MicroRNAs as the critical regulators of epithelial mesenchymal transition in pancreatic tumor cells.

Pancreatic cancer (PC), as one of the main endocrine and digestive systems malignancies has the highest cancer related mortality in the world. Lack of the evident clinical symptoms and appropriate diagnostic markers in the early stages of tumor progression are the main reasons of the high mortality rate among PC patients. Therefore, it is necessary to investigate the molecular pathways involved in the PC progression, in order to introduce novel early diagnostic methods. Epithelial mesenchymal transition (EMT) is a critical cellular process associated with pancreatic tumor cells invasion and distant metastasis. MicroRNAs (miRNAs) are also important regulators of EMT process. In the present review, we discussed the role of miRNAs in regulation of EMT process during PC progression. It has been reported that the miRNAs mainly regulate the EMT process in pancreatic tumor cells through the regulation of EMT-specific transcription factors and several signaling pathways such as WNT, NOTCH, TGF-ß, JAK/STAT, and PI3K/AKT. Considering the high stability of miRNAs in body fluids and their role in regulation of EMT process, they can be introduced as the non-invasive diagnostic markers in the early stages of malignant pancreatic tumors. This review paves the way to introduce a non-invasive EMT based panel marker for the early tumor detection among PC patients.

Wortmannin Inhibits Cell Growth and Induces Apoptosis in Colorectal Cancer Cells by Suppressing the PI3K/AKT Pathway.

BACKGROUND: Colorectal cancer (CRC) remains a significant contributor to mortality, often exacerbated by metastasis and chemoresistance. Novel therapeutic strategies are imperative to enhance current treatments. The dysregulation of the PI3K/Akt signaling pathway is implicated in CRC progression. This study investigates the therapeutic potential of Wortmannin, combined with 5-fluorouracil (5-FU), to target the PI3K/Akt pathway in CRC. METHODS: Anti-migratory and antiproliferative effects were assessed through wound healing and MTT assays. Apoptosis and cell cycle alterations were evaluated using Annexin V/Propidium Iodide Apoptosis Assay. Wortmannin's impact on the oxidant/antioxidant equilibrium was examined via ROS, SOD, CAT, MDA, and T-SH levels. Downstream target genes of the PI3K/AKT pathway were analyzed at mRNA and protein levels using RTPCR and western blot, respectively. RESULTS: Wortmannin demonstrated a significant inhibitory effect on cell proliferation, modulating survivin, cyclinD1, PI3K, and p-Akt. The PI3K inhibitor attenuated migratory activity, inducing E-cadherin expression. Combined Wortmannin with 5-FU induced apoptosis, increasing cells in sub-G1 via elevated ROS levels. CONCLUSION: This study underscores Wortmannin's potential in inhibiting CRC cell growth and migration through PI3K/Akt pathway modulation. It also highlights its candidacy for further investigation as a promising therapeutic option in colorectal cancer treatment.

G-Protein Signaling Modulator 2 as a Potential Biomarker in Colorectal Cancer: Integrative Analysis Using Genetic Profiling and Pan-Cancer Studies.

Colorectal cancer (CRC) imposes a significant healthcare burden globally, prompting the quest for innovative biomarkers to enhance diagnostic and therapeutic strategies. This study investigates the G-protein signaling modulator (GPSM) family across several cancers and presents a comprehensive pan-cancer analysis of the GPSM2 gene across several gastrointestinal (GI) cancers. Leveraging bioinformatics methodologies, we investigated GPSM2 expression patterns, protein interactions, functional enrichments, prognostic implications, genetic alterations, and immune infiltration associations. Furthermore, the expression of the GPSM2 gene was analyzed using real-time analysis. Our findings reveal a consistent upregulation of GPSM2 expression in all GI cancer datasets analyzed, suggesting its potential as a universal biomarker in GI cancers. Functional enrichment analysis underscores the involvement of GPSM2 in vital pathways, indicating its role in tumor progression. The prognostic assessment indicates that elevated GPSM2 expression correlates with adverse overall and disease-free survival outcomes across multiple GI cancer types. Genetic alteration analysis highlights the prevalence of mutations, particularly missense mutations, in GPSM2. Furthermore, significant correlations between GPSM2 expression and immune cell infiltration are observed, suggesting its involvement in tumor immune evasion mechanisms. Collectively, our study underscores the multifaceted role of GPSM2 in GI cancers, particularly in CRC, emphasizing its potential as a promising biomarker for prognosis and therapeutic targeting. Further functional investigations are warranted to elucidate its clinical utility and therapeutic implications in CRC management.

