HPV integration generates a cellular super-enhancer which functions as ecDNA to regulate genome-wide transcription.

Human papillomavirus (HPV) integration is a critical step in cervical cancer development; however, the oncogenic mechanism at the genome-wide transcriptional level is still poorly understood. In this study, we employed integrative analysis on multi-omics data of six HPV-positive and three HPV-negative cell lines. Through HPV integration detection, super-enhancer (SE) identification, SE-associated gene expression and extrachromosomal DNA (ecDNA) investigation, we aimed to explore the genome-wide transcriptional influence of HPV integration. We identified seven high-ranking cellular SEs generated by HPV integration in total (the HPV breakpoint-induced cellular SEs, BP-cSEs), leading to intra-chromosomal and inter-chromosomal regulation of chromosomal genes. The pathway analysis revealed that the dysregulated chromosomal genes were correlated to cancer-related pathways. Importantly, we demonstrated that BP-cSEs existed in the HPV-human hybrid ecDNAs, explaining the above transcriptional alterations. Our results suggest that HPV integration generates cellular SEs that function as ecDNA to regulate unconstrained transcription, expanding the tumorigenic mechanism of HPV integration and providing insights for developing new diagnostic and therapeutic strategies.

Inhibitory receptor CD47 binding to plasma TSP1 suppresses NK-cell IFN-γ production via activating the JAK/STAT3 pathway during HIV infection.

BACKGROUND: Natural killer (NK) cells play an important first-line role against tumour and viral infections and are regulated by inhibitory receptor expression. Among these inhibitory receptors, the expression, function, and mechanism of cluster of differentiation 47 (CD47) on NK cells during human immunodeficiency virus (HIV) infection remain unclear. METHODS: Fresh peripheral blood mononuclear cells (PBMCs) were collected from people living with HIV (PLWH) and HIV negative controls (NC) subjects. Soluble ligand expression levels of CD47 were measured using ELISA. HIV viral proteins or Toll-like receptor 7/8 (TLR7/8) agonist was used to investigate the mechanisms underlying the upregulation of CD47 expression. The effect of CD47 on NK cell activation, proliferation, and function were evaluated by flow cytometry. RNA-seq was used to identify downstream pathways for CD47 and its ligand interactions. A small molecule inhibitor was used to restore the inhibition of NK cell function by CD47 signalling. RESULTS: CD47 expression was highly upregulated on the NK cells from PLWH, which could be due to activation of the Toll-like receptor 7/8 (TLR7/8) pathway. Compared with NC subjects, PLWH subjects exhibited elevated levels of CD47 ligands, thrombospondin-1 (TSP1), and counter ligand signal regulatory protein-α (SIRPα). The TSP1-CD47 axis drives the suppression of interferon gamma (IFN-γ) production and the activation of the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway in NK cells. After treatment with a STAT3 inhibitor, the NK cells from PLWH showed significantly improved IFN-γ production. CONCLUSIONS: The current data indicate that the binding of the inhibitory receptor CD47 to plasma TSP1 suppresses NK cell IFN-γ production by activating the JAK/STAT3 pathway during HIV infection. Our results suggest that CD47 and its related signalling pathways could be targets for improving NK cell function in people living with HIV.

DeepEBV: a deep learning model to predict Epstein-Barr virus (EBV) integration sites.

MOTIVATION: Epstein-Barr virus (EBV) is one of the most prevalent DNA oncogenic viruses. The integration of EBV into the host genome has been reported to play an important role in cancer development. The preference of EBV integration showed strong dependence on the local genomic environment, which enables the prediction of EBV integration sites. RESULTS: An attention-based deep learning model, DeepEBV, was developed to predict EBV integration sites by learning local genomic features automatically. First, DeepEBV was trained and tested using the data from the dsVIS database. The results showed that DeepEBV with EBV integration sequences plus Repeat peaks and 2-fold data augmentation performed the best on the training dataset. Furthermore, the performance of the model was validated in an independent dataset. In addition, the motifs of DNA-binding proteins could influence the selection preference of viral insertional mutagenesis. Furthermore, the results showed that DeepEBV can predict EBV integration hotspot genes accurately. In summary, DeepEBV is a robust, accurate and explainable deep learning model, providing novel insights into EBV integration preferences and mechanisms. AVAILABILITYAND IMPLEMENTATION: DeepEBV is available as open-source software and can be downloaded from https://github.com/JiuxingLiang/DeepEBV.git. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The inhibitory receptor Tim-3 fails to suppress IFN-γ production via the NFAT pathway in NK-cell, unlike that in CD4+ T cells.

