Hossein Fahimi

The clustered regularly interspaced short palindromic repeats (CRISPR) system offers cost-effectiveness, high efficiency, precision, and ease of use compared to traditional gene editing techniques. In this study, we employed findings from prestigious investigations to develop an optimized approach for generating knockout cancer cell lines using a transient transfection method. This protocol introduces a distinctive approach that follows rigorous guidelines for designing gRNA to reduce off-target effects, a major challenge in CRISPR applications. Our step-by-step instructions allow researchers, particularly those with limited laboratory equipment and funding, as well as those undertaking CRISPR projects for the first time, to generate knockout cell lines using CRISPR technology in just ten weeks. This protocol covers all needs for enhancing various yields, such as transfection efficiency, and includes leveraging robust bioinformatics tools, conducting essential assays, isolating monoclonal cells via limiting dilution, validating knockout cells, and providing comprehensive troubleshooting recommendations. Using this method, we successfully created several new generations of colorectal cancer cell lines with monoallelic and biallelic knockouts of the epithelial cell adhesion molecule (EpCAM) gene. Our method, optimized for a wide spectrum of cancer cell lines, makes CRISPR more accessible for applications in personalized and precision medicine. It expands opportunities for novel investigations into cancer mechanisms and paves the way for potential therapeutic interventions.

Small regulatory RNAs (sRNAs) encapsulated in outer membrane vesicles (OMVs) are critical post-transcriptional regulators of gene expression in prokaryotic and eukaryotic organisms. OMVs are small spherical structures released by Gram-negative bacteria that serve as important vehicles for intercellular communication and can also play an important role in bacterial virulence and host-pathogen interactions. These molecules can interact with mRNAs or proteins and affect various cellular functions and physiological processes in the producing bacteria. This review aims to provide insight into the current understanding of sRNA localization to OMVs in Gram-negative bacteria and highlights the identification, characterization and functional implications of these encapsulated sRNAs. By examining the research gaps in this field, we aim to inspire further exploration and progress in investigating the potential therapeutic applications of OMV-encapsulated sRNAs in various diseases.

Background: Gastric cancer is one of the most prevalent human malignancy-related death worldwide, which is usually diagnosed at the advanced stages resulting in metastasis. Recent studies have revealed that long non-coding RNAs (lncRNAs), which are known as non-coding RNAs, play a significant role in creating variety of molecular pathways (e.g., growth, proliferation, differentiation, and apoptosis) and negatively contribute to many unusual processes, including human cancers. The HOX antisense intergenic RNA (HOTAIR) is one of the novel non-coding RNAs which has recently emerged as a promoter of metastasis in different types of human cancers through epithelial-to-mesenchymal transition (EMT) process. Epithelial-to-mesenchymal transition (EMT) is a cellular process where an epithelial cell could change its phenotype to mesenchymal condition, which plays a crucial role in promoting cell invasion, angiogenesis, and metastases. Methods: This study aimed to explore the effect of HOTAIR gene silencing on the expression levels of two main markers of EMT signaling pathway, fibronectin (FN1) and claudin4 (CLDN4) in MKN45 cellular model of gastric cancer. The MKN45 cells were subjected to HOTAIR specific siRNA for 48 hours, and the extracted RNAs were subjected to cDNA synthesis and real-time PCR. The expression change was calculated using 2-ΔΔct. Results: Our findings showed that FN1 and CLDN4 were upregulated in MKN45 cells. Following the transfection of cell by HOTAIR siRNA, both FN1 and CLDN4 genes were significantly downregulated. Conclusions: HOTAIR long non-coding RNA may have regulated the expression levels of FN1 and CLDN4 genes in EMT signaling pathway. However, it was recommended that further experimental analyses should be carried out to confirm this observation. In other word, our study result may not have been applied as a therapeutic access until additional experiments were conducted.

The aim of the current randomized control trial (RCT) study was to investigate the effects of fenugreek seed extract on memory, depression, quality of life, blood pressure, and serum malondialdehyde (MDA) and total antioxidant capacity (TAC) levels in adult AD patients. This randomized clinical trial was conducted in geriatric homes in Iran. The study participants included 82 AD patients with mild‐to‐moderate memory deficit. Patients in the intervention group received 5 cc of fenugreek seed extract for 4 months and subjects in the control group received a placebo. Memory, depression, quality of life, and BP levels, as well as serum MDA and TAC, were assessed before and after the intervention. There was a significant increase in serum levels of TAC (p < 0.001) and a reduction in serum MDA status (p < 0.001) after 4 months of fenugreek seed extract supplementation. In addition, increasing levels of memory (p < 0.001) and quality of life (p < 0.001), as well as reduction of depression (p = 0.002), systolic BP (p < 0.001), and diastolic BP (p < 0.001) levels were detected in the intervention group compared with baseline. Fenugreek seed extract supplementation in AD patients shows promising positive effects on memory, quality of life, BP, and selective oxidative indices levels.

