Dual silencing of tumor-intrinsic VISTA and CTLA-4 stimulates T-cell mediated immune responses and inhibits MCF7 breast cancer development.

BACKGROUND: As inhibitory immune checkpoint molecules, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and V-domain Ig suppressor of T-cell activation (VISTA) can be expressed in tumoral cells and facilitate immune evasion of tumoral cells. Herein, we studied the significance of tumor-intrinsic CTLA-4 and VISTA silencing in tumor development and inflammatory factors expression in a co-culture system with MCF7 and T-cells. METHODS: MCF7 cells were transfected with 60 pmol of CTLA-siRNA, VISTA-siRNA, and dual VISTA-/CTLA-4-siRNA. The MTT assay was performed to study the effect of CTLA-4 and VISTA knockdown on the viability of MCF7 cells. Colony formation and wound-healing assays were performed to investigate the effect of CTLA-4 and VISTA silencing on the clonogenicity and migration of MCF7 cells. Flow cytometry was used to study the significance of CTLA-4 and VISTA knockdown on the apoptosis and cell cycle of MCF7 cells. Also, a co-culture system with MCF7 and T-cells was developed to study the expression levels of IL-2, IFN-γ, TNF-α, TGF-ß, and IL-10 following CTLA-4 and VISTA knockdown. The expression levels of caspase3, Bax, Bcl2, and MMP-9 were also investigated using quantitative real-time PCR. Finally, the TCGA Breast Cancer and GSE45827 datasets were analyzed to study the potential prognostic values of VISTA and CTLA-4, their expression difference in luminal A breast cancer and non-tumoral tissues, and their correlation in luminal A breast cancer tissues. RESULTS: Combined knockdown of tumor-intrinsic VISTA and CTLA-4 is superior in upregulating IL-2, IFN-γ, and TNF-α, downregulating TGF-ß and IL-10 in T lymphocytes. Also, the combined silencing arrests the cell cycle at the sub-G1 phase, decreases migration, inhibits clonogenicity, and reduces cell viability of MCF7 cells. This combined treatment upregulates caspase 9 and BAX and downregulates MMP-9 in MCF7 cells. Our in-silico results have demonstrated a significant positive correlation between CTLA-4 and VISTA in luminal A breast cancer. CONCLUSION: The additive effect of the combined knockdown of tumor-intrinsic VISTA and CTLA-4 can substantially upregulate pro-inflammatory factors, downregulate anti-inflammatory factors, and inhibit tumor development in MCF7 cells. The significant positive correlation between VISTA and CTLA-4 in luminal A breast cancer might support the idea that a network of inhibitory immune checkpoint molecules regulates anti-tumoral immune responses; thus, combinational immune checkpoint molecules blockade can be suggested.

Unraveling dedifferentiation and metastasis traces in pancreatic ductal adenocarcinoma ductal cells: Insights from single-cell RNA sequencing analysis of ITGB4 and C19orf33.

Pancreatic Ductal Adenocarcinoma (PDAC) ranks among the most prevalent gastrointestinal malignancies, with risk factors including smoking, alcohol abuse, diabetes mellitus, obesity, age, family history, and genetic predisposition. Extensive research has focused on unraveling biomarkers and molecular intricacies associated with PDAC. Leveraging data from the Gene Expression Omnibus microarray and single-cell RNA sequencing datasets, our study identified ITGB4 and C19orf33 as potentially differentially expressed genes in PDAC samples when contrasted with non-malignant tissues. Notably, these genes exhibited a strong correlative expression pattern, primarily within ductal cells. Gene Expression Profiling Interactive Analysis corroborated our findings, further confirming the correlation between ITGB4 and C19orf33. Additionally, we conducted experiments involving two pivotal PDAC-related cell lines, MIA PaCa-2 and PANC-1, treated with oxaliplatin and 5-Fluorouracil. We also assessed the expression of these candidate genes in PDAC samples in comparison to adjacent normal tissues. Our findings revealed that C19orf33 is upregulated in PDAC samples, and treatment of PDAC cells with chemotherapeutic agents led to a correlated decrease in the expression of both ITGB4 and C19orf33. These co-expressed and correlated genes are implicated in relevant signaling pathways, suggesting shared biological activities that may contribute to the promotion of metastasis within malignant ductal cells. This study identifies ITGB4 and C19orf33 as key genes potentially shedding light on the molecular mechanisms driving tumorigenesis and metastasis in PDAC. These genes hold promise as potential diagnostic and therapeutic targets, offering valuable insights into the management of this challenging disease.

The role of probiotic supplementation in inflammatory biomarkers in adults: an umbrella meta-analysis of randomized controlled trials.

