Protein kinase Cδ expression in breast cancer as measured by real-time PCR, western blotting and ELISA

Background Oxidative stress plays crucial roles in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). Thioredoxin-interacting protein (TXNIP) is essential in the process of triggering oxidative stress. However, its role and mechanism in CRSwNP remain unclear. The present study sought to explore the role and mechanism of TXNIP in the pathogenesis of CRSwNP. Methods Western blotting, real-time PCR and immunohistochemistry (IHC) were employed to assess TXNIP, thioredoxin (TRX) expression in nasal tissue samples from patients with CRSwNP and control subjects. MDA level and SOD activity in nasal tissue homogenates were measured using MDA and SOD Assay Kit. To evaluate the role and mechanism of TXNIP in CRSwNP, human nasal epithelial cells (HNECs) were cultured and stimulated using TXNIP siRNA, with or without N-acetylcysteine (NAC, an ROS scavenger). Western blotting, real-time PCR, ROS detecting dye DCFH-DA, MDA and SOD Assay Kit were performed to assess the effects and mechanisms of stimulators on the cells. Results We found significantly increased levels of TXNIP and decreased levels of TRX protein, mRNA, positive cells, increased MDA level and decreased SOD activity in CRSwNP patients compared with control subjects. In vitro study, significantly altered levels of TXNIP, TRX, MDA, SOD and ROS in HNECs were found following treatment of TXNIP siRNA with or without NAC on HNECs. Conclusion TXNIP expression was increased and TRX expression was decreased in CRSwNP at both protein and mRNA levels. MDA levels were increased and SOD activities were decreased in CRSwNP. TXNIP may have negative association with TRX, and then decrease SOD activities and increase MDA levels, resulting in the upregulation of ROS and oxidative stress in HNECs, which may play a pivotal role in the pathogenesis of CRSwNP. Future studies are expected to further explore the role and mechanism of TXNIP in CRSwNP.

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Effects of protein kinase Cδ and phospholipase C-γ1 on monocyte chemoattractant protein-1 expression in taxol-induced breast cancer cell death

International Journal of Molecular Medicine ◽

10.3892/ijmm_00000303 ◽

2009 ◽

Vol 24 (06) ◽

Cited By ~ 1

Author(s):

Ko

Keyword(s):

Breast Cancer ◽

Cell Death ◽

Protein Kinase ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Monocyte Chemoattractant Protein 1 ◽

Monocyte Chemoattractant ◽

Monocyte Chemoattractant Protein ◽

Cancer Cell Death ◽

Protein Kinase Cδ

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P4: CIRCULATING MIRNA PROFILES POINT TO DYSREGULATION IN TGFBETA/SMAD3 TUMOR SUPPRESSOR SIGNATURE IN BRCA POSITIVE BREAST CANCER PATIENTS

Journal of Investigative Medicine ◽

10.1136/jim-2016-000080.44 ◽

2016 ◽

Vol 64 (3) ◽

pp. 818.2-818

Author(s):

I Shapira ◽

T Bhuiya ◽

S Arora ◽

N Mukhi ◽

S Datla ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Suppressor ◽

Real Time ◽

Cancer Patients ◽

Survival Data ◽

Real Time Pcr ◽

Germ Line ◽

Germline Mutations ◽

Breast Cancer Patients ◽

Mean Differences

Purpose of StudyOver 240,000 individuals are diagnosed with breast cancer (BrCa) of which 12,000 individuals carry BRCA germline mutations. MicroRNA dysregulation is common in malignancy and may correlate with germline mutations.Aims:1. Analyze microRNAs in patients with breast cancer with or without BRCA germ line mutations, with and without cancer.2. Identify molecular BRCA mutant patients to deduct reasons for accelerated malignancy.Methods UsedWe analyzed plasma miR expression from 94 br cancer patients (41 BRCA positive) relative to 24 normal controls. All samples were collected between 2010 and 2014 and survival data was known for all cancer patients. TaqMan Open Array panel was used to simultaneously run hundreds of microRNA assays in the Applied Biosystem Open array real time PCR. Using AB open array real time PCR, 756 miRNA species were detected. Two-sample t-test was used for all 2-sample comparison and ANOVA followed by Tukey HSD post-hoc test to compare the miRs mean differences. All tests were 2-tailed and results with a p<0.05 were considered statistically significant.Summary of ResultsBRCA+underexpressed hsa-mir-10a and hsa-mir-376c and over-expressed Hsa- mir- 326 and Hsa-mir-143 relative to BRCA-; p<0.05.Using Coremine data mining linking genes and diseases differentially expressed circulating miRs are linked to tumor suppressor TGFbeta/SMAD3.ConclusionsThe early onset of breast cancer in BRCA mutant patients may recapitulate the pro-oncogenic effects of TGF-β. The context dependent SMAD3 binding & tumor suppression TGF-β effects are abrogated in BRCA mutant patients. TGF-β/Smad3 tumor-suppressor signature suppresses local inflammation in the tumor microenvironment.

