الفهرس 
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Abstract
Breast cancer is the most common malignancy among females and it is the first leading cause of cancer-related death in females worldwide. One of the main obstacles in the treatment of advanced BC is anticancer drug resistance, which can be caused by inherent and acquired factors including genomic instability, tumour heterogeneity, cancer stem cells (CSCs), microenvironment of tumour through interactions occur directly between growth factors, stromal cells, cytokines and extracellular matrix, as well as epigenetic changes which alter molecular/signaling pathways leading to poor survival.
Epigenetics is the study of heritable changes in gene expression that do not affect primary DNA sequence. Epigenetic mechanisms that alter chromatin structure can be divided into four main mechanisms: covalent histone modifications, methylation of DNA, noncovalent modifications including incorporation of histone variants and nucleosome remodeling as well as ncRNAs such as miRNAs. Recent findings indicated that epigenetic alterations have emerged as fundamental players in breast cancer development and progression.
Zinc finger HIT-type containing 1, is a component of SRCAP complex, which induces DNA-end resection required for repairing DNA double-strand breaks. Specifically, this complex can alter chromatin structure by promoting the inclusion of H2AZ-H2B dimers into nucleosomes. Znhit1 is overexpressed in response to genotoxic stresses and activating transcription of numerous p53 target genes.
A hypoxic tumor microenvironment regulates the stemness phenotype in BCSCs. The activation of HIFs is one of the fundamental mechanisms of stemness maintenance triggered by hypoxia. HIF-2α plays an important role in promoting different biological processes in cancer cells, such as cell metabolism, cell proliferation, EMT, angiogenesis and drug resistance. Moreover, HIF-2α is a crucial element in stimulating a CSC state with a high specificity and selectivity for CSCs.
Breast cancer stem cells are identified as a subpopulation of cancer cells capable of self- renewal and generation of the heterogeneous populations of BC cells. This concept indicated the cellular origin, tumour maintenance and progression of BC. CD44+/CD24−/low, ALDH1+ and LGR5+ are the most commonly used biomarkers to identify the BCSC phenotype.
Aldehyde dehydrogenase 1 is an intracellular enzyme catalyzing the oxidation of intracellular aldehydes. It contributes to differentiation of normal and tumour stem cells. ALDH1A1 is the main ALDH isozyme related to SCs. ALDH1A1 also plays a crucial role as a marker of normal SCs as well as CSCs.
Leucine-rich repeat-containing G protein-coupled receptor 5 also known as GPR49, is a member of the G protein-coupled receptor family of transmembrane receptors that transduce signals from external stimuli. LGR5 promotes BC progression and CSCs maintenance partially via stimulation of Wnt/β-catenin signaling pathway.
Beta-catenin is a multifunctional protein found on the plasma membrane’s intracellular side. It plays a key structural role in cell-to-cell adhesion by connecting the cadherin and cytoskeleton and regulatory function as a transcriptional co-activator of the Wnt signal transduction pathway.
The aim of this study was to investigate Znhit1 and Hypoxia-inducible factor 2α gene expression and their impact on stemness markers LGR5, ALDH1A1 and β-catenin in tissue and serum of breast cancer patients.
The present study was conducted on eighty females. They were divided into two groups:
group I: Included 40 apparently normal healthy female volunteers served as control group.
group II: Included 40 females of matched menstrual state, age and socioeconomic status as the previous group having breast carcinoma of clinical stage II- III.
5 ml of venous blood was withdrawn from all BC patients before surgery and from the healthy control group. Blood samples were allowed to clot for 30 minutes then centrifuged for 10 minutes at approximately 3000×g for separating serum. Serum was removed and stored at -80℃ until used for assaying ALDH1A1, LGR5 and β-catenin levels by ELISA.
Human BC tissues and adjacent normal breast tissues were collected from patients who underwent surgical resection and were stored at -80 C until used for determination of the following:
Znhit1 and HIF-2α gene expression by qRT-PCR. Levels of ALDH1A1, LGR5 and β-catenin in tissue homogenate by ELISA.
The results of the present study revealed that:
1. The mean fold change of Znhit1 gene expression in BC tissues was statistically significantly lower than that in adjacent normal breast tissues.
2. The mean fold change of HIF-2α gene expression in BC tissues was statistically significantly higher than that in adjacent normal breast tissues. 3. The mean values of ALDH1A1, LGR5 and β-catenin in tissue and serum of BC patients were statistically significantly higher than that in adjacent normal breast tissue and serum of control subjects respectively.
4. Znhit1 gene expression was statistically significantly negatively correlated with clinical stage and β-catenin levels in breast cancer patients.
5. HIF-2α gene expression was statistically significantly positively correlated with Her-2/neu expression and β-catenin levels in breast cancer patients.
6. Tissue ALDH1A1 was statistically significantly positively correlated with serum ALDH1A1. Moreover, ALDH1A1 either in tissue or serum was statistically significantly positively correlated with LGR5 and β-catenin in tissue and serum of breast cancer patients.
7. Tissue LGR5 was statistically significantly positively correlated with serum LGR5. Moreover, LGR5 either in tissue or serum was statistically significantly negatively correlated with PR status, while it was positively correlated with β-catenin in tissue and serum of breast cancer patients.
8. Tissue β-catenin was statistically significantly positively correlated with serum β-catenin.
9. Each of serum ALDH1A1, LGR5 and β-catenin had high diagnostic efficacy in breast cancer.