OBJECTIVE: Despite the increasing evidence for probiotics' anti-inflammatory effects, the results of meta-analyses remain inconsistent. The present umbrella meta-analysis aimed to investigate the effects of probiotic supplementation on inflammatory biomarkers. METHODS: We performed a wide-ranging systematic search in several databases, including PubMed, Web of Science, Scopus, EMBASE, and Google Scholar up to April 2023. The overall effect sizes were calculated using effect size (ES) values and their corresponding confidence intervals (CI). RESULTS: Out of a total of 580 related articles, 39 studies were qualified for inclusion in the analysis. The results of the analysis revealed a significant reduction of C-reactive protein (CRP) (ES = -1.02; 95% CI: -1.23, -0.80, p < 0.001; I2: 94.1%, p < 0.001), TNF-α (ES = -0.35; 95% CI: -0.50, -0.20, p < 0.001; I2: 75.6%, p < 0.001), and interleukin-6 (IL-6) levels (ES = -0.36; 95% CI: -0.59, -0.13, p = 0.002; I2: 85.6%, p < 0.001), following probiotic supplementation. CONCLUSION: Probiotic supplementation significantly reduced serum concentrations of TNF-a, CRP, and IL-6. Thus, probiotic supplementation can be considered adjuvant therapy to alleviate inflammation in various inflammatory conditions.

Regulation of Dendritic Cell Functions by Vitamins as Promising Therapeutic Strategy for Immune System Disorders.

A functional immune system is crucial for a healthy life, protecting from infections, tumors, or autoimmune disorders; these are accomplished by the interaction between various immune cells. Nourishment, particularly micronutrients, are very important components in the immune system balance, therefore this review emphasizes the vitamins (D, E, A, C) and Dendritic cells' subsets due to vitamins' roles in immune processes, especially on dendritic cells' functions, maturation, and cytokine production. Current studies reveal significant benefits related to vitamins, including vitamin E, which can contribute to the control of dendritic cells' function and maturation. Furthermore, vitamin D plays an immunoregulatory and anti-inflammatory role in the immune system. Metabolite of vitamin A which is called retinoic acid leads to T cells' differentiation to T helper 1 or T helper 17, so low levels of this vitamin exacerbate the menace of infectious diseases, and vitamin C has anti-oxidant effects on dendritic cells and modulate their activation and differentiation program. Additionally, the correlation between the amount of vitamin and the occurrence or progression of allergic diseases and autoimmunity disorders is discussed according to the results of previous studies.

Targeting Nanog expression increased Cisplatin chemosensitivity and inhibited cell migration in Gastric cancer cells.

Regardless of significant advances in cancer treatment, gastric cancer (GC) incidence rate is increasing worldwide. As one of the main transcription factors participating in stemness, Nanog plays a pivotal role in various aspects of tumorigenesis, metastasis, and chemosensitivity. Given that, the current research intended to evaluate the potential effects of Nanog suppression on the GC cell Cisplatin chemosensitivity and in vitro tumorigenesis. First, bioinformatics analysis was performed to evaluate the effect of Nanog expression on GC patients' survival. The MKN-45 human GC cells were transfected with specific siRNA targeting Nanog and/or treated with Cisplatin. Then, to study cellular viability and apoptosis, MTT assay and Annexin V/PI staining were done, respectively. Also, the scratch assay was performed to investigate cell migration, and MKN-45 cell stemness was followed using colony formation assay. Western blotting and qRT-PCR were used for gene expression analysis. The findings demonstrated that Nanog overexpression was significantly correlated with poor survival of GC patients, and siRNA-mediated Nanog silencing strongly increased MKN-45 cell sensitivity to Cisplatin through apoptosis induction. Also, Nanog suppression combined with Cisplatin resulted in the upregulation of the Caspase-3 and Bax/Bcl-2 ratio at mRNA levels and increased Caspase-3 activation. Moreover, reduced expression of Nanog, separately or combined with Cisplatin, inhibited MKN-45 cell migration by downregulating MMP2 mRNA and protein expression levels. The results also evidenced CD44 and SOX-2 downregulation aligned with a decreased rate of MKN-45 cell colony formation ability through treatments. Besides, Nanog downregulation significantly decreased MDR-1 mRNA expression. Taken together, the results of this study indicated that Nanog could be suggested as a promising target combined with Cisplatin-based GC therapies for reducing drug side effects and improving patients' outcomes.

Combination of B7H6-siRNA and temozolomide synergistically reduces stemness and migration properties of glioblastoma cancer cells.

