Effects of CD44 siRNA on inhibition, survival, and apoptosis of breast cancer cell lines (MDA-MB-231 and 4T1).

BACKGROUND: Breast cancer (BC) is one of the most common cancers in the world. Despite the many advances that have been made in treating patients, many patients are still resistant to treatment. CD44 is one of the surface glycoproteins of BC cells that plays an important role in the proliferation of these cells and inhibition of their apoptosis. Therefore, targeting it can be a treatment way for BC patients. METHODS: In this study, the effect of anti-CD44 siRNA on the proliferation, apoptosis, and migration rate of MDA-MB-231 and 4T1 cells was investigated. The techniques used in this study were MTT assay, RT-PCR, and flow cytometry. RESULTS: The apoptosis and proliferation rates in CD44 siRNA-treated cells were higher and lower, respectively, compared to untreated cells. Also, cell migration was less in treated cells compared to untreated cells. CD44 siRNA also decreased the expression of CXCR4, c-myc, Vimentin, ROCK, and MMP-9. CONCLUSION: Finally, CD44 targeting can be a good treatment option to make BC cells more sensitive to apoptosis.

Dopamine receptor agonist cabergoline promotes immunogenic phenotype in human monocyte-derived dendritic cells.

Dendritic cells (DCs) are known as antigen-presenting cells that are capable of regulating immune responses. DCs and T cells can interact mutually to induce antigen-specific T-cell responses. Cabergoline, which is a dopamine (DA) receptor agonist, seems to implement anti-inflammatory properties in the immune system, and therefore in the present study the impact of a DA receptor agonist cabergoline on the monocyte-derived DCs (moDCs) was assessed. Immature moDCs were treated with lipopolysaccharide to produce mature DCs (mDCs). The expression of DCs' related surface markers namely: CD11c, HLA-DR, and CD86 was measured by utilizing of flow cytometry. Real-time PCR was the technique of choice to determine the levels at which diverse inflammatory and anti-inflammatory factors in cabergoline-treated and control mDC groups were expressed. DCs treated with cabergoline displayed a significant decrease in CD86 and HLA-DR expression, markers linked to maturation and antigen presentation, respectively. In addition, the cabergoline-mDC group showed a considerable decline in terms of the levels at which IL-10, TGF-ß, and IDO genes were expressed, and an increase in the expression of TNF-α and IL-12 in comparison to the mDC control group. Our findings revealed that cabergoline as an immunomodulatory agent can relatively shift DCs into an immunogenic state, and there is a requirement for further investigations to evaluate the effects of cabergoline-treated DCs on the T cell responses in vitro, and also in various diseases including cancer in animal models.

MicroRNA-193a and taxol combination: A new strategy for treatment of colorectal cancer.

Micro RNAs (miRNAs) show a considerable promise as a therapeutic agent for combination therapy of colorectal cancer (CRC). Given that, the current study was purposed to explore the potential therapeutic role and underlying mechanism of miR-193a as a promising tumor suppressor in human CRC cell lines in combination with Taxol. Therefore, HT-29 cells with the lowest expression levels of miR-193a were treated with miR-193a mimics and Taxol, separately or in combination. Functional analyses showed that the combination therapy inhibited migration and colony formation of HT-29 cells and arrested the cell cycle at the G1 phase. Moreover, treatment with Taxol reduced cell survival with an increase in mRNA expression of metastasis-related genes caspase-3 and caspase-9, whereas miR-193a transfection alone didn't significantly influence cell viability and apoptosis induction. Quantitative reverse transcription polymerase chain reaction results also revealed that miR-193a replacement decreased the expression levels of c-Myc, MMP-9, vimentin, and ROCK in treatment groups compared to the controls. Therefore, it could be concluded miR-193a inactivates cell migration via suppression of metastasis pathways in CRC and through downregulation of c-Myc, acts as a negative regulator of cell cycle and growth. Then, our findings imply that miR-193a replacement combined with Taxol chemotherapy could be considered as a new potential therapeutic approach for improvement of CRC treatment.

The role of microRNAs involved in PI3-kinase signaling pathway in colorectal cancer.