Background: The use of molecular methods in cancer diagnosis has led to a better prognosis. One of the important gene families in the carcinogenic pathways of various cancers is the forkhead box (FOX) family genes. Moreover, developmental transcription factors and proapoptotic proteins play critical roles in cell function and carcinogenesis. Objectives: The current study aimed to evaluate the expression of A1 FOXA1, FOXA2, SOX10, and growth arrest specific 2 (GAS2) genes in oral squamous cell carcinoma (OSCC) tumors due to biomarker discovery and early diagnosis of cancer. Methods: To evaluate the expression of FOXA1, FOXA2, SOX10, and GAS2 genes, 30 OSCC samples and 30 normal specimens were obtained from Imam Khomeini Hospital Cancer Institute. RNA extraction and cDNA synthesis were done by relevant kits. After a specific primer design for FOXA1, FOXA2, SOX10, and GAS2 genes, real-time PCR was done to evaluate the genes’ expression for molecular biomarker discovery and validation. ANOVA and independent t-test were used to analyze the data. Results: Significant differences were observed in the expression of the studied genes in tumor and control tissues (P < 0.001). The results showed that FOXA1, GAS2, and SOX10 expressions in tumor and normal cells have significant differences (P < 0.001). Regardless of FOXA1, FOXA2 and SOX10, there was a significant difference in the expression of GAS2 genes in term patients’ age (P < 0.05) and overexpressed in patients over 55 years. SOX10 gene is upregulated in grade II OSCC tumors but there is no significant difference in expression of FOXA1, FOXA2, and GAS2 in different stages and grades. The ROC curve analysis, FOXA1, and FOXA2 showed AUC = 0.66 and AUC = 0.57 respectively. Meanwhile, SOX10 and GAS2 showed AUC = 0.9 and AUC = 1 respectively. Conclusions: In general, the expression of FOXA1, GAS2, and SOX10 genes in cancer and control tissues were different, and therefore the role of these genes in OSCC is confirmed. Also, in the present study, the biomarker potential of SOX10 and GAS2 genes for OSCC diagnosis was demonstrated. In the current study, the important role of the studied genes in OSCC diagnosis was shown. However, further studies are needed to confirm this.

Background: TGF-β signaling plays a key role in the performance of leukemic stem cells (LSCs). According to the evidence, any disturbance in the TGF beta pathway function not only poses negative effects on the development of BCR-ABL-induced chronic myeloid leukemia (CML) but also depletes CML stem cells. Hence, it can be argued that the aberrant acquisition of self-renewal characteristic because of the aberrant activation of TGF-β signaling results in the CML progression, which indicates the importance of inhibiting other targets besides BCR-ABL, such as TGF-β signaling, that can regulate self-renewal of the leukemic, which in turn translates into inhibited growth of BCR-ABL-positive CD34+ LSCs. Methods: The CD34+ cells were isolated from three CML patients with no history of treatments. The cells were cultured in a Stemspan H3000 medium. After being characterized by flow cytometry, the CML CD34+ cells were transfected with miR-144. The expression levels of miR-144, Smad4, and TGFβR2 genes were evaluated by RT-PCR. The impact of miR-144 overexpression on CML CD34+ cells was determined by the evaluation of cellular proliferation. Results: Overexpression of miR-144 resulted in the down-regulation of Smad4 and TGF-βR2 and decreased cell proliferation in CML CD34+ cells. Conclusions: This study suggests the down-regulation of miR-144 as a potential contributor to CML pathogenesis by activating the oncogenic TGF-β pathway. Hence, the proliferation capability of CD34+ CML cells, as a potential source of relapse in CML patients, can be inhibited by restoring miR-144 expression.

