Mutagenesis

Expression and clinical diagnostic value of CCHE1 in breast cancer

Abstract Objective Breast cancer is a malignant tumor in the epithelial tissue of the breast gland. This study aimed to unveil the expression and clinical diagnostic value of lncRNA cervical cancer high-expressed 1 (CCHE1) in breast cancer. Methods CCHE1 expression in breast cancer tissues was evaluated by RT-qPCR. The relationship between the CCHE1 expression and clinicopathological features of breast cancer was analyzed with the chi-square test, and the survival of breast cancer patients was evaluated with the Kaplan–Meier method. The diagnostic value of CCHE1 expression for breast cancer was evaluated by using the receiver operating characteristics (ROC) curve. Breast cancer cell lines (SKBR3, T47D, BT474, and MCF-7) were cultured for detecting CCHE1 expression in the cells. MCF-7 cells were selected for the subsequent experiments, and the small interfering RNA of CCHE1 (si-CCHE1) and CCHE1 overexpression vector (pcDNA-CCHE1) were transfected into MCF-7 cells. The proliferation, migration, and invasive ability were assessed by CCK-8 and Transwell assays. The influence of CCHE1 on the growth of tumors was validated by nude mice xenograft assay. Results CCHE1 was up-regulated in breast cancer tissues and breast cancer cells. The high expression level of CCHE1 in cancer tissues of breast cancer patients was correlated with larger tumor size, advanced TNM stage, Ki-67 status, and lymph node metastasis. The area under the ROC curve for CCHE1 in the diagnosis of breast cancer was 0.983 (95% CI: 0.966–1.000), with a sensitivity of 95.00% and a specificity of 91.70%. The 5-year survival rate was higher in patients with low CCHE1 expression than those with high CCHE1 expression. Furthermore, restrained CCHE1 impeded proliferation, invasion, and migration of MCF-7 cells, as well as tumor growth in mice. Conclusion Our study highlights that elevated expression of CCHE1 in breast cancer tissues, which is closely related to clinicopathologic features, has some clinical value in the diagnosis of the disease.

Effect of BRCA1 missense variants on gene reversion in DNA double-strand break repair mutants and cell cycle-arrested cells of Saccharomyces cerevisiae

AbstractEvaluation of the functional impact of germline BRCA1 variants that are likely to be associated to breast and ovarian cancer could help to investigate the mechanism of BRCA1 tumorigenesis. Expression of pathogenic BRCA1 missense variants increased homologous recombination (HR) and gene reversion (GR) in yeast. We thought to exploit yeast genetics to shed light on BRCA1-induced genome instability and tumorigenesis. We determined the effect on GR of several neutral and pathogenic BRCA1 variants in the yeast strain RSY6wt and its isogenic DSB repair mutants, such as mre11∆, rad50∆ and rad51∆. In the RSY6wt, four out of five pathogenic and two out of six neutral variants significantly increased GR; rad51∆ strain, the pathogenic variants C61G and A1708E induced a weak but significant increase in GR. On the other hand, in rad50∆ mutant expressing the pathogenic variants localised at the BRCT domain, a further GR increase was seen. The neutral variant N132K and the VUS A1789T induced a weak GR increase in mre11∆ mutant. Thus, BRCA1 missense variants require specific genetic functions and presumably induced GR by different mechanisms. As DNA repair is regulated by cell cycle, we determined the effect on GR of BRCA1 variants in cell cycle-arrested RSYwt cells. GR is highly BRCA1-inducible in S-phase-arrested cells as compared to G1 or G2. Sequence analysis of genomic DNA from ILV1 revertant clones showed that BRCA1-induced ilv1-92 reversion by base substitution when GR is at least 6-fold over the control. Our study demonstrated that BRCA1 may interfere with yeast DNA repair functions that are active in S-phase causing high level of GR. In addition, we confirmed here that yeast could be a reliable model to investigate the mechanism and genetic requirements of BRCA1-induced genome instability. Finally, developing yeast-based assays to characterise BRCA1 missense variants could be useful to design more precise therapies.

Inhibition of homologous recombination repair by Mirin in ovarian cancer ameliorates carboplatin therapy response in vitro

Abstract Chemoresistance poses one of the most significant challenges of cancer therapy. Carboplatin (CbPt) is one of the most used chemotherapeutics in ovarian cancer (OVC) treatment. MRE11 constitutes a part of homologous recombination (HR), which is responsible for the repair of CbPt-induced DNA damage, particularly DNA crosslinks. The study’s main aim was to address the role of HR in CbPt chemoresistance in OVC and to evaluate the possibility of overcoming CbPt chemoresistance by Mirin-mediated MRE11 inhibition in an OVC cell line. Lower expression of MRE11 was associated with better overall survival in a cohort of OVC patients treated with platinum drugs (TCGA dataset, P < 0.05). Using in vitro analyses, we showed that the high expression of HR genes drives the CbPt chemoresistance in our CbPt-resistant cell line model. Moreover, the HR inhibition by Mirin not only increased sensitivity to carboplatin (P < 0.05) but also rescued the sensitivity in the CbPt-resistant model (P < 0.05). Our results suggest that MRE11 inhibition with Mirin may represent a promising way to overcome OVC resistance. More therapy options will ultimately lead to better personalized cancer therapy and improvement of patients’ survival.

Measurement of chromosomal instability and level of DNA damage in peripheral blood mononuclear cells of endometrial cancer patients

Abstract Endometrial cancer is one of the most common invasive gynecologic malignancies in developed countries. The aim of this study was to evaluate chromosomal instability and level of DNA damage in peripheral blood mononuclear cells (PBMCs) of newly diagnosed endometrial cancer patients in relation to health status (diagnosis), age, histological grade of cancer, residence, smoking, number of pregnancies, miscarriages, and abortions. The analyzed sample consisted of 60 individuals, 30 endometrial cancer patients with an average age of 64.37 ± 7.08, and 30 healthy control women with an average age of 60.23 ± 11.55. Chromosomal instability was evaluated by the cytokinesis-block micronucleus (CBMN) assay, and the level of DNA damage by the single-cell gel electrophoresis (comet) assay in PBMCs. The average frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) as well as nuclear buds (NBUDs) were significantly higher in cancer patients compared to controls (P < .0005). There was no difference in the nuclear division index (NDI) among the analyzed samples. The comet assay showed that the patients had a significantly increased genetic damage index (GDI) compared with controls (P < .0005). Using linear regression analysis, we found that health status (diagnosis) had the strongest influence on the MN frequency as well as GDI (P < .0005). Our results indicated that there is a high level of genetic damage in both the level of DNA and the level of chromosomes in the PBMCs of newly diagnosed patients with endometrial cancer, where the frequency and level of damage were significantly affected by health status, grade of cancer, residence, number of pregnancies, miscarriages, and abortions.