In recent decades, cancer has been one of the most important concerns of the human community, which affects human life from many different ways, such as breast, lung, colorectal, prostate, and other cancers. Colorectal cancer is one of the most commonly diagnosed cancers in the world that has recently been introduced as the third leading cause of cancer deaths in the world. microRNAs have a very crucial role in tumorgenesis and prevention of cancer, which plays a significant role with influencing various factors through different signaling pathways. Phosphoinositide 3 (PI3)-kinase/AKT is one of the most important signaling pathways involved in the control and growth of tumor in colorectal cancer, through important proteins of this pathway, such as PTEN and AKT, that they can perform specific influence on this process. Our effort in this study is to collect microRNAs that act as tumor suppressors and oncomirs in this cancer through PI3-kinase/AKT signaling pathway.

The effect of combined miR-200c replacement and cisplatin on apoptosis induction and inhibition of gastric cancer cell line migration.

One of the major obstacles in the treatment of cancer is resistance to standard chemotherapeutic drugs. According to the numerous reports, miR-200c is involved in many cancers, especially gastric cancer, and also miR-200c has been known as an effective factor in the elimination of chemotherapy resistance. The purpose of this study was to explore the potential function and mechanism of miR-200c and cisplatin in the inhibition of migration and induction of apoptosis in gastric cancer cells. In this study, first, miR-200c mimics and LNA-anti-miR-200c were transfected into KATOIII cells. Moreover, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay results revealed that increased miR-200c expression and cisplatin can more inhibited the proliferation of KATOIII cells. MiR-200c overexpression inhibited the movement of KATOIII cells in wound healing assay. Subsequently, miR-200c/cisplatin could suppress colony formation in KATOIII cells. To identify a potential miR-200c target, we investigated the effect of miR-200c modulation on RhoE, PTEN, VEGFR, and MMP9 expression levels. Increased miR-200c expression caused a reduction in VEGFR and MMP9 mRNA and protein, suggesting that VEGFR and MMP9 are targets of miR-200c. In addition, reverse-transcription polymerase chain reaction assays showed that RhoE is target gene of miR-200c and LNA-anti-miR-200c suppressed the expression of PTEN. Flow cytometry and 4',6-diamidino-2-phenylindole staining analysis indicated that miR-200c increased the cisplatin-induced apoptosis which may be associated with suppression of RhoE expression in KATOIII cells, also cell-cycle analysis showed the arrest of cell-cycle progression at the G2 phase. These data demonstrated that miR-200c functioned as a suppressor gene in KATOIII cells. Also, miR-200c can be a potential therapeutic approach to overcome chemoresistance during cisplatin chemotherapy.

Role of miR-142 in the pathogenesis of osteosarcoma and its potential as therapeutic approach.

Osteosarcoma (OS) is the most common primary malignant tumor of the bone with a strong tendency to early metastasis, and occurs in growing bones more commonly in children and adolescents. Considering the limited therapeutic methods and lack of 100% success of these methods, developing innovative therapies with high efficacy and lower side effects is needed. Meanwhile, miRNAs and the studies indicating the involvement of miRNAs in OS development have attracted attentions as a result of the frequent abnormalities in expression of miRNAs in cancer. miRNAs are noncoding short sequences with lengths ranging from 18 to 25 nucleotides that play a very important role in cellular processes, such as proliferation, differentiation, migration, and apoptosis. MiRNAs can have either oncogenic or tumor suppressive role based on cellular function and targets. This review aimed to have overview on miR-142 as a tumor suppressor in OS. Moreover, the genes involved in the disease, such as RAC1, HMAG1, MMP9, MMP2, and E-cadherin, which have irregularities as a result of change in miR-142 expression, and, thereby, result in increasing the proliferation, invasion, and metastasis of the cells in the tissues and OS cells will be discussed.

microRNA-193a-5p inhibits migration of human HT-29 colon cancer cells via suppression of metastasis pathway.

