Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis

Deciphering the molecular mechanism of YY1/HIF1A modulating ovarian cancer angiogenesis based on single-cell transcriptomics technology

Angiogenesis assumes an essential role in tumor development and is a fundamental condition for tumor growth. Yin Yang 1 (YY1) is highly expressed in various types of cancers and is a key player in tumor angiogenesis, but its role in ovarian cancer (OC) has not been fully elucidated. Therefore, this study will delve into the mechanism of YY1 in OC angiogenesis. Based on single-cell transcriptomics data of OC tumor samples and adjacent samples downloaded from the GEO database, differentially expressed genes (DEGs) and related signaling pathways were screened and validated in OC cells. Furthermore, co-culture technology was applied to assess the impact of YY1 expression in OC cells on angiogenesis ability. The molecular mechanism of YY1 regulation of OC angiogenesis was explored through bioinformatics analysis combined with co-immunoprecipitation, chromatin immunoprecipitation, and dual-luciferase reporter gene assays. Rescue experiments were designed, with results validated in qRT-PCR, angiogenesis assays, and Western blotting. Based on re-analysis of single-cell transcriptomics data from OC tumor samples and adjacent samples, we found that YY1 expression was significantly upregulated in OC cells, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis results showed that DEGs in YY1-positive tumor cells were significantly enriched in the HIF-1 signaling pathway. Moreover, in vitro experiments demonstrated that YY1 was highly expressed in OC to boost OC angiogenesis. Specifically, YY1 can stabilize hypoxia-inducible factor 1α (HIF1A) expression by competitively binding to WD repeat domain-containing 7 (FBXW7), thereby facilitating the transcriptional activation of angiogenesis genes. Finally, we demonstrated through rescue experiments that targeting the YY1/HIF1A axis can repress OC angiogenesis. Through single-cell transcriptomics analysis combined with cell experiments, we proved the specific mechanism by which YY1 affects the angiogenesis ability of OC. YY1 affects the expression of angiogenesis genes by modulating the FBXW7/HIF1A axis.

Agrimonolide inhibits glycolysis in ovarian cancer cells by regulating HIF1A

Ovarian cancer is one of the most common tumors affecting females, significantly disrupting their quality of life. Agrimonolide, an extract derived from Agrimony (Agrimonia pilosa Ledeb.), has been shown to exert various regulatory effects on several diseases. Notably, recent studies indicate that Agrimonolide may attenuate the progression of ovarian cancer. However, the detailed regulatory mechanisms of Agrimonolide in this context require further investigation. To determine the significance of HIF1A as a key target in ovarian cancer and its potential underlying signaling pathway. Cell viability and proliferation were assessed using CCK-8 and colony formation assays. Glucose uptake and lactate production were measured using commercial kits, and the extracellular acidification rate (ECAR) was evaluated. Protein expression levels were analyzed through western blotting. Our network pharmacology analysis identified HIF1A as a crucial target and signaling pathway in ovarian cancer. Furthermore, treatment with Agrimonolide (20 μM and 40 μM) inhibited the growth of ovarian cancer cells. Agrimonolide also reduced glycolytic activity in these cells. Additionally, Agrimonolide treatment led to decreased expression levels of HIF1A, HK2, and LDHA in ovarian cancer cells. Rescue assays revealed that glucose uptake and lactate production were diminished following Agrimonolide treatment; however, these effects were reversed upon overexpression of HIF1A. This study showed that Agrimonolide can suppress glycolysis in ovarian cancer cells by modulating HIF1A, supporting Agrimonolide as a promising therapeutic agent for ovarian cancer treatment.

Variants in exon 2 of MED12 gene causes uterine leiomyoma’s through over-expression of MMP-9 of ECM pathway

