Mechanistic insights into UHRF1­mediated DNA methylation by structure­based functional clarification of UHRF1 domains (Review).

Epigenetic modification is crucial for transmitting genetic information, while abnormalities in DNA methylation modification are primarily associated with cancer and neurological diseases. As a multifunctional epigenetic modifier, ubiquitin like with PHD and ring finger domains 1 (UHRF1) mainly affects cell energy metabolism and cell cycle control. It also inhibits the transcription of tumor suppressor genes through DNA and/or histone methylation modifications, promoting the occurrence and development of cancer. Therefore, comprehensively understanding the molecular mechanism of the epigenetic modification of UHRF1 in tumors will help identify targets for inhibiting the expression and function of UHRF1. Notably, each domain of UHRF1 functions as a whole and differently. Thus, the abnormality of any domain can lead to a change in phenotype or disease. However, the specific regulatory mechanism and proteins of each domain have not been fully elucidated. The present review aimed to contribute to the study of the regulatory mechanism of UHRF1 to a greater extent in different cancers and provide ideas for drug research by clarifying the function of UHRF1 domains.

Development of a sensitive droplet digital PCR according to the HPV infection specificity in Chinese population.

HPV16 and 18 are positively correlated with cervical carcinogenesis. However, HPV prevalence tends to vary according to region, nationality, and environment. The most prevalent high-risk (HR) HPV genotypes are HPV16, 52, 58, 56, 18, 33, and 45), while the low-risk (LR) genotypes are HPV6 and 11 in the Chinese population. Importantly, undetectable low-copy HPV DNA could be an important indicator of integration into the human genome and may be a precursor to cancer progression. The HPV viral load changes dramatically, either increasing or decreasing rapidly during carcinogenesis, and traditional quantitative real-time PCR (qPCR) cannot accurately capture this subtle change. Therefore, in this study, a reliable droplet digital PCR (ddPCR) method was developed to simultaneously detect and quantify HPV genotypes. The ddPCR quantitative results showed high accuracy, sensitivity, and specificity compared to qPCR results employing the same clinical specimens and supplemented the ddPCR assay for HPV52/56/58/6 genotypes according to the infection specificity of the Chinese population. In summary, this procedure is valuable for quantifying HPV DNA, especially under conditions of low template copy number in cervical intraepithelial neoplasia (CIN) and/or cervical cancer. Additionally, this method can dynamically observe the prognosis and outcome of HPV infection and thus be used as an effective means for real-time monitoring of tumor load.

Downregulation of MicroRNA-29-3p Following Percutaneous Coronary Intervention: An Implication of YY1/IRAK1 Pathway in the Post-Vascular Injury Inflammation.

This study explored the expression of microRNA (miR)-29b-3p following percutaneous coronary intervention (PCI) and the implication of its downstream Yin Yang 1 (YY1)/interleukin (IL)-1 receptor-associated kinase 1 (IRAK1) pathway in post-vascular injury inflammation. Blood samples were collected for analysis of plasma miR-29b-3p from patients with acute coronary syndrome before surgery, 1 day after PCI, and 30 days after PCI. Lipopolysaccharide (LPS)-treated human coronary artery endothelial cells (HCAECs) were transfected with miR-29b-3p mimic/inhibitor or YY1 shRNA and underwent viability tests. Enzyme-linked immunosorbent assay was performed to detect the levels of soluble vascular cell adhesion molecule-1 (sVCAM-1), IL-1ß, IL-6, and tumor necrosis factor (TNF)-α in serum and cell culture supernatant. Dual-luciferase reporter and RNA/chromatin immunoprecipitation were used to confirm the targeting relationships among miR-29b-3p, YY1, and IRAK1. A rat model of intraluminal injury of the common femoral artery was established to address the role of miR-29b-3p and relevant mechanisms. miR-29b-3p was lowly expressed, and sVCAM-1, IL-1ß, IL-6, and TNF-α were upregulated 1 day after PCI and 24 h after LPS treatment. miR-29b-3p overexpression or YY1 knockdown alleviated LPS-induced inflammatory responses and improved the viability of HCAECs. miR-29b-3p inhibition aggravated LPS-induced inflammatory injury in HCAECs. miR-29b-3p bound to YY1 mRNA and inhibited the expression of YY1 protein. YY1 bound to the IRAK1 promoter and activated the transcription of IRAK1. Upregulation of miR-29b-3p suppressed the inflammatory response after intraluminal injury of the common femoral artery in rats. In conclusion, dysregulation of the YY1/IRAK1 pathway via miR-29b-3p downregulation may be implicated in post-vascular injury inflammation.