PURPOSE: Altered expression of microRNAs (miRNAs) is indicated strongly in colorectal cancer (CRC). This study aims to evaluate the inhibitory role of miR-193a-5p on epithelial-mesenchymal transition markers in CRC lines. The cellular effects and potential mechanisms of miR-193a-5p were also examined. METHODS: Quantitative reverse-transcription polymerase chain reaction (RT-PCR) was performed to determine the expression of miR-193a-5p in three CRC cell lines (HCT-116, SW-480, and HT-29) and its impact on metastasis-related genes ( vimentin and CXCR4) before and after mimic transfection. Of those, the cell line with the highest changes was selected for the next upcoming experiments such as wound-healing assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and annexin-V staining tests. RESULTS: Our results revealed that miR-193a-5p was significantly downregulated in three CRC cell lines and that HT-29 displayed the most decrease ( P < 0.0001). The restoration of miR-193a-5p in human HT-29 cell line inhibited cell migration. But, miR-193a-5p transfection did not affect cell viability and had no significant effect on apoptosis induction. Also, the quantitative RT-PCR analysis of miR-193a-5p mimic transfected cells revealed a significant increase in miR-193a-5p messenger RNA (mRNA) expression level ( P < 0.0001) with reduction of vimentin and CXCR4 mRNA expression levels in HT-29 cell line ( P < 0.01 and < 0.05, respectively). CONCLUSION: Our results indicated that miR-193a-5p acts as a tumor suppressor miRNA and its downregulation plays an important role in metastasis via upregulation of metastasis-related genes in CRC. Therefore, it can be considered as a potential therapeutic target for applying in CRC management in the future.

Downregulation of miR-146a promotes cell migration in Helicobacter pylori-negative gastric cancer.

microRNAs (miRs) are short noncoding RNAs that post-transcriptionally suppress gene expression. miR-146a acts as an oncogene or a tumor suppressor in various cancers, including gastric cancer, but it is unclear what determines whether miR-146a is oncogenic or tumor suppressive and the molecular mechanisms are still largely unknown. The aim of this study was to investigate the role of miR-146a in gastric cancer, by focusing on its expression in patients who were negative for Helicobacter pylori and its reduced and increased expression effect in vitro. Twenty gastric cancer patients who were negative for H. pylori infection were selected and the expression levels of miRNA-146a in these gastric tumors, in their matched normal gastric tissues and in gastric cancer cell lines with varying tumorigenic potential was measured. Further, the impact of increased and decreased miR-146a expression levels on the expression of predicted target genes, cell migration, viability, proliferation, and apoptosis was examined, respectively. Our results for the first time indicated that miR-146a is downregulated in H. pylori-negative gastric cancers and suggests that H. pylori infection determines whether miR-146a acts as an oncogene or tumor suppressor. The level of miR-146a expression inversely correlates with the tumorigenicity of three gastric cancer cell lines and low miR-146a expression predicts poor recurrence-free survival. It was also found that miR-146a reduces the expression levels of the prometastatic genes and suppresses MKN-45 cell migration. Functional studies showed that miR-146a acts as a tumor suppressor miR and identifies miR-146a as a candidate for antimetastatic miRNA replacement therapy for gastric cancer patients.

Evaluation of Monocyte Subpopulations in Patients with Systemic Sclerosis and its Association with Clinical Manifestations of the Disease: a Cross-sectional Controlled Study.

Background: Systemic sclerosis (SSc) is a chronic autoimmune disorder characterized not only by fibrosis and vasculopathy but also by inflammation. Previous studies have demonstrated monocyte involvement in SSc development, suggesting a role for immune dysfunction in SSc pathogenesis. Objective: To investigate the relationship between SSc's clinical manifestations and altered levels of monocyte subpopulations. Methods: Twenty-six patients meeting the ACR/EULAR SSc criteria along with twenty healthy individuals as the control group, were enrolled in the study. Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized blood samples of both the SSc patients and the control group. Subpopulations of monocytes were assessed based on HLA-DR, CD14, and CD16 expression using multi-color flow cytometry. The one-way ANOVA, Student's t-test, and Mann-Whitney U test were employed for normally and non-normally distributed data. The Spearman correlation test was utilized to identify correlations between the variables. Results: The SSc patients showed a significant increase in the number of circulating peripheral blood monocytes (p<0.001). The percentage of CD16+ monocyte subpopulations was higher in the SSc cases compared to the control group. A significant decrease in the ratio of classic to non-classic monocytes was observed in SSc cases (7.43%) compared to the control group (52.09%, p<0.001). No association was observed between monocyte subpopulations and clinical characteristics of SSC. Conclusion: Our results showed an increase in the level of CD16+ monocytes in patients with SSc compared to healthy individuals. Further investigation is required to determine the clinical significance of this alteration.

