Breast cancer remains a leading cause of cancer-related mortality worldwide, with epigenetic mechanisms like N6 methyladenosine (m6A) modification playing a crucial role in tumorigenesis. The interaction between microRNAs and m6A regulators, such as the methyltransferase METTL14, is increasingly recognized as a key pathway in oncogenesis. This study investigated whether miR-30c-2-3p regulates METTL14 expression to influence global m6A levels and cell migration in breast epithelial (MCF12A) and breast cancer (MCF7) cell lines. Following transfection with miR-30c-2-3p mimics, successful overexpression was confirmed in both cell lines. Subsequent RT-qPCR and Western blotting analyses demonstrated that METTL14 mRNA and protein levels were significantly reduced at 24 and 48 h post-transfection (p < 0.05). Concurrently, global m6A RNA methylation levels decreased, with a more pronounced reduction observed in MCF12A cells (p < 0.001). Functionally, wound healing assays revealed that miR-30c-2-3p significantly inhibited migration, reducing wound closure by 30-44% in MCF7 cells and by 66-72% in MCF12A cells. These findings reveal a novel regulatory axis involving miR-30c-2-3p, METTL14, and m6A, suggesting that miR-30c-2-3p functions as a tumor suppressor and represents a promising biomarker and therapeutic target in breast cancer.

OBJECTIVES:The role of neutrophil extracellular traps (NETs) in IgA vasculitis (IgAV) pathogenesis is emerging, with NETosis-associated markers potentially linked to disease activity. This study aimed to explore the relationship between NETosis biomarkers and IgAV disease phases. METHODS:A longitudinal study involving 33 paediatric IgAV patients and 26 healthy controls was conducted. Blood and urine samples were collected from healthy controls and patients during active and inactive disease phases. NETosis markers, including cell-free DNA (cf-DNA), neutrophil elastase, MPO and citrullinated histone H3 (cit-H3) were measured using ELISA kits. Statistical analyses were conducted to compare differences for NETosis markers between groups and to evaluate correlations among variables using appropriate statistical tests. RESULTS:There was no significant difference in gender and age between the patient and control groups. The serum cf-DNA level was significantly higher in the active patient group compared with the control and inactive patient groups (P = 0.04; P = 0.04, respectively). In urine, MPO levels were significantly lower in the active phase of patients than controls (P = 0.009), while cit-H3 levels were higher in both active and inactive phases compared with controls (P = 0.01 and P = 0.03, respectively). A cf-DNA threshold of 935 ng/ml was identified, which achieved a sensitivity of 93% (correctly identifying 93% of active patients) and a specificity of 72% (correctly identifying 72% of healthy controls). CONCLUSION:Elevated serum cf-DNA and urine cit-H3 suggest a potential role for NETosis in IgAV activity, highlighting these markers as potential indicators for disease monitoring. Further studies are warranted to establish standardized protocols for NETosis marker assessment in IgAV.

OBJECTIVES:This study aimed to investigate the relationship between the neutrophil extracellular traps (NET) and NETosis findings and disease activity in Behçet disease (BD). METHODS:The study group consisted of 30 active BD patients and 10 healthy individuals as controls. Serum and saliva samples were collected from the patients during their active and remission periods. A subset of them were followed longitudinally. Serum and saliva cf-DNA, NE, MPO, and cit-H3 levels were measured as indirect NETosis findings, subsequently the results were adjusted based on the peripheral blood neutrophil counts. Unadjusted and adjusted levels of the NETosis findings were evaluated. RESULTS:In active BD, unadjusted serum cf-DNA and NE levels were significantly higher than in controls, whereas adjusted serum MPO and cit-H3 levels were found to be lower. In inactive BD, unadjusted serum NE levels remained elevated compared to controls, while unadjusted serum MPO, adjusted serum MPO, and adjusted serum cit-H3 levels were lower. No significant differences were observed in salivary NETosis findings between the patients and controls. Longitudinal follow-up revealed a decrease in both unadjusted serum cf-DNA and saliva cf-DNA levels in parallel with reduced clinical activity. Saliva and unadjusted serum cf-DNA showed a positive correlation with inflammatory markers, whereas adjusted serum MPO and cit-H3 correlated negatively. CONCLUSIONS:Indirect NETosis findings varied in relation to the systemic and/or local activity of BD patients. The changes after adjustment suggest that serum NETosis markers can be influenced by increased neutrophil turnover during the active phase of the disease.