To study the impact of Mediator complex subunit 12 (MED12) gene variants on the encoded protein's function and pathogenic relevance for genesis of uterine leiomyoma's (ULs). Mutational analysis in exon-2 of MED12 gene was performed by PCR amplification and DNA sequencing in 89 clinically diagnosed ULs tissues. Pathogenicity prediction of variation was performed by computational analysis. The functional effects of missense variation were done by quantity RT-PCR and western blot analysis. Out of 89 samples, 40 (44.94%) had missense variation in 14 different CDS position of exon-2 of MED12 gene. Out of 40 missense variation, codon 44 had 25 (62.5%) looking as a hotspot region for mutation for ULs, because CDS position c130 and c131present at codon 44 that have necleotide change G>A, T, C at c130 and c131 have necleotide change G>A and C. We also find somenovel somatic mutations oncodon 36 (T > C), 38 (G>T) of exon-2 and 88 (G>C) of intron-2. No mutations were detected in uterine myometrium samples. Our computational analysis suggests that change in Med12c .131 G>A leads to single substitution of amino acid [Glycine (G) to Aspartate (D)] which has a pathogenic and lethal impact and may cause instability of MED12 protein. Further, analysis of extracellular matrix (ECM) component (MMP-2 & 9, COL4A2 and α-SMA) mRNA and protein expression levels in the set of ULs having MED12 mutation showed significantly higher expression of MMP-9 and α-SMA. The findings of present study suggest that missense variation in codon 44 of MED12 gene lead to the genesis of leiomyoma's through over-expression of MMP-9 of ECM pathway which could be therapeutically targeted for non-surgical management of ULs.

DGKβ accelerates the progression of cervical cancer through ANGPT4-mediated tumor angiogenesis

Cervical cancer (CC) is a major cause of morbidity and mortality in women, with complex etiology and progression. Diacylglycerol kinases (DGKs) are pivotal in lipid metabolism. Although diacylglycerol kinase beta (DGKβ) is well-studied in neurology, its role in cancer, especially CC, remains underexplored. This study aimed to explore DGKβ's role and mechanism in CC. Bioinformatics analysis was employed to identify genes differentially expressed in CC, with western blot confirming DGKβ expression in CC cells. The role of DGKβ was examined through small interfering RNA-mediated gene silencing, proliferation tests, migration and invasion assays, and angiogenesis studies. In-depth bioinformatics explored DGKβ-regulated downstream targets and pathways. Pathological assessment elucidated the impact of DGKβ and angiopoietin 4 (ANGPT4) on CC samples. Our data identified DGKβ as a promising candidate gene in the context of CC. This conclusion stemmed from the notable observation that DGKβ exhibited a heightened expression in CC cell lines. Notably, the silencing of DGKβ resulted in the suppression of CC cell proliferation, invasion, migration, as well as the epithelial-mesenchymal transition processes. Additional bioinformatics analysis delving into DGKβ-associated genes revealed ANGPT4 as a downstream target gene of DGKβ, which is capable of modulating angiogenesis and possesses multiple cellular functions related to cell survival, proliferation, and migration. Most significantly, our findings also demonstrated that both DGKβ and ANGPT4 were overexpressed in clinical specimens of CC. This study uncovered an oncogenic role for DGKβ in CC and identified a potential regulatory link between DGKβ and ANGPT4 in tumor angiogenesis. These findings provided promising directions for developing new diagnostic and therapeutic approaches for CC.

APOC1, transcriptionally regulated by FOXM1, promotes M2 macrophage polarization and cervical cancer progression

Cervical cancer (CC) is a common malignant tumor in women. M2 macrophages are associated with tumor growth, metastasis, and immunosuppression. Apolipoprotein C1 (APOC1) has been confirmed as an oncogene in CC. However, the role and mechanism of APOC1 in CC progression and M2 macrophages remain to be elucidated. The effects of APOC1 on CC cell malignant phenotypes were examined by CCK-8, colony formation, wound healing, and transwell assays in vitro and mice transplant tumor model in vivo. M2 macrophage polarization was assessed by qRT-PCR and flow cytometry assays. The interaction between APOC1 and forkhead box M1 (FOXM1) was determined using chromatin immunoprecipitation (ChIP) and luciferase reporter assays. The expression of APOC1 and FOXM1 was upregulated in CC tissues and cells. Knockdown of APOC1 or FOXM1 resulted in the inhibition of cell proliferation, migration, invasion, and EMT. Moreover, the polarization of M2 macrophages was attenuated when APOC1 or FOXM1 was silenced. Mechanistically, FOXM1 transcriptionally activated APOC1 by binding to its promoter. Furthermore, overexpression of APOC1 reversed the inhibitory effects of FOXM1 knockdown on cell proliferation, metastasis, and M2 macrophage polarization. Additionally, the knockdown of APOC1 reduced tumor growth and M2 macrophage polarization in mice. FOXM1/APOC1 axis is involved in the progression of CC and the regulation of M2 macrophages polarization, bringing new hope to the treatment of CC.

