Sohair Salem

Background This study sought to uncover novel targets for fighting drug resistance in breast cancer therapy, such as miRNA that target ABC transporters and decrease the oncogenic activity of breast cancer cells. Methods MiRNA ABC transporter targets were predicted using in silico methods after evaluating the ABC transporter route. In addition, miRNA expression, gene annotation, and gene ontology concepts were investigated. The expression levels of ABC transporter genes and miRNAs were determined using QRT-PCR. Dual luciferase activity was assessed to establish the precise ABC transporter-specific interaction. Flowcytometric analysis was used to detect doxorubicin uptake in miR-4330-treated cells, as well as cell cycle analyses. The spreading capacity of miR-4330-upregulated cells was investigated. Results The current study identified miR-4330 as an anti-drug and anti-oncogenic molecule in both estrogen receptor positive and negative breast cancer cells. Furthermore, the study identified the mechanism by which miR-4330 restoration reduces drug resistance by drastically decreasing ABCG2 expression (fold change of -0.25), which was accompanied by a considerable increase in doxorubicin accumulation or influx in both cell lines. Furthermore, miRNA-4330 restoration resulted in a considerable reduction in metastatic and spreading capabilities in both cell types. Conclusion The current findings identified miR-4330 restoration as a tool for overcoming acquired drug resistance and reducing cancer activity, which might be used to improve therapeutic strategies and treatment regimens for breast cancer patients. miR-4330 may be useful as a diagnostic and prognostic marker for drug-resistant breast cancer.

Background CRISPR/Cas9 is used for editing of non-coding sequence. This study tested the involvement of downstream cis-element in regulating CD44 expression and the possibility of using CRISPR/Cas9 system to manipulate the transcription factor binding site. Bioinformatic tools predicted two binding sites (P1 and P2) for NFYA transcription factors. CRISPR/Cas9 was used to knockout NFYA gene and alter the NFYA binding site (P2). Results The data revealed decrease of CD44 gene expression and CD44⁺ CD24⁻ sub-population after editing of P2 sequence more than the decrease resulted from editing of NFYA gene itself, confirming the involvement of NFYA in regulating CD44 gene. Both editing inhibited the migration ability of MDA-MB-231 cells. Unlike editing of NFYA gene, altering P2 sequence induced apoptosis. CHIP assay revealed that NFY have the ability to bind both P1 and P2 sequences, with higher enrichment in case of P2 than P1. After performing site-directed mutagenesis and luciferase assay we confirmed the involvement of both P1 and P2 in gene regulation, with higher potential in case of P2 than P1. Conclusion The findings confirmed the regulation of CD44 by NFYA and the efficacy of using CRISPR/Cas9 in altering the binding site, and downregulation of CD44.

Purpose: Breast cancer is one of the leading types of cancer diagnosed in women. Despite the improvements in chemotherapeutic cure strategies, drug resistance is still an obstacle leading to disease aggressiveness. The small non-coding RNA molecules, miRNAs, have been implicated recently to be involved as regulators of gene expression through the silencing of mRNA targets that contributed to several cellular processes related to cancer metastasis. Hence, the present study aimed to investigate the beneficial role and mechanism of miRNA-34a-based gene therapy as a novel approach for conquering drug resistance mediated by ATP-binding cassette (ABC) transporters in breast cancer cells, besides exploring the associated invasive behaviors. Material and Methods: Bioinformatics tools were used to predict miRNA ABC transporter targets by tracking the ABC transporter pathway. After the establishment of drug-resistant breast cancer MCF-7 and MDA-MB-231 sublines, cells were transfected with the mimic or inhibitor of miRNA-34a-5p. The quantitative expression of genes involved in drug resistance was performed by QRT-PCR, and the exact ABC transporter target specification interaction was confirmed by dual-luciferase reporter assay. Furthermore, flow cytometric analysis was utilized to determine the ability of miRNA-34a-treated cells against doxorubicin uptake and accumulation in cell cycle phases. The spreading capability was examined by colony formation, migration, and wound healing assays. The apoptotic activity was estimated as well. Results: Our findings firstly discovered the mechanism of miRNA-34a-5p restoration as an anti-drug-resistant molecule that highly significantly attenuates the expression of ABCC1 via the direct targeting of its 3′- untranslated regions in resistant breast cancer cell lines, with a significant increase of doxorubicin influx by MDA-MB-231/Dox-resistant cells. Additionally, the current data validated a significant reduction of metastatic potentials upon miRNA-34a-5p upregulation in both types of breast cancer-resistant cells. Conclusion: The ectopic expression of miRNA-34a ameliorates the acquired drug resistance and the migration properties that may eventually lead to improved clinical strategies and outcomes for breast cancer patients. Additionally, miRNA-34a could be monitored as a diagnostic/prognostic biomarker for resistant conditions.