Carcinogenic roles of MAFG-AS1 in human cancers

The MAF bZIP transcription factor G-antisense RNA 1 (MAFG-AS1) is located on chromosome 17. MAFG-AS1 was upregulated in 15 human cancers. MAFG-AS1 not only suppresses 16 miRNAs but also directly impacts 22 protein-coding genes' expression. Notably, abnormal MAFG-AS1 expression is connected to clinicopathological characteristics and a worse prognosis in a variety of cancers. Moreover, MAFG-AS1 takes its part in the tumorigenesis and progression of various human malignancies by suppressing apoptosis and promoting proliferation, migration, invasion, aerobic glycolysis, ferroptosis, angiogenesis, EMT, and metastasis. Besides, it can predict treatment effectiveness in ER + breast cancer, urothelial bladder carcinoma, and liver cancer by functioning as a trigger of resistance to tamoxifen, sorafenib, and cisplatin. This study systematically presents the functions of MAFG-AS1 in various cancers, as well as the findings of bioinformatics analyses of the MAFG-AS1, which should give clear advice for future research (AU)

Design Principles of a Novel Construct for HBB Gene-Editing and Investigation of Its Gene-Targeting Efficiency in HEK293 Cells.

Beta-thalassemia is one of the most common monogenic inherited disorders worldwide caused by different mutations in the hemoglobin subunit beta (HBB) gene. Genome-editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has raised the hope for life-long gene therapy of beta-thalassemia. In a proof-of-concept study, we describe the detailed design and assess the efficacy of a novel homology-directed repair (HDR)-based CRISPR construct for targeting the HBB locus. The selected sgRNAs were designed and cloned into an optimized CRISPR plasmid. The HDR donor templates containing a reporter and a selection marker flanked by the piggyBac Inverted Tandem Repeat (ITRs), the homology arms and the delta thymidine kinase (ΔTK) gene for negative selection were constructed. The efficiency of on-target mutagenesis by the eSpCas9/sgRNAs was assessed by mismatch assays. HDR-positive cells were isolated by treatment with G418 or selection based on truncated Neuron Growth Factor Receptor (tNGFR) expression using the Magnetic Activated Cell Sorting (MACS) method followed by ganciclovir (GCV) treatment to eliminate cells with random genomic integration of the HDR donor template. In-out PCR and sanger sequencing confirmed HDR in the isolated cells. Our data showed ~ 50% efficiency for co-transfection of CRISPR/donor template plasmids in HEK293 cells and following G418 treatment, the HDR efficiency was detected at ~ 37.5%. Moreover, using a clinically-relevant strategy, HDR events were validated after selection for tNGFR+ cells followed by negative selection for ΔTK by GCV treatment. Thus, our HDR-based gene-editing strategy could efficiently target the HBB locus and enrich for HDR-positive cells.

Carcinogenic roles of MAFG-AS1 in human cancers.

The MAF bZIP transcription factor G-antisense RNA 1 (MAFG-AS1) is located on chromosome 17. MAFG-AS1 was upregulated in 15 human cancers. MAFG-AS1 not only suppresses 16 miRNAs but also directly impacts 22 protein-coding genes' expression. Notably, abnormal MAFG-AS1 expression is connected to clinicopathological characteristics and a worse prognosis in a variety of cancers. Moreover, MAFG-AS1 takes its part in the tumorigenesis and progression of various human malignancies by suppressing apoptosis and promoting proliferation, migration, invasion, aerobic glycolysis, ferroptosis, angiogenesis, EMT, and metastasis. Besides, it can predict treatment effectiveness in ER + breast cancer, urothelial bladder carcinoma, and liver cancer by functioning as a trigger of resistance to tamoxifen, sorafenib, and cisplatin. This study systematically presents the functions of MAFG-AS1 in various cancers, as well as the findings of bioinformatics analyses of the MAFG-AS1, which should give clear advice for future research.

An Update on the Application of CRISPR Technology in Clinical Practice.

