Shirin Shahbazi

Background. The PIK3CA:H1047R mutation is one of the most common driver mutations in breast cancer that leads to aberrant activation of the PI3K/AKT/mTOR signaling pathway. Rapid and low-cost detection of this mutation can significantly contribute to a better outcome and personalized treatment protocols. This study aimed to develop and optimize an allele-specific LAMP assay for the visual detection of the H1047R mutation without the need for advanced laboratory equipment. Methods. Allele-specific FIP and BIP primers were designed with a 5′ end complementary to the mutant nucleotide, along with a loop primer to enhance amplification speed and sensitivity. The assay was tested on DNA from MDA-MB-453 (mutant) and MCF-7 (wild-type) breast cancer cell lines. Sensitivity was evaluated using serial dilutions. Reaction outcomes were assessed visually using Neutral Red colorimetry and SYBR Green fluorescence. Additionally, RNA-sequencing data of H1047R-positive breast cancer samples from the TCGA database were analyzed to investigate gene expression changes within the PI3K/AKT/mTOR pathway. Results. The developed assay accurately distinguished mutant from wild-type sequences with high specificity. The loop primer reduced reaction time from 25 to 20 minutes and improved sensitivity, achieving a detection limit of 0.033 ng/µL. Neutral Red enabled clear visual discrimination between positive and negative reactions, whereas SYBR Green showed only minor fluorescence differences. RNA-seq analysis revealed significant alterations in the expression of multiple PI3K/AKT/mTOR pathway genes in H1047R-positive samples. Conclusion. The optimized PE-LAMP assay provides a rapid, sensitive, inexpensive, and naked-eye–readable method for detecting the PIK3CA:H1047R mutation. Given its simplicity and minimal equipment requirements, this approach holds strong potential for clinical application in resource-limited settings to support targeted therapy selection in breast cancer patients. Practical Implications. The application of the developed method in this study can facilitate the detection of the PIK3CA:H1047R mutation, and potentially other mutations in the future, in clinical and laboratory settings with limited resources. Its high speed, low cost, and the ability to visualize results with the naked eye make this method suitable for initial screening of the patient and rapid selection of targeted therapies. Moreover, its use in point-of-care settings can improve patient access to accurate molecular diagnostic approaches.

Introduction: As a transcription factor, FOXM1 is elaborate in diverse aspects of cancer, such as proliferation, invasion as well as metastasis. Alternative splicing creates several FOXM1 isoforms, among which FOXM1b and FOXM1c are the key transcripts evaluated. Recently, FOXM1 was reported to be direct target of miR-4521, a microRNA with a possible tumor suppressor role. In this study alterations in miR-4521 expression and its association with FOXM1b and FOXM1c were investigated in ductal carcinoma of breast. Methods: Gene expression was examined in 44 matched tumor and adjacent normal tissue samples, along with the MCF-7 cell line. By designing specific primers, the expression of two FOXM1 transcript variants and the miR-4521 gene, along with internal controls were calculated by quantitative Real-time PCR. The amplified fragments were checked via Sanger sequencing. Results: The expression of FOXM1b and FOXM1c significantly increased in tumor tissues, emphasizing the oncogenic role of both transcript variants in breast cancer. The fold change in FOXM1b and FOXM1c were 4.62 and 3.12, respectively. miR-4521 was significantly down-regulated in tumors (fold change: 0.218), which validate the tumor suppressor effect. A decrease in miR-4521 expression had a statistically significant association with the increased expression of FOXM1b and FOXM1c (P = 0.000). Conclusion: Targeting the FOXM1 transcription factor represents a promising approach to halt breast cancer progression. Our findings indicate that FOXM1b and FOXM1c might have a role in the progress of breast cancer, and miR-4521 could be a potential candidate for their suppression.

Introduction: Mitochondrial DNA (mtDNA) copy number variations have been reported in multiple human cancers. Previous studies indicate that mitochondrial retrograde signaling regulates miR663, which plays a key role in tumorigenesis, including regulating apoptosis antagonizing transcription factor (AATF). This study investigates the expression of miR663 and AATF in relation to mtDNA copy number in invasive ductal carcinoma (IDC) of the breast. Methods: Paired primary tumors and adjacent non-tumor tissues were analyzed to assess changes in miR663 and AATF expression using fold-change analysis. The mtDNA copy number was quantified using COX1 as the mitochondrial gene and COX4 as the nuclear control gene. To validate the findings, publicly available data from The Cancer Genome Atlas (TCGA) were also analyzed. Results: A significant reduction in tumor miR663 expression was observed (fold change=0.139), with a strong correlation between miR663 and AATF expression. A significant Z-score difference was also detected between miR663 and mtDNA copy number. miR663 was predominantly expressed in grade I tumors but significantly downregulated in higher-grade tumors, whereas AATF expression increased with tumor grade. In silico analysis of TCGA data confirmed elevated AATF expression, with notable variations across breast cancer subtypes. Conclusion: We observed reduced expression of miR663 and mtDNA copy number in breast tumors, along with variations in AATF levels across subtypes. The decrease in miR663 could be associated with lower mtDNA copy numbers and impaired retrograde signaling, impacting AATF expression and function. Our findings underscore the therapeutic promise of targeting the mtDNA/miR-663/AATF axis, which could lead to advancements in breast cancer treatment.

