hTERT Gene Modification Using CRISPR-dCas9-dnmt3a System as a Therapeutic Approach Against Glioma.

Background: Abnormal DNA methylation patterns have been reported in various diseases, including different cancers. CRISPR/Cas9 is a low-cost and highly effective gene editing tool that has lately revolutionized biotechnology. Studies have shown that the CRISPR/Cas9 system can effectively target and correct methylation. Objectives: Telomerase plays a survival role for cancer cells. It is encoded by the hTERT gene. The effectiveness of CRISPR/Cas9 in targeting hTERT to treat glioma cancer cells was assessed in this study. Methods: EF1a-hsaCas9-U6-gRNA vector carrying sgRNA and Cas9 hybrids were used to transfect U87 glioma cells. Four and eight µg/mL polybrene concentrations were investigated to improve transfection efficiency. The expression level of hTERT that has undergone metabisulfite modification was assessed using real-time PCR. Flow cytometry and Western blotting were also used to determine whether telomerase was present in the cells. High-resolution melting analysis (HRM) was used to examine the hTERT promoter's methylation. Finally, flow cytometry was used to measure the apoptotic rate of transfected U87 cells. Results: The findings demonstrated that gRNA significantly boosted transfection effectiveness. Significant variations were seen in the expression of hTERT in U87 cells at 4 µg/mL polybrene and 80 µg/mL transfection compared to transfection without gRNA and basal cells. Flow cytometry showed a decrease in hTERT levels in transfected cells. Furthermore, transfection with gRNA increased U87 cell apoptosis compared to transfection without gRNA. Conclusions: It appears that the designed CRISPR/Cas9 system can reduce hTERT expression and telomerase activity and thus inhibit glioma cell growth.

Reduction of GAS5 and FOXD3-AS1 long non-coding RNAs in patients with bipolar disorder.

Bipolar disorder (BD) patients suffer from severe disability and premature death because of failure in prognosis, diagnosis, and treatment. Although neural mechanisms of bipolar have not been fully discovered, studies have shown long noncoding RNAs (lncRNAs) can play an important role in signaling pathways such as PI3K/AKT pathway. There has been little study on deregulated lncRNAs and the lncRNAs' mode of action in the BD. Hence, we aimed to investigate the expression of PI3K/AKT pathway-related lncRNAs named TUG1, GAS5, and FOXD3-AS1 lncRNAs in the PMBC in 50 bipolar patients and 50 healthy controls. Our results showed that FOXD3-AS1 and GAS5 under-expressed significantly in bipolar patients compared to healthy controls (P = 0.0028 and P < 0.0001 respectively). Moreover, after adjustment, all P values remained significant (q value < 0.0001). According to the ROC curve, AUC (area under the curve), specificity, and sensitivity of these lncRNAs, GAS5 and FOXD3-AS1 might work as BD candidate diagnostic biomarkers. Taken together, the current results highlight that the dysregulation of FOXD3-AS1 and GAS5 may be associated with an increased risk of BD.

Differential gene expression of BCL-2, ZEB2-AS1 and BALR-2 in prostate cancer and benign prostatic hyperplasia.

Prostate cancer (PCa) and benign prostate hyperplasia (BPH) are highly prevalent heterogeneous disorders among men. Whereas PCa and BPH underline common pathological features, apoptotic-related genes might be differentially expressed in these diseases. This study was aimed at testing BCL-2 as well as BALR-2 and ZEB2-AS1 apoptosis-related long non-coding RNA (lncRNA) in patients with PCa and BPH. The expression levels of the BCL-2 gene and ZEB2-AS1 lncRNA were upregulated in tumoural tissues in comparison to adjacent non-cancerous tissues (ANCTs) and BPH tissues. In contrast, the expression level of BALR-2 lncRNA was significantly higher in BPH compared with tumoural tissues. Furthermore, while no association was noticed between the relative expression of ZEB2-AS1 and the tumour grade, the relative expression of BCL-2 and BALR-2 is strongly associated with a higher grade of the tumour in PCa samples compared with the ANCTs. The receiver operating characteristic (ROC) curve analysis indicated the highest specificity and diagnostic value in distinguishing PCa and ANCTs as well as PCa and BPH, respectively. In conclusion, altered expression of BCL-2 and BALR-2 was observed to be associated with tumoural progression and could be used as potential candidates for distinguishing PCa tissues from ANCTs or BPH samples.

Vitamin D changes expression of DNA repair genes in the patients with multiple sclerosis.

Oxidative stress (OS) plays an essential role in demyelination and tissue injury related to pathogenesis of multiple sclerosis (MS). On the other hand, vitamin D (VD) as an antioxidant reduces oxidative stress and has been used as adjuvant therapy in autoimmune diseases. Although VD supplementation is suggested as a protective and immunomodulation factor for MS patients, the molecular mechanisms remain unclear. Given that VD may modulate the immune system of MS patients through the DNA repair pathway, we aimed to evaluate the effects of VD supplementation in DNA repair genes expression including OGG1, MYH, MTH1, and ITPA. Transcript levels were measured using the RT-qPCR method in peripheral blood mononuclear cells (PBMCs) of relapsing-remitting multiple sclerosis (RRMS) patients before and after two months of VD supplementation. Furthermore, in silico analysis and correlation gene expression analysis was performed to find the biological binding sites and the effect of NRF2 on the regulation of DNA repair genes. Our data revealed that in MS patients, 2-month VD treatment significantly altered the expression of MYH, OGG1, MTH1, and NRF2 genes. A significant correlation was observed between DNA repair genes and NRF2 expression, which was confirmed by the presence of antioxidant response element (ARE) binding sites in the promoter of OGG1, MYH, and MTH1 genes. This study demonstrated that the impact of VD on MS patients may be mediated through the improvement of DNA repair system efficiency. This finding brought some new evidence for the involvement of DNA repair genes in the physiopathology of MS patients.

