Allelic and Genotypic Analysis of LncRNA ANRIL rs4977574 A/G Mutations in Oral Squamous Cell Carcinoma Patients: Insights into Tumor Characteristics and Genotypic Correlations.

Aim: Long noncoding RNAs (lncRNA) ANRIL and its genetic polymorphisms are shown to be associated with the risk of several cancers. However, the single nucleotide polymorphisms (SNPs) of lncRNA ANRIL are not thoroughly assessed in oral squamous cell carcinoma (OSCC) which is the most prevalent cancer in the head and neck area. Thus, this study aimed to assess the association of SNP of lncRNA ANRIL rs4977574 in patients with OSCC. Methods and Materials: 106 blood samples from the patients with OSCC were obtained with a gender- and age-matched control group to evaluate the SNP of rs4977574 of lncRNA ANRIL. The DNA was extracted using the salt-out technique and DNA genotyping was undertaken using specific primer pairs in the tetra-primer ARMS-PCR technique. Eventually, the frequency of wild-type (A) and the mutated allele (G), as well as the genotypes were estimated between the groups of patients with OSCC and healthy individuals. Results: The results of our study indicated no statistically significant difference in the frequency of rs4977574 A/G of lncRNA ANRIL among the patients with OSCC and healthy individuals (p > 0.05). Likewise, no significant difference was found in the genotypes' frequencies (p > 0.05). Nevertheless, the marked association of GG with smaller tumor size and the high level of differentiation of OSCC cells in the presence of AA or AG genotypes were interesting outcomes of this study (p < 0.05). Similarly, all the genotypes AA, AG, and GG were correlated with the site of the occurrence of OSCC. Furthermore, the association of the genotypes with the lymph node metastasis and the tumors stage was not found to be significant (p > 0.05). Conclusions: The results of our study indicate that rs4977574 A/G and its genotypes do not have any direct correlation with the presence of OSCC; however, its association with the smaller tumor size and the level of the cancer cells differentiation could imply its possible indirect role.

Poly lactic-co-glycolic acid-alginate nanocarrier for efficient drug delivery to liver cancer cells.

Efficient drug delivery systems (DDSs) can potentially replace with conventional modalities in cancer therapy, like liver cancer. In this study, a novel folic acid (FA)-functionalised and alginate (Alg)-modified poly lactic-co-glycolic acid (PLGA) nanocomposite was developed for delivery of doxorubicin (Dox) to HepG2 and Huh7 liver cancer cells. After synthesising the nanocarrier, several analytical devices, including FT-IR, DLS, TGA, and TEM, were employed for its characterisation. Nano-metric size (55 and 85 nm in diameter), close to neutral surface charge, semi-spherical morphology, and successful synthesis were approved. Dox entrapment efficiency was determined near 1%, and sustained and pH-sensitive drug release behaviours of nanocarrier were ascertained for DDS. Afterwards, the cell viability test was carried out to study the HepG2 and Huh7 cells suppression capability of FA-PLGA-Dox-Alg. About 12% and 10% cell viabilities were observed in HepG2 and Huh7 cancer cells after 24 h treatment with 400 nM concentration of FA-PLGA-Dox-Alg nanocarrier respectively. The IC50 value was observed for 100 nM after 24 h of treatment in cancer cells. These data have indicated that fabricated nanocarrier could be promising DDS against liver cancer and replace with conventional approaches in cancer treatment, like chemotherapy.

Association of Circulating Circular RNAs (hg38_circ_0008980, and CircDLGAP4) in Diagnosis, Diseases Severity, and Prognosis of Ischemic Stroke.