The CRISPR/Cas system, an innovative gene-editing tool, is emerging as a promising technique for genome modifications. This straightforward technique was created based on the prokaryotic adaptive immune defense mechanism and employed in the studies on human diseases that proved enormous therapeutic potential. A genetically unique patient mutation in the process of gene therapy can be corrected by the CRISPR method to treat diseases that traditional methods were unable to cure. However, introduction of CRISPR/Cas9 into the clinic will be challenging because we still need to improve the technology's effectiveness, precision, and applications. In this review, we first describe the function and applications of the CRISPR-Cas9 system. We next delineate how this technology could be utilized for gene therapy of various human disorders, including cancer and infectious diseases and highlight the promising examples in the field. Finally, we document current challenges and the potential solutions to overcome these obstacles for the effective use of CRISPR-Cas9 in clinical practice.

The Prognostic Value of ASPHD1 and ZBTB12 in Colorectal Cancer: A Machine Learning-Based Integrated Bioinformatics Approach.

Introduction: Colorectal cancer (CRC) is a common cancer associated with poor outcomes, underscoring a need for the identification of novel prognostic and therapeutic targets to improve outcomes. This study aimed to identify genetic variants and differentially expressed genes (DEGs) using genome-wide DNA and RNA sequencing followed by validation in a large cohort of patients with CRC. Methods: Whole genome and gene expression profiling were used to identify DEGs and genetic alterations in 146 patients with CRC. Gene Ontology, Reactom, GSEA, and Human Disease Ontology were employed to study the biological process and pathways involved in CRC. Survival analysis on dysregulated genes in patients with CRC was conducted using Cox regression and Kaplan-Meier analysis. The STRING database was used to construct a protein-protein interaction (PPI) network. Moreover, candidate genes were subjected to ML-based analysis and the Receiver operating characteristic (ROC) curve. Subsequently, the expression of the identified genes was evaluated by Real-time PCR (RT-PCR) in another cohort of 64 patients with CRC. Gene variants affecting the regulation of candidate gene expressions were further validated followed by Whole Exome Sequencing (WES) in 15 patients with CRC. Results: A total of 3576 DEGs in the early stages of CRC and 2985 DEGs in the advanced stages of CRC were identified. ASPHD1 and ZBTB12 genes were identified as potential prognostic markers. Moreover, the combination of ASPHD and ZBTB12 genes was sensitive, and the two were considered specific markers, with an area under the curve (AUC) of 0.934, 1.00, and 0.986, respectively. The expression levels of these two genes were higher in patients with CRC. Moreover, our data identified two novel genetic variants-the rs925939730 variant in ASPHD1 and the rs1428982750 variant in ZBTB1-as being potentially involved in the regulation of gene expression. Conclusions: Our findings provide a proof of concept for the prognostic values of two novel genes-ASPHD1 and ZBTB12-and their associated variants (rs925939730 and rs1428982750) in CRC, supporting further functional analyses to evaluate the value of emerging biomarkers in colorectal cancer.

Recent Advances in CRISPR/Cas9 Delivery Approaches for Therapeutic Gene Editing of Stem Cells.

Rapid advancement in genome editing technologies has provided new promises for treating neoplasia, cardiovascular, neurodegenerative, and monogenic disorders. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful gene editing tool offering advantages, including high editing efficiency and low cost over the conventional approaches. Human pluripotent stem cells (hPSCs), with their great proliferation and differentiation potential into different cell types, have been exploited in stem cell-based therapy. The potential of hPSCs and the capabilities of CRISPR/Cas9 genome editing has been paradigm-shifting in medical genetics for over two decades. Since hPSCs are categorized as hard-to-transfect cells, there is a critical demand to develop an appropriate and effective approach for CRISPR/Cas9 delivery into these cells. This review focuses on various strategies for CRISPR/Cas9 delivery in stem cells.

Mutational landscape of phenylketonuria in Iran.

To date more than 1000 different variants in the PAH gene have been identified in patients with phenylketonuria (PKU). In Iran, several studies have been performed to investigate the genetics bases of the PKU in different parts of the country. In this study, we have analysed and present an update of the mutational landscape of the PAH gene as well as the population genetics and frequencies of detected variants for each cohort. Published articles on PKU mutations in Iran were identified through a comprehensive PubMed, Google Scholar, Web of Science (ISI), SCOPUS, Elsevier, Wiley Online Library and SID literature search using the terms: "phenylketonuria", "hyperphenylalaninemia", and "PKU" in combination with "Iran", "Iranian population", "mutation analysis", and "Molecular genetics". Among the literature-related to genetics of PKU, 18 studies were on the PKU mutations. According to these studies, in different populations of Iran 1497 patients were included for mutation detection that resulted in detection of 129 different mutations. Results of genetic analysis of the different cohorts of Iranian PKU patients show that the most prevalent mutation in Iran is the pathogenic splice variant c.1066-11G > A, occurring in 19.54% of alleles in the cohort. Four other common mutations were p.Arg261Gln, p.Pro281Leu, c.168 + 5G > C and p.Arg243Ter (8.18%, 6.45%, 5.88% and 3.7%, respectively). One notable feature of the studied populations is its high rate of consanguineous marriages. Considering this feature, determining the prevalent PKU mutations could be advantageous for designing screening and diagnostic panels in Iran.