Mechanistic insights into genomic structure and functions of a novel oncogene YEATS4.

As a novel oncogene, the role of YEATS domain-containing protein 4 (YEATS4) in the occurrence, development, and treatment of tumors is now beginning to be appreciated. YEATS4 plays an important role in regulating DNA repair during replication. The upregulation of YEAST4 promotes DNA damage repair and prevents cell death, whereas its downregulation inhibits DNA replication and induces apoptosis. Additionally, accumulating evidence indicates that the aberrant activation of YEATS4 leads to changes in drug resistance, epithelial-mesenchymal transition and also in the migration and invasion capacity of tumor cells. Therefore, specific inhibition of the expression or activity of YEATS4 protein may be an effective strategy for inhibiting the proliferation, motility, differentiation, and/or survival of tumor cells. Taken together, YEATS4 has emerged as a potential target for multiple cancers and is an attractive protein for the development of small-molecule inhibitors. However, research on YEAST4 in tumor-related fields is limited and its biological functions, metabolism, and the regulatory mechanism of YEATS4 in numerous cancers remain undetermined. This review comprehensively and extensively summarizes the functions, structure and oncogenic roles of YEATS4 in cancer progression and aims to further contribute to the study of its underlying molecular mechanism and targeted drugs.

Functions and modulation of PKM2 activity by human papillomavirus E7 oncoprotein (Review).

Most tumor cells still exhibit active glucose uptake and glycolysis under aerobic conditions, a phenomenon known as the Warburg effect or aerobic glycolysis. Pyruvate kinase, one of the key enzymes in the cell glycolysis pathway, can promote the conversion of glucose to pyruvate and produce energy. Pyruvate kinase M2 (PKM2), a competitive PK subtype, is an important regulator of the aerobic glycolysis pathway in tumor cells and plays a direct role in gene expression and cell cycle regulation. Human papillomavirus (HPV) persistence is the main risk factor for cervical cancer. In recent years, it has been discovered that HPV plays an important role in malignant anal tumors and oral cancer. HPV oncoprotein E7 can promote the Warburg effect and produce a large amount of ATP, which may meet the energy requirements of cancer cell division. There appears to be a regulatory relationship between HPV E7 and PKM2, but the specific mechanism is mostly unknown. The present review article discusses the role of HPV E7 in transcriptional regulation, enzyme activity regulation, protein kinase activity regulation, post-translational modification and the immune microenvironment of PKM2 in the occurrence and development of cervical cancer.

FOXO1 represses MCL1 transcription to regulate the function of vascular smooth muscle cells in intracranial aneurysm.

Intracranial aneurysm (IA) is a pathological dilation of the cerebral arteries. Vascular smooth muscle cell (VSMC) dysfunction assumes a role in IA development. In this context, this study probed the role of FOXO1 in human brain VSMC (HBVSMC) function via MCL1. FOXO1 and MCL1 expression in arterial wall tissues from IA patients and inflammatory cytokines (IL-1ß, TNF-α, and IL-6) levels in the serum of IA patients were, respectively, detected with qRT-PCR and ELISA. Pearson's correlation analysis was utilized to analyze the correlation between FOXO1 and MCL1. After FOXO1 and/or MCL1 were overexpressed in HBVSMCs, caspase-3 and Cyt-c protein expression were examined by western blot, cell proliferation by CCK-8 and EdU assays, and cell apoptosis by flow cytometry. IL-1ß, TNF-α, and IL-6 levels were assessed in the supernatant of HBVSMCs with ELISA. Dual-luciferase gene reporter and ChIP assays were conducted to evaluate the binding of FOXO1 to MCL1. FOXO1 expression was high and MCL expression was low in arterial wall tissues from IA patients, and IL-1ß, TNF-α, and IL-6 levels were high in the serum of IA patients. There was an inverse correlation between FOXO1 and MCL1 mRNA levels. Moreover, FOXO1 bound to the MCL1 promoter to decrease MCL1 transcription. In addition, FOXO1 overexpression augmented cell apoptosis, caspase-3 and Cyt-c protein expression, and IL-1ß, TNF-α, and IL-6 secretion, while reducing cell proliferation in HBVSMCs, which was abrogated by further MCL1 overexpression. FOXO1 impeded MCL1 transcription to curb HBVSMC proliferation and facilitate their apoptosis and inflammation.