The black cumin, Nigella sativa L. is known to be effective against various diseases including cancer. Thymoquinone (TQ), active ingredient of N. sativa extracts, can inhibit proliferation metastasis and regulating immune system in different cancers as with breast cancer (BC). The mechanisms of action behind TQ are not clearly understood yet. The purpose of the current study is to investigate the effects of TQ, water (WE) and alcohol extracts (AE) of N. sativa on BC cells by focusing attention on the following genes ; CDK4 , MYC , NF- kappa B1 , VEGFA , FGF1 , N-cadherin , ULBP1 , ULBP2 and CD155 . Conventional protocols were performed in order to obtain extracts. Cell viability was measured by RTCA and MTT assay, and gene expressions were analyzed by qRT-PCR. Association was significant for CDK4 ( P =0.07), MYC ( P <0.001), NF- kappa B1 ( P =0.011), VEGFA ( P =0.013), FGF1 ( P <0.001), and ULBP1 ( P =0.021) genes. CDK4 and MYC genes may be candidate genes for mechanisms involved in reduced cell proliferation induced by AE and TQ. Increased ULBP1 expression through AE and TQ indicates that N. sativa may trigger ULBP1-mediated NK cell cytotoxicity. Our results support the idea that active ingredients in N. sativa promise an encouraging therapeutic approach in the future.

ObjectivesBeh & ccedil;et disease (BD) has heterogeneous presentations, mainly mucocutaneous, vascular, and ocular manifestations. The mechanisms associated with different phenotypes have not been clarified. We aimed to investigate the expression of innate and adaptive immunity-related cytokines in these 3 main BD phenotypes in active and untreated states and remission after treatment to be able to develop a cytokine-based treatment algorithm.MethodsSerum samples were isolated from 41 patients with newly diagnosed active BD (aBD), which consisted of 19 mucocutaneous aBD, 11 ocular aBD (o-aBD), and 11 vascular aBD patients, 35 patients in remission (rBD), and 9 healthy controls (HC). Serum levels of each cytokine were measured with sandwich enzyme-linked immunosorbent assay and analyzed as both raw measurements and corrected levels for each 1 million white blood cells.ResultsThe study included 41 aBD patients (female/male [F/M]: 9/32; median age, 29 years), 35 rBD patients (F/M: 9/26; median age, 29 years), and 9 HC (F/M: 3/6; median age, 28 years). The serum interferon gamma level was significantly higher in the aBD group than in the rBD (116 vs. 92 pg/mL, p = 0.022). The serum interleukin 35 (IL-35) level was significantly higher in the HC group compared with aBD and rBD (p = 0.05). IL-17-related cytokines were lower in o-aBD. With treatment, they increased in o-aBD but decreased in mucocutaneous aBD and vascular aBD patients.ConclusionThis study supports the involvement of both innate and TH1-predominated adaptive immune responses across all BD phenotypes. The IL-17 and TH17-related immune responses appear less prominent in ocular BD, which may explain the ineffectiveness of IL-17 blockade in treating ocular BD. These findings support the need for further studies using comprehensive gene expression analyses to develop targeted treatment strategies for BD phenotypes.

Objective: High-mobility group box-1 (HMGB1), known as an abundant and highly conserved nuclear protein, plays a pivotal role in initiating inflammation, tissue healing, and the immune response following various forms of cell damage. The chemokine C-X-C motif chemokine ligand 12 (CXCL12) forms a signaling axis known as CXCL12/ CXCR4/CXCR7, along with the receptors CXCR4 and CXCR7. Our study aimed to explore the connection between HMGB1 and the involved chemokine axis, CXCR4, CXCL12, and CXCR7, in the context of adriamycin-induced cardiotoxicity. Materials and Methods: We performed RNA interference to suppress HMGB1 expression in H9c2 cardiac myoblast cells. Adriamycin, an anti-tumor antibiotic known for causing cardiotoxicity, was used in conjunction with HMGB1 suppression. We investigated the combined and individual effects of these factors. Gene expression analysis was conducted through qRT-PCR 36 and 48 h post-treatment. Results: Adriamycin treatment increased the expression of CXCL12, CXCR4, and CXCR7. Notably, our study observed significant changes in gene expression when HMGB1 was downregulated and adriamycin was administered. These findings suggest potential molecular mechanisms associated with adriamycin-induced cardiotoxicity, emphasizing the significance of the CXCR4/CXCL12 axis and the impact of HMGB1 modulation. Conclusion: Our study provides insights into the molecular interplay between HMGB1 and the CXCL12/CXCR4/CXCR7 ligand-receptor axis in the context of adriamycin-induced cardiotoxicity. The results shed light on further research to enhance therapeutic approaches or advance new strategies to address this cardiotoxicity.