Background: Fast diagnosing ischemic stroke (IS) is a critical issue in clinical studies, as it allows more effective therapy and stops the progression of IS. The blood level of circular RNAs (CircRNAs) after stroke may be a rapid diagnostic marker. Methods: In this study, the blood level of circRNAs was evaluated using a real-time polymerase chain reaction (PCR). We used logistic and linear regression analysis to assess the potential of circRNAs levels with the risk of IS. Results: circRNA DLG associated protein 4 (CircDLGAP4) was decreased in patients compared with controls, and logistic regression showed its expression negatively associated with IS risk. The expression level of human genome version 38_Circular_0008980 (hg38_circ_0008980) was reduced significantly in patients with small vessel disease (SVD), and the linear regression analysis showed a negative relationship between hg38_circ_0008980 expressions with SVD subtype. hg38_circ_0008980 expression relative to controls showed a significant association with IS risk. Conclusion: Taken together, we found a significant decrease in the level of hg38_circ_0008980 after IS; it may act as a novel circRNA in IS pathophysiology with a positive correlation with stroke severity.

Association between Genetic Polymorphism of The lncRNA MIAT rs1894720 with Ischemic Stroke Risk and lncRNA MIAT Expression Levels in The Blood after An Ischemic Stroke: A Case-Control Study.

OBJECTIVE: Genetic aspects can play an essential role in the occurrence and development of ischemic stroke (IS). Rs1894720 polymorphism is one of the eight single nucleotide polymorphisms (SNPs) in the long non-coding RNA (lncRNA) myocardial infarction-associated transcript (MIAT) locus. The aim of study is the lncRNA MIAT rs1894720 polymorphism decreases IS risk by reducing lncRNA MIAT expression. MATERIALS AND METHODS: In this case-control study, we studied 232 Iranian patients and 232 controls. The blood samples were collected from patients admitted at different times after stroke symptoms. We enrolled 80, 78, and 74 patients who arrived at the hospital between 0-24, 24-48, and 48-72 hours after the first appearance of symptoms, respectively. DNA genotyping was done by the tetra-primer ARMS-PCR method. Circulating MIAT levels were evaluated by real-time polymerase chain reaction (PCR). RESULTS: The GT genotype of MIAT rs1894720 showed a significant association with the risk of IS (OR=3.53, 95% CI=2.13-5.84, P<0.001). MIAT expression was higher relative to the control within the first hours after IS. The MIAT levels in IS patients with rs1894720 (GT) were significantly lower relative to patients who had the GG and TT genotypes. Linear regression model indicated a significant correlation between MIAT expression with atherosclerotic risk factors and types of stroke in IS patients. Receiver operating characteristic (ROC) curve analysis showed that the level of lncRNA MIAT after IS could be diagnostic with an area under the curve (AUC) of 0.82. The sensitivity and specificity were 80.17 and 67.24%, respectively (P<0.001). CONCLUSION: Our study demonstrated that the MIAT rs1894720 polymorphism (GT) might increase the risk of IS in the Iranian population. MIAT expression was up-regulated in our IS patients. Hence, it could be a diagnostic biomarker for IS.

The effect of BKV reactivation on cytokines behavior in kidney transplanted patients.

BACKGROUND: BK virus associated nephropathy (BKVAN) is one of the common causes of graft loss among kidney transplanted recipients (KTRs). The current treatment for BKV nephropathy is decreasing the immunosuppressive regimen in KTRs. Interleukin-27 (IL-27) is a multifunctional cytokine that might be the front-runner of an important pathway in this regard. Therefore, in current study it is tried to evaluate the changes in the expression level of IL-27 and some related molecules, resulting from BKV reactivation in KTR patients. METHODS: EDTA-treated blood samples were collected from all participants. Patients were divided into two groups, 31 kidney transplant recipients with active and 32 inactive BKV infection, after being monitored by Real time PCR (Taq-Man) in plasma. Total of 30 normal individuals were considered as healthy control group. Real time PCR (SYBR Green) technique is used to determine the expression level of studied genes. RESULTS: The results of gene expression comparisons showed that the expression level of IL-27, IFN-γ, TNF-α, TNFR2 and IRF7 genes was significantly higher in inactive group in comparison to active group. The expression level of TLR4 was lower in both active and inactive groups in comparison to control group. ROC curve analysis showed that IL-27 and IRF7 are significantly different amongst other studied genes. Finally, the analyses revealed that the expression level of most of the studied genes (except for TNF-α and TLR4) have significant correlation with viral load. CONCLUSIONS: Our findings revealed that IL-27, IFN-γ, TNF-α, TNFR2 and IRF7 expression level is higher in inactive group and TLR4 expression level is lower in patients' groups in comparison to control group. Also, ROC curve analysis showed IL-27 and IRF7 can significantly differentiate studied groups (BKV active vs. inactive). Therefore, these results might help elucidating the pattern in charge of BKV reactivation in kidney transplanted patients.

