽

Clinicopathologic Characteristics ◽

Independent Prognostic Marker ◽

Potential Biomarker ◽

Mrna And Protein Expression

It was proven that PGK1 plays a vital role in the proliferation, migration, and invasion of human breast cancer. However, the correlation of PGK1 mRNA and protein expression with clinicopathologic characteristics and prognostic values according to various kinds of breast cancer patient classifications remains unsufficient. Here, we analyzed data from the Oncomine database, Breast cancer Gene-Expression Miner v4.5, TNMplot, MuTarget, PrognoScan database, and clinical bioinformatics to investigate PGK1 expression distribution and prognostic value in breast cancer patients. Our study revealed that the mRNA and protein expression levels of PGK1 were up-regulated in various clinicopathologic types of breast cancer. Moreover, the expression of PGK1 was correlated with mutations of common tumor suppressor genes TP53 and CDH1. In addition, we found that high mRNA level of PGK1 was significantly associated with poor OS, RFS, and DMFS. Notably, Cox regressionanalysis showed that PGK1 could be used as an independent prognostic marker. In summary, the aforementioned findings suggested that PGK1 might be not only explored as a potential biomarker, but also combined with TP53/CDH1 for chemotherapy in breast cancer.

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Effects of SLIRP on Sperm Motility and Oxidative Stress

BioMed Research International ◽

10.1155/2020/9060356 ◽

2020 ◽

Vol 2020 ◽

pp. 1-5

Author(s):

Dan Shan ◽

Samuel Kofi Arhin ◽

Junzhao Zhao ◽

Haitao Xi ◽

Fan Zhang ◽

...

Keyword(s):

Oxidative Stress ◽

Protein Expression ◽

Manganese Superoxide Dismutase ◽

Mrna Level ◽

Atp Content ◽

Human Male ◽

Manganese Superoxide ◽

Polymerase Chain ◽

And Oxidative Stress ◽

Relative Mrna Expression

Background. Deficient spermatozoon motility is one of the main causes of male infertility. However, there are still no accurate and effective treatments in a clinical setting for male asthenospermia. Exploring the genes and mechanism of asthenospermia has become one of the hot topics in reproductive medicine. Our aim is to study the effect of SLRIP on human spermatozoon motility and oxidative stress. Methods. Sperm samples were collected including a normospermia group (60 cases) and an asthenospermia group (50 cases). SLIRP protein expression in spermatozoa was examined by western blotting, and relative mRNA expression of SLIRP in spermatozoa was quantified by reverse transcription polymerase chain reaction. Levels of reactive oxygen species (ROS), adenosine triphosphate (ATP) content, and the activity of manganese superoxide dismutase (MnSOD) in spermatozoa were also measured. Results. The mRNA level and protein expression of SLIRP in the asthenospermia group were significantly reduced compared with those in the normospermia group. The ROS active oxygen level in the asthenospermia group significantly increased; however, the ATP content decreased significantly as well as the activity of MnSOD. Conclusion. SLIRP regulates human male fertility, and SLIRP and sperm progressive motility are positively correlated. The expression of SLIRP is declined, oxidative damage is increased, and energy metabolism is decreased in spermatozoa of asthenospermia patients compared to normospermia participants.

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Discrepancy between Jun/Fos Proto-Oncogene mRNA and Protein Expression in the Rheumatoid Arthritis Synovial Membrane

J ◽

10.3390/j3020015 ◽

2020 ◽

Vol 3 (2) ◽

pp. 181-194 ◽

Cited By ~ 2

Author(s):

René Huber ◽

Bruno Stuhlmüller ◽

Elke Kunisch ◽

Raimund W. Kinne

Keyword(s):

Rheumatoid Arthritis ◽

Protein Expression ◽

Synovial Membrane ◽

Rna Binding ◽

Mrna Level ◽

Joint Disease ◽

Protein Levels ◽

Modifying Factors ◽

Mrna And Protein Expression ◽

Joint Trauma

Rheumatoid arthritis (RA) is a chronic inflammatory and destructive joint disease characterized by overexpression of pro-inflammatory/pro-destructive mediators, whose regulation has been the focus of our previous studies. Since the expression of these proteins commonly depends on AP-1, the expression of the AP-1-forming subunits cJun, JunB, JunD, and cFos was assessed in synovial membrane (SM) samples of RA, osteoarthritis (OA), joint trauma (JT), and normal controls (NC) using ELISA and qRT-PCR. With respect to an observed discrepancy between mRNA and protein levels, the expression of the mRNA stability-modifying factors AU-rich element RNA-binding protein (AUF)-1, tristetraprolin (TTP), and human antigen R (HuR) was measured. JunB and JunD protein expression was significantly higher in RA-SM compared to OA and/or NC. By contrast, jun/fos mRNA expression was significantly (cjun) or numerically decreased (junB, junD, cfos) in RA and OA compared to JT and/or NC. Remarkably, TTP and HuR were also affected by discrepancies between their mRNA and protein levels, since they were significantly decreased at the mRNA level in RA versus NC, but significantly or numerically increased at the protein level when compared to JT and NC. Discrepancies between the mRNA and protein expression for Jun/Fos and TTP/HuR suggest broad alterations of post-transcriptional processes in the RA-SM. In this context, increased levels of mRNA-destabilizing TTP may contribute to the low levels of jun/fos and ttp/hur mRNA, whereas abundant mRNA-stabilizing HuR may augment translation of the remaining mRNA into protein with potential consequences for the composition of the resulting AP-1 complexes and the expression of AP-1-dependent genes in RA.