OBJECTIVEEndoplasmic reticulum (ER) stress, which occurs as a result of the accumulation of misfolded or unfolded proteins, has been observed in many cancers. To re-establish the ER homeostasis, pathways of unfolded protein response (UPR) are activated. The effect of ER stress-activated UPR pathways on leukemogenesis has not been elucidated. In this study, we aimed to find out whether activated UPR pathways were involved in acute myeloid leukemia (AML).METHODSExpression levels of the 2 UPR components, binding immunoglobulin protein (BiP), and X-box protein 1 (XBP1), as well as the HLA-B were analyzed in 100 newly diagnosed AML patients using quantitative and qualitative polymerase chain reaction techniques.RESULTSFifteen of the 100 newly diagnosed AML patients were shown to carry the spliced XBP1 (XBP1s) variant. Furthermore, in patients with acute promyelocytic leukemia (APL), a subtype of AML, expression levels of XBP1, and BiP were significantly higher than in non-APL patients (p<0.001 and p<0.015, respectively). It was determined that 6 of 15 (40%) AML patients carrying XBP1s variant were positive for myeloperoxidase.CONCLUSIONOur study provides adequate evidences for the involvement of activated UPR pathways in some of the AML patients by showing the presence XBP1s variant and increased expression levels for XBP1 and BiP. Thus, agents targeting XBP1s might serve as a new approach for anti-cancer therapy.

Abstract Objectives Acute myeloid leukemia (AML) is a highly heterogeneous disease. Although patients can be classified into risk groups based on their genetic changes, the prognosis of disease within these categories varies widely. This situation raises the need to search for new molecular markers related to AML. Serine peptidase inhibitor Kazal type 2 (SPINK2) has recently been reported to be upregulated in AML and associated with poor outcomes by meta-analysis and in a limited number of AML patients. Methods We analyzed SPINK2 mRNA expression in 62 patients (45 adult and 17 pediatric) with AML and 11 cell lines using quantitative Real-Time PCR (qRT-PCR). SPINK2 protein level was determined using ELISA in cell lines. Results We found that the expression of SPINK2 mRNA and protein levels in AML cell lines (HL60 and NB4) have increased compared to other cell lines (K562, Jurkat and NALM6, MCF7, HeLa, HUVEC, hFOB, 293T, U87). SPINK2 mRNA expression was upregulated in patients with AML compared to controls (p=0.004) and significantly lower in t(8;21)-positive patients compared to negative patients (p=0.0006). Conclusions Our results suggest that SPINK2 serves an important role in AML development. Further studies are needed to evaluate SPINK2 expression in AML patients with t(8.21) and investigate to clarify its prognostic value in various subgroups of AML.

OBJECTIVE:The skin pathergy test (SPT) is an important tool in the diagnosis of Behçet disease (BD), but its decreasing sensitivity over years has resulted in less frequent use in the clinical practice. This study aimed to improve the sensitivity of the SPT without compromising its specificity. METHODS:BD patients, patients with other inflammatory diseases, recurrent aphthous stomatitis, and healthy controls comprised the study group. The SPT was conducted using 20G needle and 21G lancet pricks, or with additional application of 23-valent polysaccharide pneumococcal vaccine (PS-23), Alum, or ATP to the prick site. Development of erythema and induration at 24 h/48 h were evaluated by the same observer. Induration (≥2 mm) with erythema at 48 h was accepted as a positive reaction. Proinflammatory cytokine production following stimulation with lipopolysaccharide or PS-23 was investigated by whole-blood assay (WBA) in a subgroup. RESULTS:Stimulation of the forearm skin by PS-23 and a 20G needle prick showed the highest sensitivity and specificity in BD (64.3% and 100%, respectively), especially in patients with active disease (80.3% and 100%, respectively), compared with a sensitivity of 4.8% in all and 6.1% in active patients using a single 20G prick. A positive result was associated with active disease and no use of immunosuppressives. In WBA, increased IL-1β and IL-1Ra production in response to PS-23 was observed in the group with active BD, while the cytokine response to lipopolysaccharide was similar in all groups. CONCLUSIONS:The SPT conducted using a 20G needle prick and PS-23 antigens was shown to be a promising tool for the diagnosis of BD owing to its improved sensitivity compared with the standard approach.