Quercetin Can Inhibit Angiogenesis via the Down Regulation of MALAT1 and MIAT LncRNAs in Human Umbilical Vein Endothelial Cells.

BACKGROUND: Angiogenesis is an important step in cancer metastasis since it enables the growing tumor to receive nutrients and oxygen. Quercetin is a generic flavonoid and has been investigated for its ability to inhibit angiogenesis in different types of cancers. MALAT1 and MIAT lncRNAs are associated with the angiogenesis process. MALAT1 induces hypoxia-driven angiogenesis via the overexpression of angiogenic genes. Down regulation of MIAT1 could inhibit the proliferation of endothelial cells, tube formation, and migration. In this study, we assessed the anti-angiogenic activity of quercetin on human umbilical vein endothelial cells (HUVEC) via the expression of MALAT1 and MIAT genes. METHODS: In the present study, HUVEC cells were incubated with various concentrations of quercetin for 24, 48, and 72 h. Cell proliferation was then evaluated by MTT assay. RNA was extracted by TRIzol and cDNA synthesis. The expression levels of MALAT1 and MIAT genes relative to the GAPDH gene were quantified using the highly sensitive real-time PCR method. RESULTS: Our results demonstrated that quercetin has an inhibitory impact on the cell viability of HUVEC cells. The IC50 values of quercetin after 24, 48, and 72 h were 282.05 µM, 228.25 µM, and 131.65 µM, respectively. The MALAT1/GAPDH ratio was computed as 0.21 for 24h, 0.18 for 48h, and 0.29 for 72 h. The MIAT/GAPDH ratio was computed as 0.82 for 24h, 0.84 for 48h, and 0.78 for 72 h. CONCLUSIONS: In conclusion, quercetin treatment had an anti-angiogenic effect on HUVEC cells, at least partially via the down regulation of MALAT1 and MIAT LncRNAs gene expression.

Long non-coding RNA H19 expression and functional polymorphism rs217727 are linked to increased ischemic stroke risk.

BACKGROUND: Efforts to identify potential biomarkers for the diagnosis of ischemic stroke (IS) are valuable. The H19 gene plays a functional role in increasing the prevalence of IS risk factors. We evaluated the correlation between H19 rs217727 polymorphism and the expression level of H19 lncRNA with susceptibility to IS among the Iranian population. METHODS: Blood samples were collected from IS patients (n = 114) and controls (n = 114). We concentrated on the expression pattern of H19 at different time points (i.e., 0-24, 24-48, and 48-72 h after stroke). The tetra-amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method was applied for DNA genotyping. We used the quantitative real-time PCR to evaluate H19 expression levels. We used the receiver operating characteristic (ROC) curve to evaluate the diagnosis and prognosis of IS. RESULTS: The rs217727polymorphism of H19 was related with IS susceptibility in the co-dominant (OR = 2.92, 95% CI = 0.91-10.92, P = 0.04) and recessive models (OR = 2.80, 95% CI = 0.96-8.15, P = 0.04). H19 expression was significantly upregulated in IS and remained high for 72 h after stroke. ROC curves showed that H19 expression within the first 24 h from stroke onset might serve as a biomarker for the early diagnosis of IS with 79.49% sensitivity and 80.00% specificity. H19 expression in small vessel occlusion (SVO) and large-artery atherosclerosis (LAA) patients were 3.74 and 3.34 times higher than the undetermined (UD) subtype, respectively [OR = 3.74 95% CL (1.14-12.27) P = 0.030 and OR = 3.34 95% CL (1.13-9.85) P = 0.029]. CONCLUSION: The rs217727 polymorphism of the H19 is correlated with IS susceptibility, and H19 expression levels were higher in SVO and LAA patients. The upregulation of H19 may be considered as a diagnostic biomarker in IS among the Iranian population, but it cannot serve as a useful prognostic marker.

Quercetin Arrests in G2 phase, Upregulates INXS LncRNA and Downregulates UCA1 LncRNA in MCF-7 Cells.

One of the most prevalent malignancies, which have severe effects on women's health, is breast cancer. Quercetin, a flavonoid found in vegetables, tea, and fruits, is known to have bioactive properties, such as anti-inflammatory, anti-oxidant, as well as anti-cancer. Long non-coding RNAs (lncRNAs) have been recognized to function as primary regulators of diverse cellular processes, including differentiation, development, and cell fate. INXS and UCA1 are lncRNAs that are up regulated and down regulated respectively in cancer cells. This research aimed to assess the impact of quercetin on the expression of INXS and UCA1 genes in MCF-7 cells. Various quercetin concentrations at different times were used to treat MCF-7 cells. The cell viability and IC50 values were determined using MTT assay. Then, MCF-7 cells were incubated with various quercetin concentrations for 24, 48, and 72 h. Cell cycle analyses were evaluated by flow cytometry. The levels of INXS and UCA1 gene expression compared with the GAPDH gene at different concentrations of quercetin were quantified using real-time PCR method. Based on the results, quercetin exerted a dose- and time-dependent inhibitory impact on the viability of MCF-7 cells. Furthermore, quercetin induced cell cycle arrest at the G2 phase in MCF-7 cells. Also, quercetin induced INXS upregulation and UCA1 downregulation in the MCF-7 cell line. These data suggest that quercetin might increase cell death by up regulating INXS and down regulating UCA1 lncRNAs in MCF-7 cells.

The LncRNA H19 rs217727 Polymorphism Is Associated with Oral Squamous Cell Carcinoma Susceptibility in Iranian Population.

Lack of protein-coding capacity is a main characteristic of long noncoding RNAs (lncRNAs) which, as molecular biomarkers, have found a novel pharmacological application in cancer and are reported to be important regulators of gene expression. H19 is reportedly involved in cancer progression and tumorigenesis. One of the most common types of head and neck cancers is oral squamous cell carcinoma (OSCC). The main objective of the present study was to evaluate the correlation of OSCC susceptibility with H19 gene in an Iranian population. This research was performed on 400 subjects of both sexes referred to the Namazi Hospital affiliated with the Shiraz University of Medical Sciences (SUMS). Individuals aged 15-88 years were divided into two groups: pathologically diagnosed patients with new-onset OSCC and healthy controls. After written and informed consent was obtained from the individuals, genomic DNA was extracted. The tetra-primer ARMS-PCR technique was performed for DNA genotyping by the use of specific primer pairs. The susceptibility of OSCC and H19 gene polymorphism sites was further analyzed (rs217727 and rs2107425). The allele and genotype frequencies of H19 rs2107425 polymorphism were similar between OSCC cases and controls. The H19 rs217727T allele frequency was significantly higher in OSCC cases (P = 0.002), and the polymorphism of H19 rs217727 was associated with OSCC susceptibility in the codominant (OR = 6.04, 95%CI = 1.70 - 21.42, P = 0.001 for TT genotype), dominant (OR = 1.62, 95%CI = 1.08 - 2.43, P = 0.01), and recessive (OR = 5.32, 95%CI = 1.51 - 18.69, P = 0.003) models. This study showed that rs217727 and OSCC susceptibility were statistically correlated in the Iranian population.

Association of the Risk of Dental Caries and Polymorphism of MBL2 rs11003125 Gene in Iranian Adults.

This case-control study aimed to investigate the effect of rs11003125 in dental caries. For this purpose, a total number of 404 individuals - from Fars Province in Iran - were studied. The technique of this research was the tetra-primer amplification-refractory mutation system (ARMS)-PCR. Dental caries prevalence among the 404 individuals was assessed by counting the number of decayed, missing, and filled teeth. In this research, individuals were divided into two groups: cases (n = 238) and controls (n = 166), and the peripheral blood samples were used to extract the genomic DNA. For genotyping of DNA, the tetra-primer ARMS-PCR method was conducted using specific primer pairs. While examining MBL2 rs11003125 polymorphism, we found significant differences in the genotype frequencies between the case and the control group. The pooled estimates indicated that the GG and GC genotypes of MBL2 rs11003125 polymorphism significantly increased, and therefore caries risk (OR = 2.40, 95% CI = 1.31-4.40, p = 0.004) under the dominant model. These findings suggested that polymorphism in MBL2 gene was associated with dental caries in Iranian adults. Further verification is needed with more ethnic groups and larger sample sizes to determine whether rs11003125 polymorphism is related to dental caries in other regions or not.

AMELX Gene Association with Dental Caries in Iranian Adults.

Dental decay is a disease that is greatly affected by environmental components, but recently there have been an increasing number of documents supporting a genetic factor in the development of caries. The purpose of this study was to examine the association between dental caries and single-nucleotide polymorphisms in the AMELX gene. This research was carried out on 360 individuals of both sexes, who were referred to the dental school at the Shiraz University of Medical Sciences. In this research, individuals aged 20-65 years were divided into two groups: controls (decayed, missed, or filled teeth (DMFT) ≤ 5; n = 180) and cases (DMFT ≥ 14; n = 180). The tetra-primer ARMS-PCR technique was performed for genotyping the DNA extracted from blood cells. Analysis of the AMELX rs946252 polymorphism showed that the T allele of rs946252 was a significant protective factor against dental caries in Iranian adults (T vs. C: OR = 0.70, 95% CI: 0.49-0.98, P = 0.04). We demonstrated the significant differences in the genotype frequencies under two genetic models: overdominant (TC vs. TT + CC: OR 0.35, 95% CI 0.19-0.64, P = 0.0006) and recessive (CC vs. TC + TT: OR 2.57, 95% CI 1.39-4.76, P = 0.002). Our results show that the SNPs of the AMELX gene may be related with susceptibility to dental caries in Iranian adults.

Comparison of Cytotoxicity of New Nanohybrid Composite, Giomer, Glass Ionomer and Silver Reinforced Glass Ionomer using Human Gingival Fibroblast Cell Line.

AIM: The objective of this study was to investigate the cytotoxic effects of new nanohybrid composite, giomer, conventional and resin modified and silver reinforced glass ionomer cements and compare the biocompatibility of these dental materials in cell culture. STUDY DESIGN: Five cylindrical specimens were made of each material, using a mold (2mm. thick and 5 mm in diameter). For HGF, cells were cultured in RPMI-1640 medium. After attaining 80% confluence, cells were treated with different doses of five tested materials for 24h. Then cell cytotoxicity was assessed using MTT assay. The data were analyzed using Kruskal-Wallis and Dunn test. RESULTS: The materials evaluated on HGF cells, showed significantly more cytotoxicity in silver reinforced glass ionomer but nanohybrid composite shows mild cytotoxic effect. However, giomer shows no significant cytotoxicity and conventional and resin modified glass ionomer enhance cell proliferation. CONCLUSIONS: Silver reinforced glass ionomer induced a significant high cytotoxic effect over a wide range of concentration. Therefore, higher attention should be focused on this restorative dental material, which should be chosen for further investigations.

A comparison inhibitory effects of cisplatin and MNPs-PEG-cisplatin on the adhesion capacity of bone metastatic breast cancer.

To date, high mortality in women due to malignancy breast cancer related to the metastasis to the bone is a significant challenge. As, magnetic nanoparticles (MNPs) conjugated with the biocompatible polymers was employed for the delivery of some hydrophobic anticancer agents, the main aim of the current research was to assess whether cisplatin-loaded MNPs enhanced the anticancer effect of free cisplatin in breast cancer cells. MNPs decorated with PEG were synthesized by an improved coprecipitation technique, and then cisplatin was loaded onto the MNPs via a simple mixing method. Afterward, its morphology, size, chemical structure, magnetic property, hydrodynamic diameter, zeta potential, and crystal structure were characterized by scanning and transmittance electron microscopy, Fourier transforms infrared spectroscopy, vibrating sample magnetometer, dynamic light scattering, and X-ray powder diffraction and flame atomic absorption spectroscopy respectively. Additionally, the effects of cisplatin and MNPs-PEG-cisplatin on viability, migration and adhesion capacity of T47D cells were investigated by evaluating α2-integrin and ß1-integrin; mRNAs were assessed by real-time RT-PCR. Consequently, the in vitro assay results showed a considerable dose-dependent inhibitory effect of cisplatin and MNPs-PEG-cisplatin on proliferation, migration, and adhesion of T47D cells. Finally, current research was shown that MNPs-PEG-cisplatin strongly increased anticancer effects compared with free cisplatin in the T47D cell line.