Effect of citalopram and sertraline on the expression of miRNA- 124, 132, and 16 and their protein targets in patients with depression.

Objectives: This study aimed to evaluate the effect of SSRIs on the expression of miRNAs and their protein targets. Materials and Methods: In a 100 day open-label study of citalopram (n=25) and sertraline (n=25), levels of miRNA 16, 132, and 124 and glucocorticoid receptor (GR), Brain-derived neurotrophic factor (BDNF), and serotonin transporter (SERT) protein expression were measured by QRT-PCR and western blot in healthy control (n=20), patients with depression at the baseline, and same patients after 100 days of treatment. Results: Expression levels of GR and BDNF proteins were lower in the depressed group before treatment as compared with the healthy group (P<0.0001). The SERT level was higher among the depressed group before treatment in comparison with the healthy group (P<0.0001). The level of GR and BDNF significantly increased, and SERT expression decreased after receiving sertraline (P<0.05). When the depressed group received citalopram, only SERT and GR were altered (P<0.05). Among the microRNAs' expression investigated, mir-124 and mir-132 were higher, and mir-16 was lower among the depressed compared with the healthy group (P<0.0001). Individuals receiving citalopram only showed an increase in the expression of mir-16 while administration of sertraline led to a significant increase in the expression of mir-16 and a decrease in mir-124 and mir-132 (P<0.05). Conclusion: This elucidated the relationship between antidepressant treatment and the expression of different microRNA that control gene expression in various pathways involved in depressed patients. Receiving SSRI can affect the level of these proteins and their relevant microRNAs.

MicroRNA-506 as a tumor suppressor in anaplastic thyroid carcinoma by regulation of WNT and NOTCH signaling pathways.

Objectives: Anaplastic thyroid carcinoma (ATC) is an aggressive thyroid tumor type that has a poor prognosis due to its high therapeutic resistance. Since ATC accounts for half of thyroid cancer-related deaths, it is required to introduce novel therapeutic targets to increase survival in ATC patients. WNT and NOTCH signaling pathways are the pivotal regulators of cell proliferation and migration that can be regulated by microRNAs. We assessed the role of miR-506 in the regulation of cell migration, apoptosis, and drug resistance via NOTCH and WNT pathways in ATC cells. Materials and Methods: The levels of miR-506 expressions were assessed in ATC cells and tissues. The levels of NOTCH, WNT, and EMT-related gene expressions were also assessed in miR-506 ectopic expressed cells compared with controls. Cell migration and drug resistance were also evaluated to assess the role of miR-506 in the regulation of ATC aggressiveness. Results: There were significant miR-506 down-regulations in ATC cells and clinical samples compared with normal cells and margins. MiR-506 suppressed NOTCH and WNT signaling pathways through LEF1, DVL, FZD1, HEY2, HES5, and HEY2 down-regulations, and APC and GSK3b up-regulations. MiR-506 significantly inhibited ATC cell migration and EMT (P=0.028). Moreover, miR-506 significantly increased Cisplatin (P=0.004), Paclitaxel (P<0.0001), and Doxorubicin (P=0.0014) sensitivities in ATC cells. Conclusion: MiR-506 regulated EMT, cell migration, and chemoresistance through regulation of WNT and NOTCH signaling pathways in ATC cells. Therefore, after confirmation with animal studies, it can be introduced as an efficient novel therapeutic factor for ATC tumors.

Inherited deletion of 9p22.3-p24.3 and duplication of 18p11.31-p11.32 associated with neurodevelopmental delay: Phenotypic matching of involved genes.