Background: Breast cancer is one of the most common cancers worldwide and is responsible for the death of many people. It is mainly found in women; however, it is rarely observed in men. Nowadays, extensive research has been conducted on the detection, diagnosis, and prognosis of biomarkers of this type of cancer, as well as other types. MicroRNAs play a major role in regulating the onset and progression of breast cancer. To date, many significant increases and decreases in microRNAs levels have been reported during various cancers, and also tumor inhibitory roles have been reported for these small non-coding RNA molecules. Methods: In this study, miR-30a and miR-200c were studied to find new expression patterns in breast cancer patients. For this purpose, a quantitative polymerase chain reaction was used to analyze the relationships between expression levels of miR-30a and miR-200c and several clinical and pathological features of the disease, such as tumor stage, HER2 gene expression, and lymphatic metastasis. Results: Our results showed that the expression of miR-200c and miR-30a in tumor samples was significantly lower than in normal samples (P < 0.01). Also, at higher stages of the disease, the expression of both miRNAs was remarkably reduced (P < 0.01). We also found that the expression of two miRNAs in patients with HER2 gene expression and metastasis in lymphatic regions was significantly lower than in HER2-negative and non-metastatic patients (P < 0.05 and P < 0.01, respectively). Conclusions: We identified the relationship between miR-200c and miR-30a and breast cancer. These findings indicate that these microRNAs can be considered biomarkers in breast cancer.

Breast cancer (BC) is one of the most common malignancies among women in the world. There is a global attempt to diagnose BC as early as possible. Long noncoding RNAs (lncRNAs) are emerging as novel targets and biomarkers for BC diagnosis and prognosis. Aberrant expression of lncRNAs is associated with BC development, making them a potential tumor marker for BC. To investigate this possibility, we determined the expression levels of Down syndrome cell adhesion molecule‐antisense RNA‐1 (DSCAM‐AS1) and mitotically‐associated long non‐coding RNA (MANCR) lncRNAs in BC tissues. This case–control study included 50 paired tumor and adjacent nontumor tissues from female BC patients. The total RNA was isolated and the expression levels of MANCR and DSCAM‐AS1 lncRNAs were assessed using quantitative real‐time reverse transcription‐PCR. Potential correlations between lncRNA levels and clinicopathological characteristics were also analyzed. DSCAM‐AS1 and MANCR lncRNAs were significantly upregulated in BC tumor tissues compared with the adjacent nontumor tissues. We also found the significant upregulation of DSCAM‐AS1 in advanced tumor–node–metastasis stage (TNM III) of BC tumor tissues. Furthermore, the expression of DSCAM‐AS1 and MANCR in HER‐2 positive patients was significantly higher than HER‐2 negative affected individuals. Receiver operating characteristic curve analysis showed a satisfactory diagnostic efficacy (P value < 0.0001), which means that DSCAM‐AS1 and MANCR lncRNAs can potentially serve as a biomarker. The present study might provide further approval for the clinical diagnostic significance of DSCAM‐AS1 and MANCR lncRNAs that their high expressions were associated with aggressive clinical parameters of BC.

Objective(s): Prevalence of high-fat food consumption, such as fast foods is one of the major causes of hypercholesterolemia, which can lead to cardiovascular diseases. 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) and cytochrome P450 7A1 (CYP7A1) are two key genes in cholesterol metabolism. Use of probiotics in the diet is a promising approach for modulation of serum lipid. To confirm the modulation of serum lipids by probiotics, in this study, we have examined the efficacy of Lactobacillus paracasei TD3 in improving blood cholesterol levels. Materials and Methods: 21 male Wistar rats were divided into three groups randomly (n=7). G1: negative control with normal diet, G2: positive control with high-fat diet, G3T: test group with high-fat diet plus supplementation with L. paracasei TD3 (1010 CFU). In the 21st day, the rats were anesthetized using chloroform and then sacrificed. Blood samples were collected to analyze lipid panel parameters and hepatic enzymes by the auto-analyzer system. Adipose tissue samples were analyzed using real-time PCR for HMGCR and CYP7A1 genes expression.Results: Consumption of L. paracasei TD3 could reduce serum cholesterol levels significantly (P<0.05); whereas, there was no significant difference between experimental groups for triglycerides, LDL, and HDL levels. Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) enzymes were significantly decreased in the probiotic group. Furthermore, expression of HMGCR and CYP7A1 genes was dramatically declined in the probiotic group. There was no significant change in either uric acid or urea between the control and treated groups.Conclusion: Introduction of L. paracasei TD3 in rat’s diet can modulate serum cholesterol levels.