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Comparing MicroRNA Profilings of Purified HER-2-Negative and HER-2-Positive Cells Validates miR-362-5p/Sema3A as Characteristic Molecular Change in Triple-Negative Breast Cancers

Disease Markers ◽

10.1155/2019/6057280 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12 ◽

Cited By ~ 1

Author(s):

Xiaoqing Zhang ◽

Qi He ◽

Leiqin Sun ◽

Yanfei Zhang ◽

Shengying Qin ◽

...

Keyword(s):

Breast Cancer ◽

Protein Expression ◽

Real Time ◽

Differential Expression ◽

Real Time Pcr ◽

Therapeutic Target ◽

Triple Negative ◽

Quantitative Real Time Pcr ◽

Her 2 ◽

Tnbc Subtype

Background. HER-2 is a key molecule serving as the therapeutic target, prognostic biomarker, and classification marker in breast cancer. Accurate microRNA profilings had not been conducted in purified tumor cells of HER-2-negative and HER-2-positive tissue specimens obtained from breast cancer patients. Methods. (i) Differential expression microRNA discovery using laser capture microdissection- (LCM-) assisted specimen preparation and microRNA array chips on HER-2 overexpressing and triple-negative breast carcinoma (TNBC) subtype tissues, (ii) differential expression microRNA validation by quantitative real-time PCR, and (iii) independent validation on tissue microarray. Results. Five microRNAs (miR-20a-5p, miR-221-3p, miR-362-5p, miR-502-3p, and miR-222-3p) were screened and validated as upregulated microRNAs in TNBC cells comparing to HER-2 overexpressing cells using a microRNA array (5 cases in each group) and quantitative real-time PCR (20 cases in each group). The expression difference of miR-362-5p had the most significant statistical significance (p=0.0016) among the five microRNAs. The expression of miR-362-5p and its target gene Sema3A was further analyzed using in situ hybridization (ISH) and immunohistochemistry on standard tissue sections (n=150). 70.8% of HER-2-negative cells showed moderate expression of miR-362-5p whereas 20.4% HER-2-negative cells correlated with strong expression of miR-362-5p (p<0.0001). The proportion of patients with moderate/strong miR-362-5p expression in luminal, HER-2 overexpressing, and TNBC subtypes were 53.2%, 22.2%, and 74.3%, respectively (p=0.0002). High miR-362-5p expressers had shorter overall survival in the univariate analysis (p=0.046). There was a significant negative correlation between miR-362-5p and Sema3A expression (p<0.0001). The patients with negative/weak Sema3A protein expression had poorer prognosis than those with moderate (HR: 3.723, p=0.021) or strong (HR: 3.966, p=0.013) Sema3A protein expression in the multivariate analysis. Conclusions. miR-362-5p/Sema3A might provide a promising therapeutic pathway and represents a candidate therapeutic target of the TNBC subtype.

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Abstract 4217: A real-time PCR assay for the detection of PIK3CA mutations in formalin-fixed paraffin embedded tissue (FFPET) specimens of breast cancer (BC).

10.1158/1538-7445.am2013-4217 ◽

2013 ◽

Cited By ~ 1

Author(s):

Peter Schlag ◽

Corey Hoeppner ◽

Anona Bristol ◽

Namneet Kaur ◽

Stephen Quashnick ◽

...

Keyword(s):

Breast Cancer ◽

Real Time ◽

Real Time Pcr ◽

Pcr Assay ◽

Pik3ca Mutations ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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Characterization and Localization of Calb2 in Both the Testis and Ovary of the Japanese Flounder (Paralichthys olivaceus)

Animals ◽

10.3390/ani10091503 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1503

Author(s):

Yuting Xiang ◽

Yahui Wu ◽

Haoran Zhang ◽

Jikui Wu ◽

Junling Zhang

Keyword(s):

Real Time ◽

Western Blotting ◽

Real Time Pcr ◽

Paralichthys Olivaceus ◽

Neighboring Gene ◽

Syntenic Relationship ◽

Reading Frame ◽

Cdna Sequences ◽

Its Gene ◽

And Function

Although its function in mammalian gonads has been gradually recognized, the expression and function of calretinin (CALB2)—a Ca2+-binding protein—in the testis and ovary of fish are still unclear. Here, we identified the cDNA sequences of calb2 in Paralichthys olivaceus (P. olivaceus); analyzed its gene structure and phylogenetic and syntenic relationship by bioinformatics; and investigated its tissue distribution and localization in the gonads by real-time PCR, western blotting, and immunohistochemistry. The P. olivaceuscalb2 gene has 11 exons and 10 introns, and the full-length cDNA is 1457 bp, including an open reading frame (ORF) of 816 bp encoding 271 amino acids. The CALB2 of P. olivaceus has a higher homology with Lates calcarifer (99%) compared with other species. The conserved synteny of calb2 neighboring gene loci was also detected in fish. Real-time PCR showed that the expression of calb2 mRNA is abundant not only in the brain, but also in the gonads, and exhibits a higher expression in the testis than in the ovary. Western blotting indicated that the CALB2 protein has a higher expression in the testis compared with the ovary. Immunohistochemistry demonstrated that the CALB2 protein appears in Leydig cells and the ovarian germ epithelium. These results reveal that calb2 plays an important role in the gonads of P. olivaceus.

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