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.

Glyburide-treated human monocyte-derived dendritic cells loaded with insulin represent tolerogenic features with anti-inflammatory responses and modulate autologous T cell responses in vitro.

Tolerogenic dendritic cells (TolDCs) are attractive therapeutic options for autoimmune disorders because they suppress autologous T-cell responses. Dendritic cells (DCs) are equipped with pattern recognition receptors (PRR), including nucleotide-binding and oligomerization domain-like receptors (NLRs) such as NLRP3. Abnormal NLRP3 activation has been reported to be correlated with the occurrence of autoimmune disorders. Accordingly, we hypothesized that glyburide treatment of DCs by blocking the ATP-sensitive K+ (kATP) channels generates TolDCs by inhibiting NLRP3. Insulin was even loaded on a group of glyburide-treated mature DCs (mDCs) to investigate the antigen (Ag) loading effects on glyburide-treated mDCs' phenotypical and functional features. Consequently, T lymphocytes' mediated responses ensuing co-culture of them with control mDCs, insulin loaded and unloaded glyburide treated mDCs were evaluated to determine generated TolDCs' capacity in inhibition of T cell responses that are inducer of destruction in insulin-producing pancreatic beta cells in Type 1 Diabetes Mellitus (T1DM). Our findings indicated that glyburide generates desirable TolDCs with decreased surface expression of maturation and Ag presentation related markers and diminished level of inflammatory but increased level of anti-inflammatory cytokines, which even insulin loading demonstrated more anti-inflammatory functions. In addition, co-cultured T cells showed regulatory or T helper 2 phenotype instead of T helper 1 features. Our findings suggested that insulin-loaded and unloaded glyburide-treated DCs are promising therapeutic approaches for autoimmune patients, specifically DCs loaded with insulin for T1DM patients. However, further research is required before this technique can be applied in clinical practice.

Everolimus treatment enhances inhibitory immune checkpoint molecules' expression in monocyte-derived dendritic cells.

BACKGROUND: Antigen-specific T-cell immunity is provided by dendritic cells (DCs), which are specialized antigen-presenting cells. Furthermore, they establish a link between innate and adaptive immune responses. Currently, DC modification is a new approach for the therapy of several disorders. During solid organ transplantation, Everolimus, which is a mammalian target of rapamycin (mTOR) inhibitor, was initially utilized to suppress the immune system's functionality. Due to the intervention of Everolimus in various signaling pathways in cells and its modulatory properties on the immune system, this study aims to investigate the effect of treatment with Everolimus on the maturation and expression of immune checkpoint genes in monocyte-derived DCs. METHODS: To isolate monocytes from PBMCs, the CD14 marker was used via the MACS method. Monocytes were cultured and induced to differentiate into monocyte-derived DCs by utilizing GM-CSF and IL-4 cytokines. On the fifth day, immature DCs were treated with Everolimus and incubated for 24 h. On the sixth day, the flow cytometry technique was used to investigate the effect of Everolimus on the phenotypic characteristics of DCs. In the end, the expression of immune checkpoint genes in both the Everolimus-treated and untreated DCs groups was assessed using the real-time PCR method. RESULTS: The findings of this research demonstrated that the administration of Everolimus to DCs led to a notable rise in human leukocyte antigen (HLA)-DR expression and a decrease in CD11c expression. Furthermore, there was a significant increase in the expression of immune checkpoint molecules, namely CTLA-4, VISTA, PD-L1, and BTLA, in DCs treated with Everolimus. CONCLUSION: The findings of this study show that Everolimus can target DCs and affect their phenotype and function in order to shift them toward a partially tolerogenic state. However, additional research is required to gain a comprehensive understanding of the precise impact of Everolimus on the activation status of DCs.