Glioblastoma multiforme (GBM) is among the malignant brain tumors of the central nervous system (CNS). The survival of this disease is about 14 months after diagnosis. To date, temozolomide is known as first-line therapy for glioma. Drug resistance and severe side effects against this drug are important obstacles to the effective treatment of this cancer. Small interfering RNA (siRNA) can adjust the expression of several genes and is used as a new method of gene therapy. Recent studies have shown that siRNAs can increase the sensitivity of cancer cells to chemotherapy drugs. This study aimed to understand the potential role and molecular mechanism of the combination therapy of B7H6-siRNA and temozolomide in glioblastoma cancer. U87 cells were treated with B7H6-siRNA and temozolomide, separately and in combination. Cell viability, stemness, cell migration, and apoptosis were measured. The results of this work presented the synergistic effect of B7H6-siRNA and temozolomide in inhibiting the cancerous features of the U87 cell line. Down-regulating B7H6-siRNA expression inhibited the cell viability of U87 glioblastoma cancer cells and increased their sensitivity to temozolomide. In addition, a noteworthy decrease in cell migration ability and stemness, an increase in apoptosis were observed in the combined groups compared to B7H6-siRNA and temozolomide individually. According to the results, a combination of B7H6-siRNA and temozolomide can be a promising strategy in glioblastoma cancer therapy.

The emerging role of noncoding RNAs in systemic lupus erythematosus: new insights into the master regulators of disease pathogenesis.

Auto-immune diseases are a form of chronic disorders in which the immune system destroys the body's cells due to a loss of tolerance to self-antigens. Systemic lupus erythematosus (SLE), identified by the production of autoantibodies in different body parts, is one of the most well-known examples of these diseases. Although the etiology of SLE is unclear, the disease's progression may be affected by genetic and environmental factors. As studies in twins provide adequate evidence for genetic involvement in the SLE, other phenomena such as metallization, histone modifications, and alterations in the expression of noncoding RNAs (ncRNAs) also indicate the involvement of epigenetic factors in this disease. Among all the epigenetic alterations, ncRNAs appear to have the most crucial contribution to the pathogenesis of SLE. The ncRNAs' length and size are divided into three main classes: micro RNAs, long noncoding RNAs (LncRNA), and circular RNAs (circRNAs). Accumulating evidence suggests that dysregulations in these ncRNAs contributed to the pathogenesis of SLE. Hence, clarifying the function of these groups of ncRNAs in the pathophysiology of SLE provides a deeper understanding of the disease. It also opens up new opportunities to develop targeted therapies for this disease.

Regulatory Effects of Long Non-coding RNAs on Th17/Treg Differentiation and Imbalance.

Scientific research over the past decades has proven the pivotal role of long non-coding RNAs (LncRNAs) in regulating gene expression. The immune responses are controlled through the interaction of pro-inflammatory (predominance of T helper 17 cells (Th17)) and anti-inflammatory cytokines excretion (predominance of Regulatory T cells (Treg)). Recent studies have marked the impact of many diverse LncRNAs on Treg/Th17 imbalances. Moreover, some of the roots and causes of human diseases can be associated with the alterations in the Th17/Treg ratio. In this review study, we overviewed the association between LncRNAs and Th17/Treg, with the potential of providing novel prognostic and diagnostic biomarkers and promising therapeutic targets in various diseases, particularly cancer.

The modulatory role of dendritic cell-T cell cross-talk in breast cancer: Challenges and prospects.

Antigen recognition and presentation are highlighted as the first steps in developing specialized antigen responses. Dendritic cells (DCs) are outstanding professional antigen-presenting cells (APCs) responsible for priming cellular immunity in pathological states, including cancer. However, the diminished or repressed function of DCs is thought to be a substantial mechanism through which tumors escape from the immune system. In this regard, DCs obtained from breast cancer (BC) patients represent a notably weakened potency to encourage specific T-cell responses. Additionally, impaired DC-T-cell cross-talk in BC facilitates the immune evade of cancer cells and is connected with tumor advancement, immune tolerance, and adverse prognosis for patients. In this review we aim to highlight the available knowledge on DC-T-cell interactions in BC aggressiveness and show its therapeutic potential in BC treatment.

CTLA-4 silencing in dendritic cells loaded with colorectal cancer cell lysate improves autologous T cell responses in vitro.