PAX8-AS1/microRNA-25–3p/LATS2 regulates malignant progression of ovarian cancer via Hippo signaling

Ovarian cancer (OC) is a frequent malignancy of the female reproductive system. Recently, the aberrant expression of numerous lncRNAs has been confirmed as a key factor for cancer development. The regulatory role of PAX8-AS1 in some cancers has been investigated, but its role in OC progression remains unclear. This study focuses on the role and molecular mechanism of PAX8-AS1 in the malignant progression of OC. Bioinformatics means were adopted to analyze the expression of PAX8-AS1, microRNA-25-3p, and LATS2 in OC tissues and the binding sites between the three. qRT-PCR was employed to determine the expression of these genes in OC cells. CCK-8, colony formation, scratch healing, and Transwell assays were used to see cell viability, proliferation, migration, and invasion, respectively. Fluorescence in situ Hybridization was performed to probe the subcellular localization of PAX8-AS1. Western blot was applied to evaluate the expression and phosphorylation levels of YAP and TAZ, and an immunofluorescence assay was used to detect the translocation of them. Dual luciferase assay was applied to validate the binding relationship between PAX8-AS1 and microRNA-25-3p, as well as between microRNA-25-3p and LATS2. PAX8-AS1 and LATS2 were lowly expressed. MicroRNA-25-3p was highly expressed in OC. PAX8-AS1 was expressed in cytoplasm and regulated LATS2 expression by sponging microRNA-25-3p. Overexpressing PAX8-AS1 can suppress the malignant behaviors of OC cells, whereas treatment with microRNA-mimic can reverse these results. In addition, the phosphorylation levels of YAP and TAZ increased upon oe-LATS2 treatment, and oe-LATS2 could promote YAP and TAZ translocate from the nucleus to cytoplasm. Rescue experiments demonstrated that sh-PAX8-AS1 fostered malignant progression of OC, which was reversed by simultaneous oe-LATS2. In summary, PAX8-AS1/microRNA-25-3p/LATS2 regulated the malignant progression of OC through Hippo signaling, which suggested that PAX8-AS1/microRNA-25-3p/LATS2 axis may be a novel target for OC treatment.

MiR-210-3p accelerates tumor-relevant cell functions of endometrial carcinoma by repressing RUNX1T1

Biological mechanism of miR-210-3p in endometrial carcinoma (EC) remains unclear. Here, our purpose is to study effects of miR-210-3p on malignant progression of EC. Bioinformatics analysis showed miRNA and mRNA are abnormally expressed in EC tissues. Quantitative real-time fluorescence polymerase chain reaction (qRT-PCR) was utilized to compare miR-210-3p mRNA level in EC cells and tissues. qRT-PCR and western blot were used to measure RUNX1T1 and NCAM1 at mRNA and protein levels, and western blot for p-AKT and AKT proteins related to PI3K/AKT signaling pathway. Furthermore, EC cell behaviors were assayed via Cell Counting Kit-8, cell colony formation assay, wound healing, transwell and flow cytometry experiments. Interaction between RUNX1T1 and miR-210-3p was verified through dual-luciferase assay. Immunohistochemistry was used to analyze RUNX1T1 expression in clinical samples RESULTS: MiR-210-3p was considerably upregulated and RUNX1T1 was significantly under-expressed in EC. Overexpression of miR-210-3p stimulated cell proliferation, migration, invasion, and restrained cell apoptosis in EC. Dual-luciferase assay proved that RUNX1T1 was a target gene of miR-210-3p. The level of RUNX1T1 in EC was downregulated after overexpressing miR-210-3p. Rescue assay showed that overexpression of RUNX1T1 had an inhibitory impact on tumor-relevant cell behaviors, whereas overexpression of miR-210-3p rescued such inhibition. Overexpression of RUNX1T1 reduced p-AKT expression, which was restored with concomitantly overexpressed miR-210-3p. In general, miR-210-3p behaves as an oncogene in EC by down-regulating the expression of RUNX1T1. This study elucidates a new functional mechanism in EC, and indicates miR-210-3p an underlying target.