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Mechanisms of CCK regulation of monitor peptide mRNA expression in pancreatic acinar AR42J cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1997.272.4.g794 ◽

1997 ◽

Vol 272 (4) ◽

pp. G794-G801 ◽

Cited By ~ 1

Author(s):

T. Kinouchi ◽

S. Tsuzuki ◽

C. Minami ◽

Y. Hayashi ◽

E. Sugimoto ◽

...

Keyword(s):

Mrna Expression ◽

Actinomycin D ◽

Mrna Level ◽

Chain Reaction ◽

Independent Manner ◽

Ar42j Cells ◽

Kinase C ◽

A 23187 ◽

Polymerase Chain ◽

Stimulation Of

We explored the mechanism(s) by which cholecystokinin (CCK) stimulation of AR42J rat pancreatoma cells results in increased mRNA expression of a CCK-releasing peptide [monitor peptide (MP)]. With the use of a newly established reverse transcription-polymerase chain reaction assay system, CCK was shown to increase the level of MP mRNA by about ninefold. When protein synthesis was blocked by addition of cycloheximide, the MP mRNA level remained unchanged in the presence of CCK. Inhibition of transcription with actinomycin D resulted in a half-life for MP mRNA of approximately 17 h, and this rate remained unchanged after CCK treatment, suggesting that CCK may regulate the MP mRNA level by influencing gene transcription. A-23187, bombesin, substance P, and carbachol increased the MP mRNA level. CoCl(2) abolished actions of both CCK and A-23187 on MP mRNA expression. Dibutyryl-adenosine 3',5'-cyclic monophosphate, forskolin, secretin, and vasoactive intestinal polypeptide had no effect on MP mRNA expression. 12-O-tetradecanoylphorbol-13-acetate and phorbol 12,13-dibutyrate also failed to increase MP mRNA. It was therefore proposed that CCK stimulates MP mRNA expression of AR42J cells in a Ca2+-dependent and protein kinase C-independent manner.

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Differential between Protein and mRNA Expression of CCR7 and SSTR5 Receptors in Crohn's Disease Patients

Mediators of Inflammation ◽

10.1155/2009/285812 ◽

2009 ◽

Vol 2009 ◽

pp. 1-9 ◽

Cited By ~ 10

Author(s):

Nathalie Taquet ◽

Serge Dumont ◽

Jean-Luc Vonesch ◽

Didier Hentsch ◽

Jean-Marie Reimund ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Protein Expression ◽

Mrna Expression ◽

Large Scale ◽

Mononuclear Cells ◽

Biologically Active ◽

Chronic Inflammatory Bowel Disease ◽

Peripheral Blood Mononuclear ◽

Mrna And Protein Expression

Crohn's disease (CD) is a multifactorial chronic inflammatory bowel disease of unknown cause. The aim of the present study was to explore if mRNA over-expression of SSTR5 and CCR7 found in CD patients could be correlated to respective protein expression. When compared to healthy donors, SSTR5 was over-expressed 417±71 times in CD peripheral blood mononuclear cells (PBMCs). Flow cytometry experiments showed no correlation between mRNA and protein expression for SSTR5 in PBMCs. In an attempt to find a reason of such a high mRNA expression, SSTR5 present on CD PBMCs were tested and found as biologically active as on healthy cells. In biopsies of CD intestinal tissue, SSTR5 was not over-expressed but CCR7, unchanged in PBMCs, was over-expressed by 10±3 times in the lamina propria. Confocal microscopy showed a good correlation of CCR7 mRNA and protein expression in CD intestinal biopsies. Our data emphasize flow and image cytometry as impossible to circumvent in complement to molecular biology so to avoid false interpretation on receptor expressions. Once confirmed by further large-scale studies, our preliminary results suggest a role for SSTR5 and CCR7 in CD pathogenesis.

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Parathyroid hormone stimulates endothelial expression of atherosclerotic parameters through protein kinase pathways

AJP Renal Physiology ◽

10.1152/ajprenal.00406.2006 ◽

2007 ◽

Vol 292 (4) ◽

pp. F1215-F1218 ◽

Cited By ~ 77

Author(s):

Gloria Rashid ◽

Jacques Bernheim ◽

Janice Green ◽

Sydney Benchetrit

Keyword(s):

Parathyroid Hormone ◽

Protein Kinase ◽

Protein Expression ◽

Mrna Expression ◽

Umbilical Vein ◽

Protein Levels ◽

Glycation End Products ◽

Mrna And Protein Expression ◽

Vascular Structures ◽

The Impact

Parathyroid hormone (PTH), the major systemic calcium-regulating hormone, has been linked to uremic vascular changes. Considering the possible deleterious action of PTH on vascular structures, it seemed logical to evaluate the impact of PTH on the receptor of advanced glycation end products (RAGE) and interleukin 6 (IL-6) mRNA and protein expression, taking into account that such parameters might be involved in the pathogenesis of vascular calcification, atherosclerosis, and/or arteriolosclerosis. Human umbilical vein cord endothelial cells (HUVEC) were stimulated for 24 h with 10−12–10−10 mol/l PTH. The mRNA expression of RAGE and IL-6 was established by reverse transcriptase/PCR techniques. RAGE protein levels were determined by Western blot and IL-6 secretion was measured by ELISA. The pathways by which PTH may have an effect on HUVEC functions were evaluated. PTH (10−11–10−10mol/l) significantly increased RAGE mRNA and protein expression. PTH also significantly increased IL-6 mRNA expression without changes at protein levels. The addition of protein kinase (PKC or PKA) inhibitors or nitric oxide (NO) synthase inhibitors significantly reduced the RAGE and IL-6 mRNA expression and the RAGE protein expression. PTH stimulates the mRNA expressions of RAGE and IL-6 and the protein expression of RAGE. These stimulatory effects are probably through PKC and PKA pathways and are also NO dependent. Such data may explain the possible impact of PTH on the atherosclerotic and arteriosclerotic progression.

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