Dendritic cell (DC)-based immunotherapy has increased interest among anti-cancer immunotherapies. Nevertheless, the immunosuppressive mechanisms in the tumor milieu, e.g., inhibitory immune checkpoint molecules, have been implicated in diminishing the efficacy of DC-mediated anti-tumoral immune responses. Therefore, the main challenge is to overcome inhibitory immune checkpoint molecules and provoke efficient T-cell responses to antigens specifically expressed by cancerous cells. Among the inhibitory immune checkpoints, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression on DCs diminishes their maturation and antigen presentation capability. Accordingly, we hypothesized that the expression of CTLA-4 on DCs inhibits the T cell-mediated anti-tumoral responses generated following the presentation of tumor antigens by DCs to T lymphocytes. In this study, we loaded colorectal cancer (CRC) cell lysate on DCs and inhibited the expression of CTLA-4 by small interfering RNA (siRNA) in them to investigate the DCs' functional and phenotypical features, and T-cell mediated responses following DC/T cell co-culture. Our results demonstrated that blockade of CTLA-4 could promote stimulatory properties of DCs. In addition, CTLA-4 silenced CRC cell lysate-loaded DCs compared to the DCs without CTLA-4 silencing resulted in augmented T cell proliferation and cytokine production, i.e., IFN-γ and IL-4. Taken together, our findings suggest CTLA-4 silenced CRC cell lysate-loaded DCs as a promising therapeutic approach however further studies are needed before this strategy can be used in clinical practice.

Inflammatory reflex disruption in COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified in Wuhan, China, in late 2019 and caused coronavirus disease 2019 (COVID-19), which is still a global pandemic. In most infected people, SARS-CoV-2 can only cause moderate symptoms, while in other patients, it leads to severe illness and eventually death. Although the main clinical manifestation of COVID-19 is often seen in the lungs, this disease affects almost all body organs. The excessive and prolonged release of inflammatory cytokines that may occur in COVID-19 patients, known as cytokine storms, stimulates undesired immune responses and can cause various tissues damage. In the current review article, we focus on the potential advantages of the intrinsic cholinergic anti-inflammatory pathway (CAP) as the efferent arm of inflammatory reflex in COVID-19 management. Considering this endogenous protective mechanism against chronic inflammation, we focused on the effects of SARS-CoV-2 in the destruction of this anti-inflammatory system. Several studies indicated the interaction of SARS-CoV-2 with the alpha7 subtype of the nicotinic acetylcholine receptor as the effector molecule of the inflammatory reflex. On the other hand, neurological manifestations have increasingly been identified as significant extrapulmonary manifestations of COVID-19. The rational connection between these findings and COVID-19 pathogenesis may be an important issue in both our understanding and dealing with this disease. COVID-19 is deeply rooted in our daily life and requires an urgent need for the establishment of effective therapeutic options, and all the possible treatments must be considered for the control of such inflammatory conditions.

The regulatory role of autophagy-related miRNAs in lung cancer drug resistance.

Autophagy is conserved cellular machinery that degrades un-usable proteins and cellular components and has a crucial role in the pathogenesis and drug resistance of various diseases such as lung cancer (LC). Multiple types of endogenous molecules (i.e. miRNAs) have been found to regulate multiple biological processes, such as autophagy. Dysfunction of these molecules is associated with the onset and progression of a variety of human malignancies. Several studies had shown that some miRNAs could mediate autophagy activity in LC cells, which would affect drug resistance as a major problem in LC therapy. Therefore, identifying the underlying molecular targets of miRNAs and their function in autophagy pathways could develop new treatment interventions for LC patients. In this review, we will summarize the interplay between miRNAs, autophagy, and drug resistance of LC patients, as well as the genes and molecular pathways that are involved.

Enhancement of chemosensitivity by simultaneously silencing of Mcl-1 and Survivin genes using small interfering RNA in human myelomonocytic leukaemia.

Acute myeloid leukaemia (AML) is a genetically heterogeneous, severe and rapidly progressing disease triggered by blocking granulocyte or monocyte differentiation and maturation. Overexpression of myeloid cell leukaemia-1 (Mcl-1) and Survivin is associated with drug resistance, tumour progression and inhibition of apoptotic mechanisms in leukaemia and several cancers. In the present study, we examined the combined effect of etoposide and dual siRNA-mediated silencing of Mcl-1 and Survivin on U-937 AML cells. The AML cells were co-transfected with Mcl-1 and Survivin-specific siRNAs and genes silencing were confirmed by quantitative real-time PCR and Western blotting. Subsequently, MTT assay was used for the evaluation of cytotoxic effects by dual siRNA and etoposide on their own and in combination. For the studying of apoptosis, DNA-histone ELISA and annexin-V/FITC assays were performed. Co-transfection of Mcl-1 and Survivin siRNA significantly blocked their expression at the mRNA and protein levels, leading to the induction of apoptosis and strong inhibition of growth (p < .05). Besides, combined treatment of etoposide with Mcl-1 and Survivin siRNAs co-transfection leads to synergistically enhance etoposide-induced cytotoxic and apoptotic effects (p < .05). The results showed that Mcl-1 and Survivin play a major role in the U937 cells survival and their resistance relative to etoposide. Thus, Mcl-1 and Survivin can be considered as promising molecular targets for the treatment of AML. The combination treatment with etoposide, and siRNA-mediated silencing of corresponding genes may be a novel strategy in chemoresistance AML treatment.