The Simultaneous Effects of miR-145-5p and hsa-let-7a-3p on Colorectal Tumorigenesis: In Vitro Evidence.

Purpose: MicroRNAs (miRNAs) are a group of small regulatory non-coding RNAs, which are dysregulated through tumor progression. let-7 and MIR-145 are both tumor suppressor microRNAs that are downregulated in a wide array of cancers including colorectal cancer (CRC). Methods: This study was aimed to investigate the effect of simultaneous replacement of these two tumor suppressor miRNAs on proliferation, apoptosis, and migration of CRC cells. HCT-116 with lower expression levels of hsa-let-7a-3p and MIR-145-5p was selected for functional investigations. The cells were cultured and transfected with hsa-let-7a and MIR-145, separately and in combination. Cell viability and apoptosis rates were assessed by MTT assay and flow cytometry, respectively. Cell cycle status was further evaluated using flow cytometry and qRT-PCR was employed to evaluate gene expression. Results: The obtained results showed that exogenous overexpression of MIR-145 and hsa-let-7a in HCT-116 cells could cooperatively decrease CRC cell proliferation and induce sub-G1 cell cycle arrest. Moreover, hsa-let-7a and MIR-145 co-transfection significantly increased apoptosis induction compared to separate transfected cells and control through modulating the expression levels of apoptosis-related genes including Bax, Bcl-2, P53, Caspase-3, Caspase-8, and Caspase-9. Furthermore, qRT-PCR results illustrated that hsa-let-7a and MIR-145 combination more effectively downregulated MMP-9 and MMP-2 expression, as the important modulators of metastasis, compared to the controls. Conclusion: Taken together, considering that exogenous overexpression of MIR-145 and hsa-let-7a showed cooperative anti-cancer effects on CRC cells, their combination may be considered as a novel therapeutic strategy for the treatment of CRC.

The Effect of miR-4800 Restoration on Proliferation and Migration of Human Breast Cancer Cells In Vitro.

Purpose: MicroRNAs (miRNAs) can contribute to cancer initiation, development, and progression. In this study, the effect of miRNA-4800 restoration on the growth and migration inhibition of human breast cancer (BC) cells was investigated. Methods: For this purpose, transfection of miR-4800 was performed into MDA-MB-231 BC cells using jetPEI. Subsequently, the expression levels of miR-4800 and CXCR4, ROCK1, CD44, and vimentin genes were measured using quantitative real-time polymerase chain reaction (q-RT-PCR) and specific primers. Also, the proliferation inhibition and apoptosis induction of cancer cells were evaluated by MTT and flow cytometry (Annexin V-PI method) techniques, respectively. Additionally, cancer cell migration after miR-4800 transfection was assessed by wound-healing (scratch) assay. Results: The restoration of miR-4800 in MDA-MB-231 cells resulted in the decreased expression level of CXCR4 (P ˂ 0.01), ROCK1 (P ˂ 0.0001), CD44 (P ˂ 0.0001), and vimentin (P ˂ 0.0001) genes. Also, MTT results showed restoration of miR-4800 could significantly reduce cell viability rate (P ˂ 0.0001) compared with the control group. Cell migration remarkably inhibited (P ˂ 0.001) upon miR-4800 transfection in treated BC cells. Flow cytometry data demonstrated that miR-4800 replacement considerably induced apoptosis in cancer cells (P ˂ 0.001) compared with control cells. Conclusion: Taken together, it seems that miR-4800 can act as a tumor suppressor miRNA in BC and play an essential role in modulating apoptosis, migration, and metastasis in BC. Therefore, it may be suggested as a potential therapeutic target in treating BC by performing additional tests in the future.

Photodynamic therapy with zinc phthalocyanine enhances the anti-cancer effect of tamoxifen in breast cancer cell line: Promising combination treatment against triple-negative breast cancer?