STAT3 plays an important role in DNA replication by turning on WDHD1.

BACKGROUND: Signal transducers and activators of transcription 3 (STAT3) is a transcription factor that plays a key role in many cellular processes such as cell growth and cancer. However, the functions and mechanisms by which STAT3 regulates cellular processes are not fully understood. RESULTS: Here we describe a novel function of STAT3. We demonstrated that STAT3 plays an important role in DNA replication. Specifically, knockdown of STAT3 reduced DNA replication while activation and ectopic expression of STAT3 promoted DNA replication. We further identified the WD repeat and HMG-box DNA-binding protein 1 (WDHD1), which plays an important role in DNA replication initiation, as a novel STAT3 target gene that mediated the DNA replication function of STAT3. We showed that STAT3 bind the promoter/up regulatory region of WDHD1 gene. CONCLUSIONS: These studies identified a novel function of STAT3 that is mediated by its newly identified target gene WDHD1 and have important implications.

A CRISPR-Cas12a-based specific enhancer for more sensitive detection of SARS-CoV-2 infection.

BACKGROUND: Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. METHODS: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. FINDINGS: The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. INTERPRETATION: SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. FUNDING: Detailed funding information is available at the end of the manuscript.

WDHD1 facilitates G1 checkpoint abrogation in HPV E7 expressing cells by modulating GCN5.

BACKGROUND: Genomic instability is a hallmark of cancer. The G1 checkpoint allows cells to repair damaged DNA that may lead to genomic instability. The high-risk human papillomavirus (HPV) E7 gene can abrogate the G1 checkpoint, yet the mechanism is still not fully understood. Our recent study showed that WDHD1 (WD repeat and high mobility group [HMG]-box DNA-binding protein 1) plays a role in regulating G1 checkpoint of E7 expressing cells. In this study, we explored the mechanism by which WDHD1 regulates G1 checkpoint in HPV E7 expressing cells. METHODS: NIKS and RPE1 derived cell lines were used. Real-time PCR, Rescue experiment, FACS and BrdU labeling experiments were performed to examine role of GCN5 in G1 checkpoint abrogation in HPV-16 E7 expressing cells. RESULTS: In this study, we observed that WDHD1 facilitates G1 checkpoint abrogation by modulating GCN5 in HPV E7 expressing cells. Notably, depletion of WDHD1 caused G1 arrest while overexpression of GCN5 rescued the inhibitory effects of WDHD1 knockdown on G1/S progression. Furthermore, siWDHD1 significantly decreased cell cycle proliferation and DNA synthesis that was correlated with Akt phosphorylation (p-Akt), which was reversed by GCN5 overexpression in HPV E7 expressing cells. CONCLUSIONS: In summary, our data identified a WDHD1/GCN5/Akt pathway leading to the abrogation of G1 checkpoint in the presence of damaged DNA, which may cause genomic instability and eventually HPV induced tumorigenesis.

Patients With LR-HPV Infection Have a Distinct Vaginal Microbiota in Comparison With Healthy Controls.

Condyloma acuminatum (CA) is a benign epithelium hyperplasia mainly caused by human papillomavirus (HPV), which is now the second most common viral sexually transmitted infection (STI) in China. In total, 90% of CA patients are caused by the low-risk HPV 6 and 11. Aside from low-risk HPV infection there are likely other factors within the local microenvironment that contribute to CA and there has been related research before. In this study, 62 vaginal specimens were analyzed using 16S rRNA gene sequencing. The diversity of the vaginal microbiota was higher and the composition was different with LR-HPV infection. While the relative abundance of dominant Firmicutes was lower, Actinobacteria, Proteobacteria, and Fusobacteria phyla were significantly higher; at the genus level Gardnerella, Bifidobacterium, Sneathia, Hydrogenophilus, Burkholderia, and Atopobium were higher. This study firstly confirmed a more accurate and comprehensive understanding of the relationship between low-risk HPV infection and vaginal microbiota, in order to provide a theoretical basis for further research on the occurrence and development of CA.