MiR-132-3p acts in normal breast development and its downregulation has been documented in breast cancer. One of the targets of miR-132-3p is RB1 which is also inactivated in breast cancer. The interactions between RB1 and miR-132 have been reported in several pathological conditions. We aimed to investigate the correlation between expression levels of miR-132 and RB1 in ductal carcinoma of the breast. The study was carried out on tissues obtained from female patients with primary breast cancer. Tumor samples were classified using clinical and pathological data. Following RNA extraction and cDNA synthesis, relative gene expressions in tumors were compared to non-cancerous adjacent tissues. The link between RB1 and miR-132 was assessed by the correlation coefficient test. Our findings revealed a significant decrease in miR-132 and RB1 expressions with a ratio of 0.165 and 0.365, respectively. Tumor grade showed an association with miRNA-132 levels. The expression of miR-132 in grade I tumors was almost equal to that of normal adjacent tissues, but was intensely decreased in grades II and III. The correlation analysis showed a small linear association between RB1 and miR-132 levels. The reduction of miR-132 and RB1 expression confirmed the tumor-suppressive role of both genes in breast cancer. Considering that RB1 is one of the miR-132 targets, further studies are required to discover any miRNA-mediated upregulation role for miR-132. Our finding discovered a small linear association between miR-132 and RB1, which can be concluded towards their independent function in breast cancer pathogenesis.

Background: K-Ras mutations rarely occur in breast cancer. However, studies have supported that K-Ras upregulation is involved in breast cancer pathogenesis. Two main K-Ras transcript variants; K-Ras4A and K-Ras4B, originate from the alternative splicing of exon 4. In this study, we aimed to evaluate variations in the expression of K-Ras4A ...

Background: K-Ras mutations rarely occur in breast cancer. However, studies have supported that K-Ras upregulation is involved in breast cancer pathogenesis. Two main K-Ras transcript variants; K-Ras4A and K-Ras4B, originate from the alternative splicing of exon 4. In this study, we aimed to evaluate variations in the expression of K-Ras4A ...

Background: Lack of knowledge around underlying mechanisms of gliomas mandates intense research efforts to improve the disease outcomes. Identification of high-grade gliomas pathogenesis which is known for poor prognosis and low survival is of particular importance. Distinguishing the differentially expressed genes is one of the core approaches to clarify the causative factors. Methods: Microarray datasets of the treatment-naïve gliomas were provided from the Gene Expression Omnibus considering the similar platform and batch effect removal. Interacting recovery of the top differentially expressed genes was performed on the STRING and Cytoscape platforms. Kaplan–Meier analysis was piloted using RNA sequencing data and the survival rate of glioma patients was checked considering selected genes. To validate the bioinformatics results, the gene expression was elucidated by real-time RT-qPCR in a series of low and high-grade fresh tumor samples. Results: We identified 323 up-regulated and 253 down-regulated genes. The top 20 network analysis indicated that PTX3, TIMP1, CHI3L1, LTF and IGFBP3 comprise a crucial role in gliomas progression. The survival was inversely linked to the levels of all selected genes. Further analysis of RNA sequencing data indicated a significant increase in all five genes in high-grade tumors. Among them, PTX3, TIMP1 and LTF did not show any change in low-grade versus controls. Real-time RT-qPCR confirmed the in-silico results and revealed significantly higher expression of selected genes in high-grade samples compared to low-grade. Conclusions: Our results highlighted the role of PTX3 and TIMP1 which were previously considered in glioma tumorigenesis as well as LTF as a new potential biomarker.

Prenatal diagnosis of hereditary diseases has substantially altered the way medical geneticists are helping families affected by genetic disorders. However, the risk of miscarriage and fear of invasive diagnostic procedures may discourage many couples from seeking prenatal diagnosis. With the discovery of maternal plasma cell-free fetal DNA, prenatal diagnosis has entered a new era of progress. Cell-free DNA is released during normal physiological functions as well as through the cell death programs of apoptosis and necrosis. It can be found in the plasma and other body fluids. Although this method has the advantage of being noninvasive, it is still rather expensive and requires advanced hardware and comprehensive data analysis. Promising implications of noninvasive prenatal diagnosis methods for the diagnosis of common trisomy disorders have paved the way for the development of more complicated assays of single-gene disorders. Relative mutation dosage and relative haplotype dosage are the most widely implemented assays for noninvasive prenatal diagnosis of single-gene disorders. However, each assay has its own advantages and disadvantages. Relative mutation dosage is based on the droplet digital polymerase chain reaction (PCR) technique which includes quantification features of real-time PCR assays. Relative haplotype dosage is based on next-generation sequencing that includes analysis of the maternal and paternal genome followed by sequencing of maternal plasma cell-free DNA. Co-amplification at a lower denaturation temperature PCR is another approach that is based on forming heteroduplexes between alleles to selectively amplify paternal mutations. In this review, we have described the most common noninvasive prenatal diagnosis approaches and compared their applications in genetic disorder diagnosis with different inheritance patterns. Key words: Cell-free nucleic acids, Prenatal diagnosis, Noninvasive prenatal testing, Single-gene diseases, Non-invasive techniques.