The role of microRNA-30c in targeting interleukin 6, as an inflammatory cytokine, in the mesenchymal stem cell: a therapeutic approach in colorectal cancer.

INTRODUCTION: Colorectal cancer (CRC) is the third most prevalent cancer and the second significant cause of cancer-associated death worldwide. The microRNA-30 is a substantial member of the miRNA family and plays a vital role in expanding several cancers. This microRNA potentially targets interleukin 6 as an inflammatory cytokine in CRC. MATERIALS AND METHODS: MSCs were isolated and identified from mice bone marrow and then transduced with lentiviruses containing miR-30C. Transfected MSCs were collected to evaluate IL-6 levels, CT-26 cells were also co-cultured with MSCs, and the effect of apoptosis and IL-6 on the supernatant was assessed. RESULTS: Our result showed the expression of IL-6 mRNA and the level of protein were decreased in the supernatant of miR-30-transduced MSC cells compared to the control group. In addition, the rate of apoptosis was assessed, and the obtained data revealed the induction of apoptosis in CT-26 cells when they are in the vicinity of miR-30c-transduced MSCs. DISCUSSION AND CONCLUSION: We demonstrated that downregulation of miR-30c was significantly correlated with CRC progression and survival. So, the present study elucidated the anticancer effects of miR-30c in CRC and presented a novel target for CRC therapy.

Decrease of Tumor-infiltrating Regulatory T Cells Using Pentoxifylline: An Ex Vivo Analysis in Triple-negative Breast Cancer Mouse Model.

Triple-negative breast cancer (TNBC) is the most aggressive type of BC with the highest percentage of tumor-infiltrating lymphocytes (TILs). Hence, TIL therapy is considered a promising approach to target TNBC. Depletion of regulatory T cells (Tregs) in TILs can improve the antitumor function of TIL therapy. Pentoxifylline (PTXF) is a xanthine derivative that can modulate the nuclear factor kappa B (NF-κB) signaling and probably affect the Treg proportion in TILs. We aimed to evaluate the ex vivo effect of PTXF on the proportion of Treg cells in the TILs derived from a mouse model of TNBC. The 4T1 cells were inoculated subcutaneously to BALB/c mice to induce TNBC. TILs were isolated from tumor tissue by enzymatic digestion and cultured alone or with 4T1 cells for 24, 48, and 72 h in the presence of interleukin (IL)-2 and different concentrations of PTXF. The toxicity of PTXF and its effects on Tregs proportion as well as cytokine production was evaluated using MTT assay, flow cytometry, and ELISA, respectively. PTXF had no significant impact on the viability of TILs. Both 500 and 1000 mg/mL of PTXF decreased the proportion of Tregs in a dose-dependent manner. The level of interferon-g and tumor growth factor-b in TILs supernatant was increased and decreased, respectively. Our data suggest that ex vivo treatment of TILs with pentoxifylline could decrease the proportion of Tregs in the conventional IL-2-mediated TIL expansion and change the cytokine balance of TILs in favor of antitumor immune response.

MicroRNA-30c delivered by bone marrow-mesenchymal stem cells induced apoptosis and diminished cell invasion in U-251 glioblastoma cell line.

BACKGROUND: Glioblastoma multiform (GBM) is the most belligerent and prevalent brain malignancy among adults. Due to the blood-brain barrier (BBB), drug administration is confronted by massive challenges, making resectional surgery the only treatment pipeline. MicroRNAs have recently absorbed the attention of studies for correlating with the progression of various malignancies. miR-30c has been reported to play a role in cell proliferation, metabolism, and apoptosis process. For instance, miR-30c has been reported to regulate apoptosis through the TNF-related apoptosis-inducing ligand (TRAIL). miR-30c also targets IL-6, which further induces apoptosis. Besides, miR-30c inhibits glioma proliferation and its migratory ability. Besides, the overexpression of miR-30c arrested cells at G0 as well as dampening their migration and invasion. However, it has been shown that the expression level of miR-30c was low in glioma. MSCs can migrate toward tumor cells which is called tumor-tropism, in which they are capable of delivering engineered miR-30c based on gap junction and non-intimacy mechanisms. MATERIAL AND METHODS: MiR-30c was cloned into pCDH-CMV-MCS-EF1-copGFP vector utilizing XbaI and EcoRI in order to construct pCDH-miR-30c. Then psPAX2, pMD2.G, and pCDH-miR-30c were co-transfected into Hek-293T to yield lenti-miR-30c virus particles. Next, bone marrow-mesenchymal stem cells (BM-MSCs) were Transduced with lenti-miR-30c. Thereafter, we co-cultured U-251 cell line with BM-MCSs-miR-30c and evaluated the apoptosis rate and the relative expression level of IL-6, Klf4, Sox2, c-Myc, and Oct4 using Real-Time PCR and flow cytometry. RESULTS: Wound healing assays represented low migratory ability in U-251 cells treated with BM-MSCs-miR-30c. Plus, apoptosis assay using Annexin V/7AAD showed an increased number of apoptotic U-251 cells following the treatment. miR-30 targeted IL-6 and induced apoptosis. It also impacted on the self-renewal and the anti-apoptotic cluster of genes, namely Klf4, Sox2, c-Myc, and Oct4, to induce apoptosis and dwindle the migration and invasion.

Candida albicans Beta-Glucan Induce Anti- Cancer Activity of Mesenchymal Stem Cells against Lung Cancer Cell Line: An In-Vitro Experimental Study.

OBJECTIVE: ß-glucan, glucopyranosyl polymers of fungi cell wall, represent an immune stimulating effects with potential anti-cancer activity. Mesenchymal stem cells (MSC) have immunomodulating properties in cancer microenvironment. The aim of this study was to investigate the anti-cancer effect of Candida albicans (C. albicans) beta-glucan on MSCs supernatant for apoptosis assay of lung cancer cells in vitro. METHODS: Beta-glucan was extracted from cell wall of C.albicans. MSC isolated from adipose tissue of patients and confirmed using specific surface markers expression which examined by flow cytometry. MSCs treated with various concentrations of ß-glucans for 48 hours. Cytotoxic effect of ß-glucans was evaluated using MTT assay. MSC and lung cancer line cocultured and treated with ß-glucans and apoptosis assay was done by flow cytometry. RESULTS: Cytotoxicity findings showed a significant decrease in MSC viability during 48h, however it was dose-dependent (P<0.05). According to the obtained findings, supernatant of mesenchymal stem cells treated with ß-glucans increased cancer cells apoptosis (P<0.05). CONCLUSION: Beta glucan may highlight a potential and novel promising candidate in future strategies to cause apoptosis of cancer cells and consider as therapeutic  agent against tumor growth as well. Definitely, more in vitro and in vivo studies are required to understand its functions.

Clinical Value of Serum S100A8/A9 and CA15-3 in the Diagnosis of Breast Cancer.

BACKGROUND AND OBJECTIVE: S100A8/A9 is a heterodimer calcium-binding protein which is involved in tumor cell proliferation, adhesion and invasion, and is proposed as a biomarker for better diagnosis and prognosis in many cancers. The aim of this study was to evaluate the simultaneous serum-based level of S100A8/A9 and CA15-3 as well-illustrated cancer biomarkers, as well as their prognostic value in breast cancer patients and healthy matched controls. MATERIAL AND METHODS: Thirty breast cancer patients at different stages of disease and healthy matched controls with no history of inflammatory, autoimmune diseases, or cancer, were enrolled in the study. The levels of S100A8/A9 and CA15-3 were assessed serologically using the Enzyme-linked immunosorbent assay (ELISA) method, and the relevance of these markers with patients' clinicopathological features were subsequently assessed. RESULTS: Based on our data, the serum levels of both S100A8/A9 and CA15-3 were significantly higher in patients compared to the healthy controls, and thus positively correlated with tumor size. Also, statistical analysis shows that the serum level of S100A8/A9 has 100% specificity and sensitivity (AUC = 1.00, 95% CI) for the diagnosis of breast cancer patients. CONCLUSION: According to our data as well as other observations, the S100A8/A9 heterodimer can be considered as a potential biomarker for the proper diagnosis and prognosis of breast cancer.

The Evaluation of Diagnostic and Predictive Values of Helicobacter pylori Stool Antigen Test in Iranian Patients with Dyspepsia.

BACKGROUND AND OBJECTIVES: Iran, as a developing country, is experiencing high burdens of Helicobacter pylori (Hp)-associated non-communicable diseases. Hp stool antigen test (HpSA) is widely used as an inexpensive and feasible noninvasive method to diagnose Hp infection, instead of invasive approaches. The current study aimed at evaluating the diagnostic and predictive values of HpSA test for Hp infection in Iranian patients with dyspepsia. METHODS: The current cross sectional study was performed on 100 patients with dyspepsia. Gastric mucosal specimens were taken, processed, and examined according to the standard protocols. Simultaneously, stool samples were obtained and sent to laboratory for further analyses. Hp stool antigen titers were assessed using enzyme-linked immunosorbent assay (ELISA) technique. RESULTS: Stool antigen titers were not associated with gender (P-value=0.284), but correlated to age (r=0.213, P-value=0.034). Considering 0.385 as a cutoff point, the HpSA test had 80.4% sensitivity and 85.7% specificity. CONCLUSION: Based on cost-effectiveness of HpSA test, the current study findings corroborated the use of HpSA test to detect and follow-up patients with Hp infection, as an alternative method to detect Hp rather than invasive procedures.

Sesame seeds essential oil and Sesamol modulate the pro-inflammatory function of macrophages and dendritic cells and promote Th2 response.

Background: Herbal medicine is becoming progressively accepted treatment for management of different diseases worldwide. Recognition of the active ingredients and mechanisms of herbal medicine against the immune system and related anomalies is highly favorable. This experimental study aimed to investigate the effects of Sesame (Sesamum indicum L.) essential oil and sesamol as effective components on mouse splenocytes subsets, macrophages and dendritic cells (DCs). Methods: Effective components of sesame were extracted and used to treat splenocytes, PHA (5µg/ml) and LPS (10 µg/ml) stimulated splenocytes, macrophages and DCs in different concentration (0.01-100 µg/ml). The cell proliferation/viability was measured using the MTT assay and nitrite levels were measured by the diazotization method. Moreover, TNF-α and IL-1ß cytokines concentration were assayed by ELISA. Treated DCs also analysed for maturation marker levels and cytokine production. Results: Analysis of the results indicated that sesame components suppress PHA-stimulated splenocytes with no effect on LPS-stimulated subsets. Furthermore, the sesame ingredients reduced the release of IFN-γ and increased secretion of IL-4 from lymphocytes. Macrophages viability was not affected and production of NO, TNF-α, and IL-1ß were inhibited using sesame essential oil and sesamol. DCs phenotype skewed to immature and release of TNF-α and IL-1ß were abrogated form DCs. Conclusion: These results indicate that sesame essential oil and its effective component as sesamol may capable of suppressing the response of cellular immunity with the domination of Th2 responses and also could modulate macrophages and the dendritic cells proinflammatory functions.

MicroRNA-146a induces immune suppression and drug-resistant colorectal cancer cells.

Recent studies underline the involvement of microRNAs in cancer development through induction of immune suppression milieu and evolution of drug resistance. The goal of this study was to evaluate the effects of miR-146a on regulatory T cells' frequencies, T-lymphocyte proliferation, and cytokine expression as well as drug resistance in cancer cells. We found that miR-146a was overexpressed in colon cancer HT-29 cells. Peripheral blood mononuclear cells were obtained from healthy donors and were co-cultured with transfected HT-29 cells. Afterward, peripheral blood mononuclear cell proliferation, expression of anti-inflammatory cytokines, and regulatory T cells' frequencies were assayed. Also, drug resistance in transfected HT-29 cells was analyzed following treatment with 5-fluorouracil and irinotecan. Overexpression of miR-146a increased transforming growth factor-ß and interleukin-10 expressions and enhanced regulatory T cells' frequencies in peripheral blood mononuclear cells. Also, the number of cells undergoing cell cycle arrest and apoptosis significantly decreased in transfected HT-29 cancer cells. In conclusion, upregulation of miR-146a plays an important role in enhancing immune suppression through increasing the regulatory T cells' population. Also, our data indicated that colon cancer drug resistance is possibly associated with miR-146a overexpression.

Association of HLA-G*01:01:02:01/G*01:04:01 polymorphism with gastric adenocarcinoma.

Human leukocyte antigen-G (HLA-G) plays an important role in tumor cell escape from immune surveillance and HLA-G polymorphisms might service as a potential risk factor for clinical outcomes in GAC (gastric adenocarcinoma). We investigated the association between HLA-G polymorphisms as well as soluble HLA-G level and accordance of GAC. This case-control study included 100 GAC patients and 102 unrelated Iranian individual's samples as control. The clinical stages ranged from I to IV. PCR-RFLP method was carried out in order to specify the genotypes of the HLA-G gene. Concentrations of sHLA-G in serum were determined with the sHLA-G-specific enzyme linked immunosorbent assay (ELISA) kit. The G*01:04:01 and G*01:01:02:01 alleles were the predominant alleles in GAC patients and healthy controls. The G*01:01:03:01 and G*01:01:08 allele distributions are significantly higher among controls comparing to cases and seem to have protective effect (P value=0.026 and 0.007 respectively). There is a substantial differences in G*01:01:02:01/G*01:04:01 genotype frequencies between cases and controls (OR=2.8, P value<0.001). The G*01:01:03:01/G*01:04:01 and G*01:01:02:01/G*01:01:08 genotypes frequency are higher among controls in comparison to patients (P value=0.028 and 0.007 respectively). The polymorphisms in HLA-G could affect GAC induction and its outcome. Also, increased sHLA-G levels in serum might be a useful biomarker for diagnosis.

Verification of ALDH Activity as a Biomarker in Colon Cancer Stem Cells-Derived HT-29 Cell Line.

BACKGROUND: Recent evidence has suggested that epithelial cancers including colorectal cancer (CRC) have driven by a small population of self-renewing, multi-potent cells termed cancer stem cells (CSCs) which could be responsible for recurrence of cancer. Aldehyde dehydrogenase 1 (ALDH1) activity has used as a functional stem cell biomarker to isolate CSCs in different cancers such as colorectal cancer. OBJECTIVES: The main aim of this research was to determine the utility of ALDH1 activity along with CD44 and EPCAM in identifying stem cell-like cells in human HT-29 colonic adenocarcinoma cell line. MATERIALS AND METHODS: In this experimental study, colon CSCs biomarkers including CD44, EPCAM and ALDH1 in colonospheres and parent cells have analyzed by flow cytometry. The expression levels of stemness genes in spheroid and parental cells have investigated using SYBR Green real-time PCR. In addition, in vivo xenografts assay has performed to determine tumorigenic potential of tumor spheroid cells in nude mice. RESULTS: According to results, over 92% of spheroids were CD44+/EpCAM+, while parent cells only have expressed 38% of CD44/EpCAM biomarkers (P < 0.001). Controversially, ALDH activity was about 2-fold higher in the parent cells than spheroid cells (P < 0.05). In comparison with the parental cells, expression levels of ''stemness'' genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 have significantly increased in colonosphere cells (P < 0.05). Further, administration of 2500 spheroids could be sufficient to initiate tumor growth in nude mice, while 1x106 of parental cells has needed to form tumor. CONCLUSIONS: For the first time, we have shown that colonospheres with low ALDH1 activity has indicated increased tumorigenic potential and stemness properties. So, it hasn't seemed that ALDH1 could become a useful biomarker to identify CSCs population in HT-29 cell line.

Negative Association of Plasma Levels of Vitamin D and miR-378 With Viral Load in Patients With Chronic Hepatitis B Infection.

BACKGROUND: Chronic Hepatitis B (CHB) is accompanied by inflammation of liver because of infection with Hepatitis B Virus (HBV). Previous studies revealed an inverse association between vitamin D and HBV DNA levels. OBJECTIVES: The current study aimed to investigate the levels of 25 (OH) D3 (the steady form of vitamin D), miR-378 and HBV DNA in the patients with CHB. PATIENTS AND METHODS: One hundred and seventy three patients with HBeAg negative CHB were recruited for the study. Plasma levels of HBVDNA and 25 (OH) D3 were quantified. The expression level of miR-378 in plasma was measured by a relative quantitative Real Time Polymerase Chain Reaction (qRT-PCR) assay. RESULTS: In the pathway regression analysis, the plasma level of 25 (OH) D3 showed a significant inverse correlation with plasma levels of HBV DNA (-0.198, P = 0.008) and direct correlation with miR-378 (0.188, P = 0.013). Similarly plasma level of miR-378 had inverse association with HBV DNA level (-0.177, P = 0.020). CONCLUSIONS: These results suggest that vitamin D could involve in a miRNA- mediated regulatory pathway in control of HBV replication. Further studies are recommended to understand the effects of miR-378 and anti-infective action of vitamin D on Hepatitis B Virus.

Simultaneous Underexpression of let-7a-5p and let-7f-5p microRNAs in Plasma and Stool Samples from Early Stage Colorectal Carcinoma.

Colorectal cancer (CRC) is the third most common malignancy and the second most common cause of cancer death worldwide. Early detection of CRC can improve patient survival rates; thus, the identification of noninvasive diagnostic markers is urgently needed. MicroRNAs (miRNAs) have extensive potential to diagnose several diseases, including cancer. In this study, we compared the expression pattern of miRNAs from plasma and stool samples of patients with early stages of CRC (I, II) with that of healthy subjects. We performed miRNA profiling using microarrays on plasma and stool samples of eight patients with CRC and four healthy subjects. Seven miRNAs were found to be underexpressed in both plasma and stool samples of patients with CRC versus healthy subjects. Then, we aimed to verify two out of these seven differentially expressed miRNAs (let-7a-5p and let-7f-5p) by quantitative reverse transcriptase polymerase chain reaction on a larger set of plasma and stool samples of 51 patients with CRC and 26 healthy subjects. We confirmed the results of microarray analysis since their expression was significantly lower in stool and plasma samples of patients with CRC. Moreover, receiver operating characteristic curve analysis demonstrated that fecal let-7f expression levels have significant sensitivity and specificity to distinguish between patients with CRC and healthy subjects. In conclusion, if the results are confirmed in larger series of patients, underexpressed let-7a-5p and let-7f-5p miRNAs in both plasma and stool samples of patients with CRC may serve potentially as noninvasive molecular biomarkers for the early detection of CRC.

The relationship between HLA-G and viral loads in non-responder HCV-infected patients after combined therapy with IFN-α2α and ribavirin.

Hepatitis C disease is a virus mediated infection causing major health problem worldwide. Conversions of immune surveillance play an important role in response to virus clearance. Immune modulating molecules such as HLA-G and IL-10 that convert immune response toward Th2 may play a role to inhibit response from combined therapy with IFN-α2α and ribavirin. The objective of this study was to investigate the expression of HLA-G and IL-10 in responder and non-responder HCV positive patients. In this study, characteristics of the virus and 48 responder and non-responder patients in response to the combined therapy with IFN-α2α and ribavirin were analyzed. The expression levels of HLA-G and IL-10 were conducted using real-time PCR. Also, soluble HLA-G in both groups of patients and healthy individuals were determined by enzyme-linked immunosorbent assay. According to the obtained data, HCV 1a was the predominant genotype in responder and non-responder patients. Expression levels of HLA-G and IL-10 in non-responder group was significantly more than responder and control groups (P<0.001). Additionally, expression levels of HLA-G and IL-10 were remarkably higher compared to healthy individuals at the beginning of treatment. Soluble HLA-G in non-responder patients was noticeably increased in comparison to responder patients after treatment (P<0.05). These findings suggest that elevation of HLA-G and IL-10 in HCV infected patients may play an important role in response to combined therapy with IFN-α2α and ribavirin.