Role of Cdc6 in re-replication in cells expressing human papillomavirus E7 oncogene.

The E7 oncoprotein of high-risk human papillomavirus (HPV) types induces DNA re-replication that contributes to carcinogenesis; however, the mechanism is not fully understood. To better understand the mechanism by which E7 induces re-replication, we investigated the expression and function of cell division cycle 6 (Cdc6) in E7-expressing cells. Cdc6 is a DNA replication initiation factor and exhibits oncogenic activities when overexpressed. We found that in E7-expressing cells, the steady-state level of Cdc6 protein was upregulated and its half-life was increased. Cdc6 was localized to the nucleus and associated with chromatin, especially upon DNA damage. Importantly, downregulation of Cdc6 reduced E7-induced re-replication. Interestingly, the level of Cdc6 phosphorylation at serine 54 (S54P) was increased in E7-expressing cells. S54P was associated with an increase in the total amount of Cdc6 and chromatin-bound Cdc6. DNA damage-enhanced upregulation and chromatin binding of Cdc6 appeared to be due to downregulation of cyclin-dependent kinase 1 (Cdk1) as Cdk1 knockdown increased Cdc6 levels. Furthermore, Cdk1 knockdown or inhibition led to re-replication. These findings shed light on the mechanism by which HPV induces genomic instability and may help identify potential targets for drug development.

Role of WDHD1 in Human Papillomavirus-Mediated Oncogenesis Identified by Transcriptional Profiling of E7-Expressing Cells.

UNLABELLED: The E7 oncoprotein of the high-risk human papillomavirus (HPV) plays a major role in HPV-induced carcinogenesis. E7 abrogates the G1 cell cycle checkpoint and induces genomic instability, but the mechanism is not fully understood. In this study, we performed RNA sequencing (RNA-seq) to characterize the transcriptional profile of keratinocytes expressing HPV 16 (HPV-16) E7. At the transcriptome level, 236 genes were differentially expressed between E7 and vector control cells. A subset of the differentially expressed genes, most of them novel to E7-expressing cells, was further confirmed by real-time PCR. Of interest, the activities of multiple transcription factors were altered in E7-expressing cells. Through bioinformatics analysis, pathways altered in E7-expressing cells were investigated. The upregulated genes were enriched in cell cycle and DNA replication, as well as in the DNA metabolic process, transcription, DNA damage, DNA repair, and nucleotide metabolism. Specifically, we focused our studies on the gene encoding WDHD1 (WD repeat and high mobility group [HMG]-box DNA-binding protein), one of the genes that was upregulated in E7-expressing cells. WDHD1 is a component of the replisome that regulates DNA replication. Recent studies suggest that WDHD1 may also function as a DNA replication initiation factor as well as a G1 checkpoint regulator. We found that in E7-expressing cells, the steady-state level of WDHD1 protein was increased along with the half-life. Moreover, downregulation of WDHD1 reduced E7-induced G1 checkpoint abrogation and rereplication, demonstrating a novel function for WDHD1. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications. IMPORTANCE: The high-risk HPV types induce cervical cancer and encode an E7 oncoprotein that plays a major role in HPV-induced carcinogenesis. However, the mechanism by which E7 induces carcinogenesis is not fully understood; specific anti-HPV agents are not available. In this study, we performed RNA-seq to characterize transcriptional profiling of keratinocytes expressing HPV-16 E7 and identified more than 200 genes that were differentially expressed between E7 and vector control cells. Through bioinformatics analysis, pathways altered in E7-expressing cells were identified. Significantly, the WDHD1 gene, one of the genes that is upregulated in E7-expressing cells, was found to play an important role in E7-induced G1 checkpoint abrogation and rereplication. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications.