Osteosarcoma (OS) is an aggressive primary bone malignancy with peak incidence in children and adolescents. Despite current multimodal treatments, there has been little change in overall survival outcomes in the last two decades. The canonical Wnt/β-catenin pathway is known to be a critical pathway in OS progression. To better understand the molecular basis of OS and potentially provide target/s for new therapies or diagnostics, we investigated the relationship between β-catenin, more specifically, the transcriptionally active form of β-catenin, Activated β-Catenin (ABC), and OS progression. We previously reported an association between ABC and aggressive OS whereby, cellular/nuclear ABC levels, but not cellular/nuclear β-catenin levels, increase with the degree of aggressiveness. However, a direct role for ABC in promoting OS progression has not been shown. In order to directly determine the role/impact of ABC in OS progression, we generated a pEGFP-ABC fusion construct which simulates ABC's phosphorylation pattern. Transfection of pEGFP-ABC, pEGFP-β-catenin, or an empty vector (pEGFP) into OS cell lines showed that wnt pathway transcriptional activity in GFP-ABC-expressing cells was significantly higher than that in both GFP-β-catenin and empty-vector-transfected cells. We also show that the in vitro invasive potential of the pEGFP-ABC-transfected cells was significantly higher compared to both pEGFP-β-catenin and pEGFP-transfected cells. Immunohistochemistry of clinical pediatric OS specimens supported these findings, showing a significant correlation between high ABC levels and invasive disease. To the best of our knowledge, this is the first report that suggest that ABC drive transcriptional activity to enhance invasiveness in OS and could serve as a biomarker of aggressive or metastatic OS.
BACKGROUND:Epstein-Barr Virus (EBV), a potent viral carcinogen, plays a crucial role in the development of various malignancies. Among its proteins, EBV nuclear antigen-1 (EBNA1) stands out for its ability to modulate gene expression. In this study, we explored the impact of EBNA1 on the expression patterns of four cellular genes-Derlin1, ZEB1, CNN3, and PSMD10-in HeLa cells. MATERIALS AND METHODS:Three distinct categories of HeLa cells were established: EBNA1-Transfected Cells: These cells were transfected with the EBNA1 gene.Control Plasmid-Transfected Cells: These cells received transfection with a control plasmid.Non-Transfected Cells (Control Group): These cells were not subjected to any transfection. After RNA extraction, we employed real-time PCR to evaluate the transcriptional levels of four specific genes-Derlin 1, ZEB1, CNN3, and PSMD10-in each of the three cell groups. The Mann-Whitney U-test was subsequently utilized to compare means, and statistical significance was determined based on p-values below 0.05. Data were meticulously recorded in an Excel 2016 spreadsheet. RESULTS:The results demonstrated that HeLa cells transfected with the EBNA1 plasmid exhibited significantly increased expression levels of Derlin1 (p = 0.028) and PSMD10 (p = 0.028) genes compared to cells transfected with the control plasmid. However, the expression changes observed in CNN3 and ZEB1 were not statistically significant (p = 0.99 and p = 0.2, respectively). CONCLUSIONS:Our findings suggest that increase expression levels of Derlin1 and PSMD10 genes in HeLa cells by the EBV-EBNA1 might induce cancer cell survival and accelerates the development of cervical cancer (CC). However, to establish a conclusive link between EBV-EBNA1 and CC progression, further investigations are warranted.
BACKGROUND:Gastric cancer (GC) is a multifactorial disease with a high death rate due to the unknown mechanisms involved in the developing, progressing, and late diagnosing GC. Several cancers have been linked to Long non-coding RNAs (lncRNAs), including GC, through differential expression. They play a crucial role in tumorigenesis pathways as modulatory factors, making them intriguing clinical and diagnostic biomarkers for many malignancies. This study's objective is to compare the lncRNAs CBR3-AS1 and PCA3 expression levels in tumoral tissues to marginal tissues and the clinicopathological features of patients. METHODS AND RESULTS:100 GC patients' tumoral and marginal tissue samples from Tabriz's Valiasr Hospital were gathered for this case-control research. To determine the expression level of PCA3 and CBR3-AS1 lncRNAs in GC, total RNA was extracted, and the qRT-PCR technique was employed. Compared to adjacent marginal tissues, the tumor tissue of patients with GC showed a significant increase in the expression levels of PCA3 and CBR3-AS1 (P < 0.0001). The expression ratio of lncRNA CBR3-AS1 and PCA3 did not significantly correlate with clinicopathological variables. The ROC curve's findings lead to the conclusion that the genes lncRNAs PCA3 and CBR3-AS1, with AUC values of 0.68 and 0.79, respectively, suggest that they could play carcinogenic roles in GC and may act as moderate diagnostic biomarkers for GC. CONCLUSIONS:In GC, CBR3-AS1 and PCA3 may be utilized as therapeutic targets and prognostic biomarkers, respectively.
Site-directed mutagenesis is a basic molecular tool required for protein, RNA and plasmid engineering. For mutagenesis methods, an ideal goal is to reach the efficiency of 100%. Towards this goal, we have recently taken the first step by adopting an innovative strategy using primer pairs with 3'-overhangs, thereby developing P3 site-directed mutagenesis, with an average efficiency of ~50%. As the second step towards the ideal goal, we report here P3a site-directed mutagenesis with an efficiency reaching ~100%. We systematically evaluated this new method by engineering >100 point mutations and small deletions (or insertions) on >20 mammalian expression vectors encoding various epigenetic regulators and the spike protein of SARS-CoV-2. As all known mutagenesis methods are limited to point mutations and small deletions/insertions (up to a dozen nucleotides), a technical problem is how to carry out cassette mutagenesis for replacement, deletion or insertion of large DNA fragments. The high efficiency of P3a mutagenesis and the 'handshaking' feature of primer pairs with 3'-overhangs inspired us to adapt this new method for seamless cassette mutagenesis, including highly efficient epitope tagging and untagging, deletion of small or large DNA fragments (up to 5 kb) and insertion of gene fragments (up to ~0.4 kb), LoxP sites and sequences encoding degrons, sgRNA and tigRNA. Thus, this new site-specific and cassette mutagenesis method is highly efficient, fast and versatile, likely resulting in its wide use for typical biomedical research, as well as for engineering and refining synthetic or mutant proteins from AI-assisted design.
Retinoblastoma ( Rb1) is a gene that codes for a tumour suppressor protein involved in various types of cancer.It was first described in retinoblastoma and is segregated as an autosomal dominant trait with high penetrance.In 1971, Knudson proposed his hypothesis of the two hits, where two mutational events are required to initiate tumour progression.We analysed three different point mutations present in patients' retinoblastoma.We produced three cell lines with retinoblastoma protein (RB) mutated in various regions: the missense pN328H, pD718N, and the nonsense early stop codon pR552*.We studied the effect of these point mutations on levels of mRNA and protein expression, proliferation, viability, localisation, and migration using an RBKO cell line.All three affected their localisation patterns and proliferation.However, the pR552* mutation also increases viability and migration.Moreover, when this mutation is simultaneously expressed with a wild-type RB, the phenotype and proliferation parameters are as with the mutant alone, suggesting that maybe only one mutated allele is needed to trigger the characteristic cancer phenotype.In other words, the pR552* mutant behaves more like a gain-of-function or oncogenic mutant.Indeed, a family carrying this mutation showed complete penetrance and high expressivity.