Photodynamic therapy (PDT) is a light-based anti-neoplastic therapeutic approach. Growing evidence indicates that combining conventional anti-cancer therapies with PDT can be a promising approach to treat malignancies. Herein, we aimed to investigate anti-cancer effects of the combination treatment of zinc phthalocyanine (ZnPc)-PDT with tamoxifen (TA) on MDA-MB-231 cells (as a triple-negative breast cancer (TNBC) cell line). For this purpose, we investigated the cytotoxicity of TA and ZnPc-PDT on MDA-MB-231 cells performing the MTT assay. The effect of TA and ZnPc-PDT on the apoptosis of MDA-MB-231 cells was studied using Annexin V/PI and DAPI staining. The wound-healing assay, and colony formation assay were performed to study the effect of TA and ZnPc-PDT on the migration, and clonogenicity of MDA-MB-231 cells, respectively. The qRT-PCR was done to study the gene expression of caspase-8, caspase-9, caspase-3, ZEB1, ROCK1, SNAIL1, CD133, CD44, SOX2, and ABCG2 (ATP-binding cassette sub-family G member 2). Based on our results, monotherapies with TA and ZnPc-PDT can remarkably increase cell cytotoxicity effects, stimulate apoptosis via downregulating Bcl-2 and upregulating caspase-3 and caspase-9, inhibit migration via downregulating SNAIL1 and ZEB1, and suppress clonogenicity via downregulating SOX2 and CD44 in MDA-MB-231 cells. Besides, these monotherapies can downregulate the expression of ABCG2 in MDA-MB-231 cells. Nevertheless, the combination treatment can potentiate the above-mentioned anti-cancer effects compared to monotherapy with TA. Of interest, the combined treatment of TA with ZnPc-PDT can synergically increase cell cytotoxicity effects on MDA-MB-231 cells. In fact, synergistic effects were estimated by calculation of Combination Index (CI); that synergistic outcomes were observed in all groups. Also, this combination treatment can significantly upregulate the caspase-8 gene expression and downregulate ROCK1 and CD133 gene expression in MDA-MB-231 cells. Overall, our results show that ZnPc-PDT can more sensitize the MDA-MB-231 cells to TA treatment. Based on our knowledge and experiment, the synergistic effects of ZnPc-PDT and TA deserve further evaluation in cancer research.

STA-21, a small molecule STAT3 inhibitor, ameliorates experimental autoimmune encephalomyelitis by altering Th-17/Treg balance.

BACKGROUND: Numerous studies have demonstrated the role of T helper (Th) 17 and T regulatory (reg) cells and pro-inflammatory and anti-inflammatory cytokines related to these cells in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). STAT3 is one of the downstream signaling proteins of IL-23, IL-6, and IL-21 that are required for Th17 cells differentiation. STA-21 is a STAT3 inhibitor that functions by inhibiting STAT3 dimerization and binding to DNA impairing the expression of STAT3 target genes including, RORγt, IL-21 and IL-23R that are also required for Th17 cell differentiation. AIM: In this study, we evaluated the effect of STA-21 on EAE Model and investigated how this small molecule can change Th17/Treg balance leading to amelioration of disease. METHODS: After EAE induction and treatment with STA-21, its effects were assessed. Major assays were H&E and LFB staining, Flow cytometric analysis, Reverse transcription-PCR (RT-PCR), and ELISA. RESULTS: STA-21 ameliorated the EAE severity and decreased the EAE inflammation and demyelination. It also decreased STAT3 phosphorylation, the proportion of Th17 cells and the protein level of IL-17. In contrast, the balance of Tregs and the level of anti-inflammatory cytokine, IL-10 increased in STA-21-treated mice. Moreover, STA-21 significantly decreased the expression of Th17 related transcription factors, RORɣt and IL-23R while FOXP3 expression associated with Treg differentiation was increased. CONCLUSION: This study showed that STA-21 has therapeutic effects in EAE by reducing inflammation and shifting inflammatory immune responses to anti-inflammatory and can be used as a suitable treatment strategy for the treatment of EAE. The effectiveness of inhibiting or strengthening the functional cells of the immune system by these small molecules in terms of easy to access, simple construction and inexpensive expansion make them as a suitable tool for the treatment of inflammatory and autoimmune diseases.

siRNA-Mediated B7H7 Knockdown in Gastric Cancer Lysate-Loaded Dendritic Cells Amplifies Expansion and Cytokine Secretion of Autologous T Cells.