We describe a 3.5-year-old Iranian female child and her affected 10-month-old brother with a maternally inherited derivative chromosome 9 [der(9)]. The postnatally detected rearrangement was finely characterized by aCGH analysis, which revealed a 15.056 Mb deletion of 9p22.3-p24.3p22.3 encompassing 14 OMIM morbid genes such as DOCK8, KANK1, DMRT1 and SMARCA2, and a gain of 3.309 Mb on 18p11.31-p11.32 encompassing USP14, THOC1, COLEC12, SMCHD1 and LPIN2. We aligned the genes affected by detected CNVs to clinical and functional phenotypic features using PhenogramViz. In this regard, the patient's phenotype and CNVs data were entered into PhenogramViz. For the 9p deletion CNV, 53 affected genes were identified and 17 of them were matched to 24 HPO terms describing the patient's phenotypes. Also, for CNV of 18p duplication, 22 affected genes were identified and six of them were matched to 13 phenotypes. Moreover, we used DECIPHER for in-depth characterization of involved genes in detected CNVs and also comparison of patient phenotypes with 9p and 18p genomic imbalances. Based on our filtration strategy, in the 9p22.3-p24.3 region, approximately 80 pathogenic/likely pathogenic/uncertain overlapping CNVs were in DECIPHER. The size of these CNVs ranged from 12.01 kb to 18.45 Mb and 52 CNVs were smaller than 1 Mb in size affecting 10 OMIM morbid genes. The 18p11.31-p11.32 region overlapped 19 CNVs in the DECIPHER database with the size ranging from 23.42 kb to 1.82 Mb. These CNVs affect eight haploinsufficient genes.

Construction and Quantitative Evaluation of a Tissue-Specific Sleeping Beauty by EDL2-Specific Transposase Expression in Esophageal Squamous Carcinoma Cell Line KYSE-30.

Gene delivery to esophageal tissue could provide novel treatments for diseases, such as cancer. The Sleeping Beauty (SB) transposon system, as a natural and non-viral tool, is efficient at transferring transgene into the human genome for human cell genetic engineering. The plasmid-based SB transposon can insert into chromosomes through an accurate recombinase-mediated mechanism, providing long-term expression of transgene integrated into the target cells. In this study, we aimed to investigate the activity of ED-L2 tissue-specific promoter that was engineered from the Epstein-Barr Virus (EBV) and combined with the hyperactive SB100X transposase to achieve the stable expression of T2-Onc3 transposon in esophageal squamous epithelial cells. Here we constructed an SB transposon-based plasmid system to obtain the stable expression of transposon upon introduction of a hyperactive SB transposase under the control of tissue-specific ED-L2 promoter via the lipid-based delivery method in the cultured esophageal squamous cell carcinoma cells. Among established human and mouse cell lines, the (ED-L2)-SB100X transposase was active only in human esophageal stratified squamous epithelial and differentiated keratinocytes derived from skin (KYSE-30 and HaCaT cell lines), where it revealed high promoter activity. Data offered that the 782 bp sequence of ED-L2 promoter has a key role in its activity in vitro. The (ED-L2)-SB100X transposase mediated stable integration of T2-Onc3 in KYSE-30 cells, thereby providing further evidence of the tissue specificity of ED-L2 promoter. The KYSE-30 cells modified with the SB system integrate on average 187 copies of the T2-Onc3 transposon in its genome. In aggregate, the (ED-L2)-SB100X transposase can be efficiently applied for the tissue-specific stable expression of a transgene in human KYSE-30 cells using SB transposon.

EZH2 deregulates BMP, Hedgehog, and Hippo cell signaling pathways in esophageal squamous cell carcinoma.

PURPOSE: Cell signaling pathways play central roles in cellular stemness state, and aberrant activation of these cascades is attributed to the severity of esophageal squamous cell carcinoma (ESCC). In this study, we aimed to determine the potential impact of enhancer of zeste homolog 2 (EZH2) gene on different cell signaling pathways including bone morphogenesis protein (BMP), Hedgehog, and Hippo in ESCC, and to illuminate EZH2-mediated gene regulatory networks in this aggressive malignancy. MATERIALS AND METHODS: EZH2 silencing was performed in two ESCC lines, KYSE-30 and YM-1, followed by gene expression analysis of BMP, Hedgehog, and Hippo signaling using RT-qPCR. EZH2 enforced expression was induced in both cell lines and gene expression of the pathways was evaluated in parallel. The contribution of EZH2 in epithelial-mesenchymal transition (EMT) and cell migration were also evaluated. RESULTS: EZH2 downregulation decreased expression of the vital components of the Hedgehog and Hippo signaling, while EZH2 upregulation significantly increased its levels in both ESCC cell lines. The expression of BMP target genes was either reduced in EZH2-expressing cells or increased in EZH2-silencing cells. Enforced expression of EZH2 stimulated downregulation of epithelial markers and upregulation of mesenchymal markers in KYSE-30 and YM-1 â€‹cells. Significant downregulation of mesenchymal markers was detected following the silencing of EZH2 in the cells. Knocking down EZH2 decreased migration, while enforced expression of EZH2 increased migration in both ESCC lines. CONCLUSIONS: These results may support the promoting role of EZH2 in ESCC tumorigenesis through the recruitment of important cell signaling pathways.