The role of long non-coding RNA MALAT1 in patients with bipolar disorder.

Bipolar disorders are known as chronic, recurrent, and heterogenic diseases. Regarding, diagnosis and treatment of them are very complex. The molecular mechanism and pathophysiology of bipolar disorder are slightly known. Accordingly, long noncoding RNAs are considered as one of the main factors that are dysfunctional in many diseases such as the nervous system diseases. Hence, we aim to investigate the expression of two long non coding RNAs, MALAT1 and UCA1, in patients in bipolar disorder. The levels of MALAT1 and UCA1 lncRNA were evaluated in peripheral blood mononuclear cells (PBMCs) of 50 bipolar patients and 50 healthy controls with real-time PCR. Also, ROC curve analysis and correlation analysis were performed between the gene expression and some clinical features of bipolar individuals. The significant decline of MALAT1 expression level was found in the patients compared to controls; but no significant difference was observed in the UCA1 expression level between the patients and controls. Furthermore, computational analysis of CpG Islands and miRNAs binding sites on LncRNAs, MALAT1, and UCA1 was conducted. Also, The ROC curve area (AUC) of MALAT1 was 0.80. The current results suggest that the expression level of MALAT1 could serve as a potential diagnostic biomarker for bipolar patients.

Significant increasing of DISC2 long non-coding RNA expression as a potential biomarker in bipolar disorder.

Bipolar disorder (BD) is a mental disorder that is often misdiagnosed with ineffective treatment. It has strong genetic component but unknown pathophysiology. Long non-coding RNAs (lncRNAs) have been recently recognized as one of the important genetic factors and are considered as one of the regulatory mechanisms of nervous system. Given that lncRNAs may be diagnostic biomarkers for BD, we aimed to quantify the levels of DISC1 and DISC2 lncRNA transcripts. The levels of DISC1 and DISC2 lncRNA were tested in peripheral blood mononuclear cells (PBMCs) of 50 BD and 50 controls by real-time PCR. In addition, we performed ROC curve analysis as well as correlation analysis between the gene expression and some clinical features of BD cases. Computational analysis of miRNAs binding sites and CpG Islands on DISC1 and DISC2 lncRNA was performed as well. Significant down-regulation of DISC1 and up-regulation of DISC2 were observed in BD cases compared with controls. The areas under the ROC curve (AUC) for DISC1 and DISC2 lncRNA were 0.76 and 0.68 respectively. There was no significant correlation between the levels of mRNA expression in PBMCs of BD patients and clinical features. These data demonstrated that DISC1 and DISC2 lncRNA expression was potentially associated with an increased risk of bipolar disorder and might involve several molecular mechanisms. Our results revealed that the transcript levels of DISC1 and DISC2 lncRNA could be considered as a good putative biomarker for individuals with bipolar disorder.

Protein-Protein Interaction Analysis of Common Top Genes in Obsessive-Compulsive Disorder (OCD) and Schizophrenia: Towards New Drug Approach Obsessive-Compulsive disorder (OCD) and Schizophrenia Comorbidity Gene Analysis.

Comorbidty is common among psychiatric disorders including obsessive-compulsive disorder and schizophrenia with a high rate. Many studies suggested that the disorders may have same etiological bases. In this regard, shared pathways of glutamate, dopaminergic, and serotonin are the known ones. Here, the common significant genes are examined to understand the possible molecular origin of the disorders in terms of sequence and functional features. Exploring the underling mechanisms of OCD and schizophrenia is important to achieve a better treatment options. Methods of Cytoscape software following R statistical software were applied for this purpose. Needleman-Wunsch global alignment algorithm was used to determine pair-wise similarities followed by clustering methods, AGNES and PAM in R statistical programming software. The results indicate that SLC1A1, DRD2, DRD4, BDNF, ESR1, CDH2, GRIN2B, TNFa, GABBR1, and OLIG2 are significantly common for the two disorders and PPI network analysis showed the important key genes in the interaction profile. ESR1 (estrogen receptor α) as a key hub-bottleneck gene regulates many underling mechanisms of the brain. Application of global alignments indicates some of the genes with sequence similarities also elucidate similar biological terms.

Vitamin D supplementation up-regulates IL-6 and IL-17A gene expression in multiple sclerosis patients.

Vitamin D regulates gene expression and affects target cell functions. IL-6 and IL-17A are pro-inflammatory cytokines associated with MS pathogenesis. The aim of this study was to investigate the vitamin D effects on the expression level of IL-6 and IL-17A in peripheral blood mononuclear cells (PBMCs) of multiple sclerosis (MS) patients. Also, we performed a correlation analysis between the gene expression and some clinical features such as serum level of vitamin D and the expanded disability status scale (EDSS). Significant up-regulation of IL-6 and IL-17A gene expression was shown under vitamin D treatment. Also, some gender specific correlations between the gene expression with vitamin D levels were detected in female RR-MS patients.