Resumen Objectivos La leucemia mieloide aguda (AML, por sus siglas en inglés) es una enfermedad muy heterogénea. Aunque se puede clasificar a los pacientes en grupos de riesgo según sus mutaciones genéticas, el pronóstico dentro de cada categoría varía sustancialmente. Es perentorio identificar nuevos marcadores moleculares de la AML. Recientemente, se ha descrito la elevación del inhibidor de la serina peptidasa Kazal tipo 2 ( SPINK2 ) en la AML, habiendo sido relacionada con peores resultados clínicos en metaanálisis, así como en un número limitado de pacientes con AML. Métodos Analizamos la expresión de SPINK2 en 62 pacientes (45 adultos y 17 niños) con AML y en 11 líneas celulares mediante PCR cuantitativa (qRT-PCR). Los niveles de la proteína SPINK2 se determinaron en líneas celulares mediante ELISA. Resultados Observamos un aumento de expresión del ARNm de SPINK2 y de los niveles de la proteína en las líneas celulares de AML (HL60 y NB4), frente a otras líneas celulares (K562, Jurkat y NALM6, MCF7, HeLa, HUVEC, hFOB, 293T, U87). Los pacientes con AML mostraron una expresión elevada de ARNm de SPINK2 frente a los controles (p=0,004) y esta fue significativamente menor en los pacientes t(8;21) positivos, frente a los pacientes negativos (p=0,0006). Conclusions Estos resultados sugieren que el gen SPINK2 tiene un papel relevante en el desarrollo de la AML. Son necesarios más estudios para evaluar la expresión de SPINK2 en los pacientes con AML con la mutación t(8.21) e investigar su valor pronóstico en varios subgrupos de pacientes con AML.

PURPOSEAdriamycin (ADR) is a commonly used anti-cancer drug. ADR has toxic effects on cardiomyocytes and leads to heart failure. However, the underlying mechanism(s) by which ADR causes heart failure is still not clarified exactly. The aim of present study is to investigate whether ADR-induced heart failure is mediated via HMGB1/TLR4 to initiate the apoptosis through MAPK/AMPK pathways.METHODSH9c2 cell line was used to create four groups as a control, HMGB1 inhibition, ADR, ADR+HMGB1 inhibition. Silencing HMGB1 was performed with specific small interfering RNA. ADR was used at 2 µM concentration for 36 and 48 hours. Protein and genes expressions, apoptosis was measured.RESULTSAlthough ADR decreased AMPK, pAMPK, ERK1/2, pERK1/2, p38, JNK protein expression, ADR+HMGB1 inhibition led to change those protein expressions. The effect of silencing of HMGB1 prevented apoptosis induced by ADR in the cells. HMGB1 caused changes a kind of posttranscriptional modification on the TLR4 receptor. This posttranscriptional modification of TLR4 receptor led to decreased AMPK protein level, but phosphorylated-AMPK. This alternation of AMPK protein caused enhancing of JNK protein, resulting from the decline of p38 and ERK protein levels. Eventually, JNK triggered apoptosis by a caspase-dependent pathway. The number of TUNEL positive and active caspase 8 cells at ADR was high, although HMGB1 silencing could decrease the cell numbers.CONCLUSIONSInhibition of HMGB1 might prevent the lose of the cardiac cell by inhibition of apoptotic pathway, therefore HMGB1 plays an essential role as amplifying on ADR toxicity on the heart by TLR4.

The promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) fusion gene is present in 98% of the patients with acute promyelocytic leukemia (APL), the M3 subtype of acute myeloid leukemia (AML). All-trans retinoic acid (ATRA) is widely used to treat patients with APL. Nucleolar Protein 7 (NOL7) is a tumor suppressor gene regulated by retinoid X receptor (RXR). The 6p23 chromosomal region, where NOL7 is located, is associated with many cancers, including AML. Here, we aimed to investigate the effect of NOL7 in two AML cell lines (the PML-RARA-positive NB4 cell line and the PML-RARA-negative HL60 cell line) and in the resistance to ATRA. For this purpose, we knocked down NOL7 expression by using short interfering RNA (siRNA) and stimulated the cells with ATRA. Apoptosis, cell viability, proliferation, and granulocytic differentiation analyses were performed. Our findings show that granulocytic differentiation was blocked in ATRA-treated NB4 cells 24 hours after NOL7 siRNA transfection when the expression of NOL7 had been reduced by 81%. Downregulation of NOL7 had no apparent effect on the cellular proliferation and apoptosis. Our study suggests that ATRA-induced granulocytic differentiation is inhibited in NOL7-downregulated NB4 cells. These results suggest that NOL7 expression contributes to ATRA-induced granulocytic differentiation and loss of NOL7 might be involved in the development of ATRA resistance.