Role of TiF4 in Microleakage of Silorane and Methacrylate-based Composite Resins in Class V Cavities.

AIM: This study investigated the effect of TiF4 solution pretreat-ment on microleakage of silorane and nanofilled methacrylate-based composites in class V cavities. MATERIALS AND METHODS: Forty-eight intact premolar teeth were randomly allocated to four groups of 12 teeth. Restorative techniques after standard class V tooth preparations were as follows: Group 1, Filtek P90 composite; group 2, Filtek Z350 XT; group 3, TiF4 solution pretreatment and Filtek P90 composite; group 4, TiF4 solution pretreatment and Filtek Z350 XT. After storing the specimens in distilled water at 37°C for 24 hours and followed by immersion of the specimens in a 0.5% basic-fuchsin solution for 24 hours, they were sectioned buccolingually to obtain four surfaces for each specimen for analysis of microleakage using a stereomicroscope. Data analysis was performed using Kruskal-Wallis test to compare the four groups and the Mann-Whitney test for paired comparisons with Statistical Package for the Social Sciences (SPSS) version 17 software. RESULTS: At the enamel margins, microleakage score of the Filtek Z350 XT group was lower than those of the Filtek P90 with and without the application of the TiF4 (p = 0.009 and p = 0.031 respectively). At the dentin margins, groups 3 and 4 (TiF4+Filtek P90 and TiF4+Filtek z350 XT respectively) showed significantly lower microleakage than group 1 (Filtek P90). However, there was no significant difference between other groups (p > 0.05). CONCLUSION: At the enamel margins, microleakage score of the silorane-based composite was more than that of the nanofilled composite. No significant differences were observed between the other groups. At the dentin margins, for the silorane-based composite restorations, TiF4 solution pretreatment resulted in significantly lower microleakage. However, the similar result was not observed for Filtek Z350 XT. Also, no significant difference was observed between microleakage scores of Filtek P90 and Filtek Z350 XT with or without TiF4 pretreatment. CLINICAL SIGNIFICANCE: In spite of better mechanical and physical properties of modern composites than earlier methacrylate-based composites, polymerization shrinkage has been remaining as one of the main shortcomings of them. Different methods, such as using new low shrinkage resin composites and different dentin pretreatments, have been suggested to overcome this problem. This study evaluated the effect of TiF4 as pretreatment on microleakage of class V tooth preparations restored with a nanocomposite and a silorane-based resin composite.

Evaluation of silibinin on the viability, migration and adhesion of the human prostate adenocarcinoma (PC-3) cell line.

Prostate cancer (PCA) is the most common cancer diagnosed in men and the second most common cause of death due to cancers after lung cancer. Metastasis of cancer cells involves multiple processes and various cytophysiological changes, including changed adhesion capability between cells and extracellular matrix (ECM) and damaged intercellular interaction. Silibinin, a naturally occurring flavonoid antioxidant found in the milk thistle, has recently been shown to have potent antiproliferative effect against various malignant cell lines. In the present study, PC-3 cells were incubated with various concentrations of silibinin for different times; then, cell cytotoxicity, cell adhesion and cell motility were assessed using MTT assay, cell-matrix adhesion assay and cell migration assay, respectively. The results showed that silibinin exerted a dose- and time-dependent inhibitory effect on the viability, motility and adhesion of highly metastatic PC-3 cells. These observations indicate that silibinin can probably inhibit metastasis in PCA.