TWIST1 activates cancer stem cell marker genes to promote epithelial-mesenchymal transition and tumorigenesis in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers worldwide. Overexpression of EMT master transcription factors can promote differentiated cells to undergo cancer reprogramming processes and acquire a stem cell-like status. METHODS: The KYSE-30 and YM-1 ESCC cell lines were transduced with retroviruses expressing TWIST1 or GFP and analyzed by quantitative reverse transcription PCR (qRT-PCR), chromatin immunoprecipitation (ChIP), and immunostaining to investigate the correlation between TWIST1 and stemness markers expression. Cells expressing TWIST1 were characterized for mRNA candidates by qRT-PCR and for protein candidates by Flow cytometry and Immunocytochemistry. TWIST1-ESCC cells were also evaluated for apoptosis and drug resistance. RESULTS: Here we identify a role for TWIST1 in the establishment of ESCC cancer stem cell (CSC)-like phenotype, facilitating the transformation of non-CSCs to CSCs. We provide evidence that TWIST1 expression correlates with the expression of CSC markers in ESCC cell lines. ChIP assay results demonstrated that TWIST1 regulates CSC markers, including CD44, SALL4, NANOG, MEIS1, GDF3, and SOX2, through binding to the E-box sequences in their promoters. TWIST1 promoted EMT through E-cadherin downregulation and vimentin upregulation. Moreover, TWIST1 expression repressed apoptosis in ESCC cells through upregulation of Bcl-2 and downregulation of the Bax protein, and increased ABCG2 and ABCC4 transporters expression, which may lead to drug resistance. CONCLUSIONS: These findings support a critical role for TWIST1 in CSC-like generation, EMT progression, and inhibition of apoptosis in ESCC. Thus, TWIST1 represents a therapeutic target for the suppression of esophageal cell transformation to CSCs and ESCC malignancy.

Contribution of TWIST1-EVX1 Axis in Invasiveness of Esophageal Squamous Cell Carcinoma; a Functional Study.

Background: Epithelial-mesenchymal transition (EMT) is a biological process in embryonic development and cancer progression, and different gene families, such as HOX genes, are closely related to this process. Objectives: Our aim in this study was to investigate the correlation between TWIST1 and EVX1 mRNA expression in ESCC patients and also examine the probable regulatory function of TWIST1 on EVX1 expression in human ESCC cell line. Materials and Methods: TWIST1 and EVX1 gene expression patterns were assessed in ESCC patients by relative comparative Real-time PCR then correlated with their clinical characteristics. In silico analysis of the EVX1 gene was conducted. KYSE-30 cells were transduced by a retroviral system to ectopically express TWIST1, followed by qRT-PCR to reveal the correlation between TWIST1 and EVX1 gene expression. Results: The expression of TWIST1 and EVX1 was correlated to each other significantly (p=0.005) in ESCC. Of 28 patients with under/normal expression of TWIST1, 22 samples (78.57%) had over/normal expression of EVX1. TWIST1 overexpression was correlated with advanced stages of the tumor (III, IV) (P = 0.019) and lymph node metastasis. However, EVX1 under expression was associated with lymph node metastasis (p = 0.027) and invasiveness of the disease (P = 0.037) in ESCC. Furthermore, retroviral transduction enforced significant overexpression of TWIST1 in GFP-hTWIST-1 approximately 9-fold compared to GFP control cells, causing a - 8.83- fold reduction in EVX1 mRNA expression significantly. Conclusions: Our results indicated the repressive role of TWIST1 on EVX1 gene expression in ESCC. Therefore, our findings can help dissect the molecular mechanism of ESCC tumorigenesis and discover novel therapeutic targets for ESCC invasion and metastasis.