BACKGROUND: Gastric cancer, ranked as the fifth most common cancer worldwide, presents multiple treatment challenges. These obstacles often arise due to cancer stem cells, which are associated with recurrence, metastasis, and drug resistance. While dendritic cell (DC)-based immunotherapy has shown promise as a therapeutic strategy, its efficacy can be limited by the tumor microenvironment and certain inhibitory immune checkpoint molecules, such as B7H7. SiRNA-medicated knockdown of B7H7 in tumor cell lysate-pulsed DCs can increase cytokine secretion and autologous T lymphocyte expansion. This study aimed to evaluate the impact of B7H7 suppression in gastric cancer cell lysate-pulsed DCs on the stimulatory potential of autologous CD3+ T lymphocytes. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated and monocytes were obtained; then, they were differentiated to immature DCs (iDCs) by GM-CSF and IL-4. Tumor cell lysates from human gastric cancer cell lines were harvested, and iDCs were transformed into mature DCs (mDCs) by stimulating iDCs with tumor cell lysate and lipopolysaccharide. B7H7-siRNA was delivered into mDCs using electroporation, and gene silencing efficiency was assessed. The phenotypic characteristics of iDCs, mDCs, and B7H7-silenced mDCs were evaluated using specific surface markers, an inverted light microscope, and flow cytometry. CD3+ T cells were isolated via magnetically activated cell sorting. They were labeled with CFSE dye and co-cultured with mDCs and B7H7-silenced mDCs to evaluate their ability to induce T-cell proliferation. T-cell proliferation was assessed using flow cytometry. The concentration of TGF-ß, IL-4, and IFN-γ secreted from CD3+ T cells in the co-cultured supernatant was evaluated to investigate the cytokine secretory activity of the cells. RESULTS: Transfection of B7H7 siRNA into mDCs was performed in optimal conditions, and the siRNA transfection effectively reduced B7H7 mRNA expression in a dose-dependent manner. SiRNA-mediated B7H7 knockdown in mDCs enhanced maturation and activation of the DCs, as demonstrated by an increased surface expression of CD11c, CD86, and CD40. Co-culture experiments revealed that B7H7-silenced mDCs had more capacity to induce T cell proliferation compared to non-transfected mDCs. The cytokine production patterns of T cells were also altered. Upon examining the levels of TGF-ß, IL-4, and IFN-γ released by CD3+ T cells in the co-culture supernatant, we found that silencing B7H7 in mDCs resulted in a rise in IL-4 secretion and a reduction in TGF-ß levels compared to mDCs that were not transfected. CONCLUSIONS: The study found that suppressing B7H7 expression in DCs significantly enhances their maturation and stimulatory activity when exposed to gastric cancer cell lysate. These B7H7-silenced DCs can substantially increase cytokine production and promote co-cultured T-cell expansion. Consequently, inhibiting B7H7 in DCs may offer a practical strategy to enhance the ability of DCs to initiate T lymphocyte responses and improve the effectiveness of DC-based cell therapy for cancer patients.

Downregulation of HMGA2 by Small Interfering RNA Affects the Survival, Migration, and Apoptosis of Prostate Cancer Cell Line.