BACKGROUND:Acute myeloid leukemia (AML) is an aggressive hematological malignancy caused by a variety of genetic abnormalities and epigenetic dysregulation. The incidence of AML is strongly related to age, with the highest incidence rates being in older adults. The loss of function mutations in BCOR and BCORL1 genes have been identified in AML. BCL6 corepressor (BCOR) and BCL6 corepressor like 1 (BCORL1) are important epigenetic regu-lators as a member of Polycomb repressive complex 1 (PRC1.1), involved in histone modification processes. METHODS:We analyzed the BCOR and BCORL1 mRNA expression in 74 adult and 22 pediatric patients with AML by Real-Time quantitative PCR in this study. RESULTS:Our results indicated that both BCOR and BCORL1 mRNA expressions decrease with age (p = 0.009 and p = 0.008, respectively) and there is a positive correlation between BCOR and BCORL1 mRNA expression (p < 0.001). BCOR and BCORL1 mRNA expressions were not significantly different in both adult and pediatric patients with AML compared to control (p > 0.05). CONCLUSIONS:Our findings indicate that expression of BCOR and BCORL1 mRNA are down-regulated with age. The increase in AML incidence with age suggests that age-associated BCOR and BCORL1 down-regulation might potentially contribute to age-related epigenetic alterations and form a predisposing condition for the development of elderly AML.

Objective: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease resulting from the accumulation of genetic changes that affect the development of T-cells. The precise role of lymphoid enhancer-binding factor 1 (LEF1) in T-ALL has been controversial since both overexpression and inactivating LEF1 mutations have been reported to date. Here, we investigate the potential gene targets of LEF1 in the Jurkat human T-cell leukemia cell line. Materials and Methods: We used small interfering RNA (siRNA) technology to knock down LEF1 in Jurkat cells and then compared the gene expression levels in the LEF1 knockdown cells with nontargeting siRNA-transfected and non-transfected cells by employing microarray analysis. Results: We identified DHRS2, a tumor suppressor gene, as the most significantly downregulated gene in LEF1 knockdown cells, and we further confirmed its downregulation by real-time quantitative polymerase chain reaction (qRT-PCR) in mRNA and at protein level by western blotting. Conclusion: Our results revealed that DHRS2 is positively regulated by LEF1 in Jurkat cells, which indicates the capability of LEF1 as a tumor suppressor and, together with previous reports, suggests that LEF1 exhibits a regulatory role in T-ALL via not only its oncogenic targets but also tumor suppressor genes.

Background: Behcet's disease (BD) is a rare, chronic autoinflammatory disorder of unknown origin. Natural killer (NK) cells are one of the major immunoregulatory cell groups of the innate immune system, but their role in BD pathogenesis is not well documented.Objectives: We aimed to investigate the role of NK cell subsets and their cytokine secretion and cytotoxic activity in patients with BD.Patients and methods: The study group consisted of BD patients who had only mucocutaneous involvement, and they were compared with healthy subjects. BD patients were divided into two groups according to their frequencies of oral ulcerations. NK cell cytotoxicity was determined using CD107a expression and a CFSE-based cytotoxicity test. Expression of NK cell receptors and surface markers and the intracellular IL-5, IL-10, IL-17, and IFN- levels in CD16(+) NK cells were assessed by flow cytometry.Results: Although the cytokine secretion pattern was different, no difference was obtained in cytotoxic activity, expression of activatory receptors, or degranulation of NK cells.Conclusion: Increases in NK1/NK2 ratio and CD16(+)IFN-(+) NK1 cells might support the idea of a biased IFN- dominant immune response in the mucocutaneous involvement of BD pathogenesis. Although the cytokine secretion pattern was different, no difference was obtained in cytotoxic activity, expression of activatory receptors, or degranulation of NK cells.