Rapid Isolation of Gastric Adenocarcinoma Cancer Stem Cells as a Target for Autologous Dendritic Cell-Based Immunotherapy.

Background: Gastric cancer (GC) is a malignancy cause associated with a high death rate in the world. Cancer stem cells (CSCs) are a rare immortal subpopulation of cells within tumors with characteristics of the ability to self-renew, initiate tumor, and differentiate into defined progenies as well as and high resistance to conventional therapies. Objectives: Despite the use of surgery and chemotherapy for GC therapy, there are no efficient therapeutic protocols for it to date. Therefore, rapid isolation of CSCs in order to therapeutic targets, especially immunotherapy is very important. Materials and Methods: Cancerous cell suspension isolated from patients with GC was cultured in the serum-free medium containing EGF, bFGF, LIF, and heparin under non-adherent culture conditions to generate spheres. Expression of mRNA level stemness transcription factors (OCT4, SOX2, SALL4, and Cripto-1), CD44 variable isoforms (CD44s, CD44v3, CD44v6, CD44V8-10) of spheroid-forming single cells compared with gastric normal tissue cells using real time PCR and molecules of CD44, CD54, and EpCAM as gastric CSC markers, and stemness factor Oct4 using flow cytometry, as well as tumorgenicity using subcutaneous injection of sphere-forming cells to nude mice were investigated. Results: Few cancerous cells isolated from patients with GC were able to generate three-dimensional spheroid colonies in the serum-free medium containing EGF, bFGF, LIF, and heparin under non-adherent culture conditions, and form xenograft tumors in immunodeficient nude mice after subcutaneous injection. Spheroid-forming single cells upregulated stemness transcription factors OCT4, SOX2, SALL4, and Cripto-1 that are associated with pluripotency and self-renewal and CD44 isoforms (CD44s, CD44v3, CD44v6, CD44V8-10) compared with gastric normal tissue cells. Finally, molecules of CD44, CD54, and EpCAM as gastric CSC markers and stemness factor Oct4 were expressed in sphere-forming cells. Conclusion: We suggested that the sphere formation and tumorigenicity assays are two procedures, leading to the rapid isolation of cancer cells with certain stem-like properties in order to target CSCs using autologous dendritic cell therapy, especially in patients with advanced disease.

INPP5A/HLA-G1/IL-10/MMP-21 Axis in Progression of Esophageal Squamous Cell Carcinoma

Background: Background: Type I inositol polyphosphate-5-phosphatase A (INPP5A) is involved in different cellular events, including cell proliferation. Since INPP5A, HLAG1, IL-10, and matrix metalloproteinases (MMP)-21 genes play fundamental roles in esophageal squamous cell carcinoma (ESCC) tumorigenesis, we aimed in this study to clarify the possible interplay of these genes and explore the potential of these chemistries as a predictor marker for diagnosis in ESCC disease. Methods: Methods: Gene expression analysis of INPP5A, HLAG-1, IL-10, and MMP-21 was performed using relative comparative real-time PCR in 56 ESCCs compared to their margin normal tissues. Immunohistochemical staining was accomplished for INPP5A in ESCCs. Analysis of ROC curves and the AUC were applied to evaluate the diagnostic capability of the candidate genes. Results: Results: High levels of HLA-G1, MMP-21, and IL-10 were detected in nearly 23.2%, 62.5%, and 53.5% of ESCCs compared to the normal tissues, respectively, whereas INPP5A underexpression was detected in 19.6% of ESCCs, which all tested genes indicated significant correlations with each other. The protein expression level of INPP5A in ESCC tissues was significantly lower than that of the non-tumor esophageal tissues (p = 0.001). Interestingly, the concomitant expression of the INPP5A/HLA-G1, INPP5A/MMP-21, INPP5A/IL-10, HLA-G1/MMP-21, HLA-G1/IL-10, and MMP-21/IL-10 was significantly correlated with several clinicopathological variables. INPP5A, HLA-G1, MMP-21, and IL-10 showed to be the most appropriate candidates to discriminate tumor/non-tumor groups due to the total AUCs of all combinations (>60%). Conclusion: Conclusion: Our results represent a new regulatory axis containing INPP5A/HLAG-1/IL-10/MMP-21 markers in ESCC development and may provide novel insight into the mechanism of immune evasion mediated by the INPP5A/HLAG-1/IL-10/MMP-21 regulatory network in the disease.