Genes and Cancer最新文献

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.

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.

[This corrects the article DOI: 10.18632/genesandcancer.236.].

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.

Background: In some breast cancers, altered estrogen-sulfotransferase (SULT1E1) and its inactivation by oxidative-stress modifies E2 levels. Parallelly, hypoxia-inducible tissue-damaging factors (HIF1α) are induced. The proteins/genes expressions of these factors were verified in human-breast-cancer tissues. SULT1E1 inducing-drugs combinations were tested for their possible protective effects.

Methods: Matrix-metalloproteases (MMP2/9) activity and SULT1E1-HIF1α protein/gene expression (Western-blot/RTPCR) were assessed in breast-cancers versus adjacent-tissues. Oxidant-stress neutralizer, chalcone (trans-1,3-diaryl-2-propen-1-ones) and SULT1E1-inducer pure dialyl-sulfide (garlic; Allium sativum) were tested to prevent cancer causing factors in rat, in-vitro and in-vivo. The antioxidant-enzymes SOD1/catalase/GPx/LDH and matrix-degenerating MMP2/9 activities were assessed (gel-zymogram). Histoarchitecture (HE-staining) and tissue SULT1E1-localization (immuno-histochemistry) were screened. Extensive statistical-analysis were performed.

Results: Human cancer-tissue expresses higher SULT1E1, HIF1α protein/mRNA and lower LDH activity. Increase of MMP2/9 activities commenced tissue damage. However, chalcone and DAS significantly induced SULT1E1 gene/protein, suppressed HIF1α expression, MMP2/9 activities in rat tissues. Correlation and group statistics of t-test suggest significant link of oxidative-stress (MDA) with SULT1E1 (p = 0.006), HIF1α (p = 0.006) protein-expression. The non-protein-thiols showed negative correlation (p = 0.001) with HIF1α. These proteins and SULT1E1-mRNA expressions were significantly higher in tumor (p < 0.05). Correlation data suggest, SULT1E1 is correlated with non-protein-thiols.

Conclusions: Breast cancers associate with SULT1E1, HIF1α and MMPs deregulations. For the first time, we are revealing that advanced cancer tissue with elevated SULT1E1-protein may reactivate in a reducing-state initiated by chalcone, but remain dormant in an oxidative environment. Furthermore, increased SULT1E1 protein synthesis is caused by DAS-induced mRNA expression. The combined effects of the drugs might decrease MMPs and HIF1α expressions. Further studies are necessary.

The MYC gene is a regulatory and proto-oncogenic gene that is overexpressed in the majority of prostate cancers (PCa). Numerous studies have indicated that aberrant expression of microRNAs is involved in the initiation and progression of prostate cancer. In this investigation, we assessed the impact of miR-377 on MYC through luciferase assay. Real-time PCR was employed to determine whether miR-377 could reduce the levels of MYC mRNA in transfected PCa cell lines (PC-3 and DU145) and change in the mRNA levels of BCL-2/Bax, PTEN, and CDK4 as a consequence of MYC downregulation. Moreover, we analyzed the effects of miR-377 on apoptosis, proliferation, cell cycle, and wound healing. Our findings demonstrate that miR-377 effectively targets MYC mRNA, as confirmed by luciferase assay and Real-time PCR. We observed a significant reduction in BCL-2 and CDK4 expression, along with an increase in Bax and PTEN, in prostate cancer cell lines upon MYC suppression. Additionally, elevated levels of miR-377 in PCa cell lines induced apoptosis, inhibited proliferation and migration, and arrested the cell cycle. Taken together, these results unveil the inhibitory role of miR-377 in MYC function within PCa, thereby suggesting its potential as a therapeutic target for the treatment of this malignancy.

Ewing sarcoma is a cancer of bone and soft tissue in children and young adults that is driven by the EWS-ETS fusion transcription factor, most commonly EWS-FLI1. We previously reported that Ewing sarcoma harbors two populations of cells, the CD133^high population displaying higher growth rate and the CD133^low population displaying chemotherapy resistance. We now find that the ubiquitin-specific protease 1 (USP1) is a transcriptional target of the EWS-FLI1 fusion oncoprotein, expressed at high and low levels in the CD133^high and the CD133^low populations, respectively, and determines chemo-sensitivity. We also find that USP1 inhibits cdc42, increases EWS-FLI1 transcriptional output, and simulates Ewing sarcoma growth. We show that chemo-sensitization by USP1 is independent of cdc42. A pharmacological inhibitor of USP1 was able to activate cdc42 and inhibit Ewing sarcoma growth. These results uncover critical roles for USP1 in Ewing sarcoma, which regulates growth and chemo-sensitivity via distinct mechanisms.

Hepatocellular carcinoma (HCC) is the third leading cause of death from cancer worldwide but is often diagnosed at an advanced incurable stage. Yet, despite the urgent need for blood-based biomarkers for early detection, few studies capture ongoing biology to identify risk-stratifying biomarkers. We address this gap using the TGF-β pathway because of its biological role in liver disease and cancer, established through rigorous animal models and human studies. Using machine learning methods with blood levels of 108 proteomic markers in the TGF-β family, we found a pattern that differentiates HCC from non-HCC in a cohort of 216 patients with cirrhosis, which we refer to as TGF-β based Protein Markers for Early Detection of HCC (TPEARLE) comprising 31 markers. Notably, 20 of the patients with cirrhosis alone presented an HCC-like pattern, suggesting that they may be a group with as yet undetected HCC or at high risk for developing HCC. In addition, we found two other biologically relevant markers, Myostatin and Pyruvate Kinase M2 (PKM2), which were significantly associated with HCC. We tested these for risk stratification of HCC in multivariable models adjusted for demographic and clinical variables, as well as batch and site. These markers reflect ongoing biology in the liver. They potentially indicate the presence of HCC early in its evolution and before it is manifest as a detectable lesion, thereby providing a set of markers that may be able to stratify risk for HCC.

Background: In the pediatric patient, central venous catheterization may be associated with relevant complications. Though, most of them may be prevented by a wise choice of materials, methods, and techniques. Evidence-based insertion bundles for central venous catheterization have been developed in the adult patient, but not in neonates and children.

Methods: The Italian Group for Long Term Venous Access Devices (GAVeCeLT) has developed an insertion bundle for central venous catheterization in neonates, infants, and children, which includes seven evidence-based strategies: (1) preprocedural ultrasound evaluation, (2) appropriate aseptic technique, (3) ultrasound guided venipuncture, (4) intraprocedural tip location by non-radiological methods, (5) proper choice of the exit site by tunneling, (6) sutureless securement, and (7) protection of the exit site using glue and transparent membranes. The effectiveness and safety of this bundle has been tested in a prospective study.

Results: All neonates, infants and children requiring a non-emergency central line (except for umbilical venous catheters and epicutaneo-cava catheters) were included in the study. Out of 729 central line insertions, there were no immediate complications (no pneumothorax, no arterial puncture, no malposition); the incidence of early and late complications (local ecchymosis, dislodgment, local pain, exit site infection) was 3.7%; in the first 2 weeks after insertion, no catheter-related bacterial infection or catheter-related thrombosis was recorded.

Conclusion: The results of this prospective study strongly validate the hypothesis that an insertion bundle is highly effective in optimizing the safety of the maneuver, reducing immediate, early, and late complications.