Autism is associated with genomic instability, which is regulated by telomere length (TL) and index of global methylation (LINE-1). This study will determine relative TL (RTL) and LINE-1 methylation percentage for 69 patients and 33 control subjects to evaluate their potential role as biomarkers for autism. The results displayed a significant decrease of both RTL and LINE-1 methylation in autistic cases relative to controls (P < 0.001). Analysis of receiver operating characteristics curve revealed that both of RTL and LINE-1 methylation percentage have the ability to serve as autism biomarkers (area under the curve = 0.817 and 0.889, respectively). The statistical analysis revealed positive correlation between the two biomarkers (correlation coefficient = 0.439 and P < 0.001).

Epigenetic modifications play a significant role in the host's immune response to viral infection. Two epigenetic events, DNA methylation and histone acetylation, are crucial for modifying the chromatin architecture and the location of regulatory elements such as promoters and enhancers. In this case-control study, we evaluated the expression of genes involved in epigenetic machinery (DNMT1, DNMT3A, DNMT3B, HDAC2, and HDAC3) and the degree of methylation of promoters of immune response genes (IFITM1/2/3, TLR3/4, TNF-α, NF-κB, and MYD88) as well as global methylation (LINE-1 and global 5–mC) in blood samples from 120 COVID-19 patients (30 mild, 30 moderate, 30 severe, and 30 critical) and 30 healthy subjects without COVID-19. In contrast to previous reports, DNMT3A and DNMT3B expression was found to be significantly downregulated in COVID-19 cases, whereas DNMT1, HDAC2, and HDAC3 expression did not change. DNMT1 and DNMT3A were negatively correlated with COVID-19 severity. Critically ill patients had lower HDAC3 expression levels. TLR4 and TNF-α had increased promoter methylation, whereas IFITM1/2/3, TLR3, NF-κB, MYD88, and LINE-1 did not differ between cases and controls. Methylation of the TNF-α promoter increased as disease severity increased. Significantly less methylation of the TLR3 promoter was observed in patients with a positive outcome (recovery). We also found a correlation between the expression of DNMT3B and the methylation level of the TLR4 promoter. In milder cases, the global 5–mC levels were lower than that in more severe cases. Our findings suggest the exclusion of DNMTs inhibitors previously recommended for COVID-19 treatment and the need for additional research in this area.

The ability of individual and combined extracts of ginger and persimmon to target BCSCs subpopulation and restore the expression of tumor suppressive miRNAs, miR-200c, miR-30a and miR-128, is evaluated in this study. Combined treatment led to decreasing CD44+ CD24- subpopulation in MDA-MB-231 to a percent lower than both individual extracts, however, the combined treatment didn’t affect the expression of pluripotency-associated genes, OCT3/4, SOX2 and NANOG, which are down-regulated as a consequence of both individual extracts treatment. Bioinformatics predicted DNMT3A as the target of miR-200C and miR-30a, and ABCB9 as the target of miR-128a. Both individual extracts exerted an activating effect on miRNAs (except persimmon extract on miR-30a) and an inhibitory effect on their targets DNMT3a and ABCB9 expression. Additionally, combined treatment keeps the activating effect of individual extracts on miRNAs, decrease the inhibitory effect of individual extracts on ABCB9, and reversed it into activating effect in the case of DNMT3a. Moreover, we found a significant decrease in miR-200c promoter methylation after individual treatments, which could represent a positive feedback loop between miR-200c and its target DNMT3a, however, this effect on methylation status is absent in the case of combined treatment. The methylation of LINE1, the index of global DNA methylation, was increased in response to ginger extract treatment, predicting increasing in genomic stability. Our finding highlighted the role of individual and combined aqueous ginger extract and methanolic persimmon extract to subdue BCSCs by modulating the expression of tumor-suppressive miRNAs, despite the variation of molecular genetics and epigenetic response of candidate genes