Purpose: To investigate the downregulation of high mobility group AT-hook 2 (HMGA2)expression by small interfering RNAs (siRNAs) in PC3 prostate cancer cell line. HMGA2belongs to the non-histone chromatin-binding protein family that serves as a crucial regulator ofgene transcription. The overexpression of this gene is positively correlated with various prostatecancer (PC)-related properties. Thus, HMGA2 is an emerging target in PC treatment. This studyaimed to examine the impact of siRNAs targeting HMGA2 on the viability, migration, andapoptosis processes of the PC3 PC cell line. Methods: siRNA transfection was conducted with a liposome-mediated approach. The mRNAand protein expression levels for HMGA2 are evaluated by real-time polymerase chain reaction(qRT-PCR) and western blot analysis. The cytotoxic properties of HMGA2-siRNA were measuredby MTT assay on PC3 cells. The migration of PC3 cells was measured by implementing awound-healing assay. Apoptosis measurement was also quantified by TUNEL assay. Results: Transfection with siRNA significantly decreased both mRNA and protein levels of theHMGA2 gene in a dose-dependent manner after 48 hours. Also, we demonstrated that theknockdown of HMGA2 led to a reduction in cell viability, migration ability, and enhancedapoptosis of PC3 cells in vitro. Conclusion: Our findings recommend that the specific siRNA of HMGA2 may efficiently beable to decrease PC progression. Therefore, it may be a promising adjuvant treatment in PC.

microRNA-193a-5p Suppresses the Migratory Ability of Human KATO III Gastric Cancer Cells through Inhibition of Vimentin and MMP-9.

Purpose: microRNA-193a-5p is one of the well-known tumor suppressor miRNAs in the body but in many cases, its expression became reduced in patients suffering from gastric cancer (GC). The main purpose of this study was to restore the function of this miRNA in human GC cells and investigating the effects of enhanced expression of miR-193a-5p on proliferation, apoptosis, and migration of GC cells upon in vitro transfection. Methods: The KATO III GC cells were treated with 100 nM of miR-193a-5p or negative control sequences. Following that, the MTT assay, flow cytometry assay, and wound-healing assay were applied to estimate the impacts of enhanced expression of this miRNA on the viability, apoptosis, and migration rate of the cells, respectively. Moreover, the total RNA was isolated and alterations in the mRNA expression ratio of migratory genes were measured by qRT-PCR techniques. Results: The findings designated that enhanced expression of miR-193a-5p suppressed the migratory ability of the cells, but had no significant effects on cell survival or apoptosis of the transfected cells. In addition, this inhibitory function of miR-193a-5p on the migration rate of the KATO III cell line occurs with concurrent suppression of vimentin and MMP-9 gene expression. Conclusion: It can be concluded that miR-193a-5p negatively influences the migratory ability of the cancerous cells and restoring its effects can be regarded as a promising target of future therapeutic interventions, especially for GC metastasis.

Immunomodulatory and clinical responses to zinc gluconate supplementation in patients with Behçet's disease: A double-blind, randomized placebo-controlled clinical trial.

BACKGROUND & AIMS: Toll-like receptor (TLR) 2 and 4 are involved in the pathogenesis of Behçet's disease (BD). The current study aimed to investigate the effect of zinc supplementation on TLR-2/4 expression and the clinical manifestations of BD. METHODS: In this double-blind placebo-controlled randomized clinical trial, 50 BD patients were randomly allocated into either zinc gluconate (30 mg/day) or placebo groups for 12 weeks. Before and after the intervention, the surface and mRNA expression level of TLR-2 and TLR-4 in the leukocytes, serum level of zinc and tumor necrosis factor-α (TNF-α), quality of life, anthropometric measures, and blood pressure of patients were collected. BD activity was studied using the nonocular Iranian Behçet's disease dynamic activity measure (IBDDAM), Behçet's disease current activity form (BDCAF), and total inflammatory activity index (TIAI) at the pre-and post-intervention phases. The effect sizes were compared between two groups using analysis of covariance. RESULTS: There were significant decrease in TLR-2 mRNA (P = 0.038) and protein expression (P = 0.034) and nonocular IBDDAM score (P = 0.046) in the zinc group compared to placebo at the endpoint. The serum level of zinc was increased in the zinc group (P < 0.001). Zinc supplementation significantly decreased the TLR-4 surface (P = 0.012) and mRNA expression (P = 0.028) within the group. However, this decrease was not significant compared to the placebo group. There was no significant difference between the two groups regarding the serum level of TNF-α, BDCAF, TIAI, quality of life, anthropometric measures, and blood pressure (P > 0.05). CONCLUSIONS: The present study revealed that zinc supplementation significantly improved nonocular IBDDAM score and TLR-2 expression in BD patients. GOV REGISTRATION NUMBER: NCT05098678.