BACKGROUND: T cell immunoglobulin and mucin domain-containing-3 (Tim-3) is a negative regulator expressed on T cells, and is also expressed on natural killer (NK) cells. The function of Tim-3 chiefly restricts IFNγ-production in T cells, however, the impact of Tim-3 on NK cell function has not been clearly elucidated. RESULTS: In this study, we demonstrated down-regulation of Tim-3 expression on NK cells while Tim-3 is upregulated on CD4+ T cells during HIV infection. Functional assays indicated that Tim-3 mediates suppression of CD107a degranulation in NK cells and CD4+ T cells, while it fails to inhibit the production of IFN-γ by NK cells. Analyses of downstream pathways using an antibody to block Tim-3 function demonstrated that Tim-3 can inhibit ERK and NFκB p65 signaling; however, it failed to suppress the NFAT pathway. Further, we found that the NFAT activity in NK cells was much higher than that in CD4+ T cells, indicating that NFAT pathway is important for promotion of IFN-γ production by NK cells. CONCLUSIONS: Thus, our data show that the expression of Tim-3 on NK cells is insufficient to inhibit IFN-γ production. Collectively, our findings demonstrate a potential mechanism of Tim-3 regulation of NK cells and a target for HIV infection immunotherapy.

Long non-coding RNA tumor suppressor candidate 7 advances chemotherapy sensitivity of endometrial carcinoma through targeted silencing of miR-23b.

Endometrial carcinoma is the most common malignant tumor of the female genital tract worldwide. TUSC7 (tumor suppressor candidate 7) is an antisense long non-coding RNA and is downregulated and acts as a potential tumor suppressor in several malignant tumors. In this study, the low expression of TUSC7 was confirmed in endometrial carcinoma tissues and was associated with high pathological stages of endometrial carcinoma, which revealed that TUSC7 might be involved in tumorigenesis and progression of endometrial carcinoma. Moreover, the expression of TUSC7 in endometrial carcinoma tissues and cell lines resistant to CDDP and Taxol was lower than that in sensitive endometrial carcinoma tissues and cell lines, which indicated that the TUSC7 expression level was positively correlated with the response of endometrial carcinoma patients to chemotherapy with CDDP and Taxol. TUSC7 upregulation inhibited proliferation, blocked cells at G1 phase, and advanced apoptosis and chemotherapy sensitivity to CDDP and Taxol in HEC1A/CR cell line. Furthermore, miR-23b was upregulated in endometrial carcinoma and negatively correlated with the expression of TUSC7. RNA pull-down assay indicated that TUSC7 could specifically silence the expression of miR-23b in HEC1A/CR cell line; miR-23b was a target gene of TUSC7. MiR-23b upregulation mostly reversed the TUSC7-induced regulatory effects on HEC1A/CR cell line. In summary, long non-coding RNA TUSC7 was underexpressed in endometrial carcinoma, especially in endometrial carcinoma chemotherapy-resistant tissues and cell lines and acted as a potential tumor suppressor gene to inhibit cell growth as well as advance the chemotherapy sensitivity through targeted silencing of miR-23b, which might provide a new therapeutic target to endometrial carcinoma.

Clinical characteristics of PRKACA mutations in Chinese patients with adrenal lesions: a single-centre study.

CONTEXT: Recent studies have identified that the somatic PRKACA L206R mutation can cause cortisol-producing adenomas (CPAs). This study investigated the prevalence and characteristics of PRKACA, GNAS and CTNNB1 mutations in adrenal lesions in patients from a single centre in China. DESIGN, PATIENTS AND MEASUREMENTS: We sequenced PRKACA, GNAS and CTNNB1 genes in 108 patients, including 60 patients with CPAs (57 with unilateral and three with bilateral adenomas), 13 with nonfunctional adenomas, 12 with adrenocortical carcinomas (ACCs), 15 with primary bilateral macronodular hyperplasia (PBMAH) and eight with aldosterone and cortisol cosecreting adenomas. Mutations in PRKACA, GNAS and CTNNB1 were examined, and clinical characteristics were compared. RESULTS: Among the unilateral CPAs, we identified somatic mutations in PRKACA (L206R) in 23 cases (40·4%), GNAS (R201C and R201H) in six cases (10·5%), CTNNB1 (S45C, L46P and S45P) in six cases (10·5%) and CTNNB1 plus GNAS in two cases (3·5%). PRKACA and GNAS mutations were mutually exclusive. Among the patients with nonfunctional adenoma, two carried CTNNB1 mutations. Among the patients with ACC, two carried GNAS and CTNNB1 mutations but none carried PRKACA mutations. One patient showed bilateral CPA, and one PBMAH patient carried PRKACA mutations. No mutations in PRKACA, GNAS or CTNNB1 were identified in the eight patients with aldosterone and cortisol cosecreting adenomas. PRKACA-mutant adenomas were associated with young age, overt Cushing's syndrome and high cortisol levels compared with non-PRKACA-mutant or CTNNB1-mutant lesions. CONCLUSIONS: PRKACA mutations are present in CPAs and bilateral adrenal macronodular hyperplasia. PRKACA mutation is associated with more severe autonomous cortisol secretion.

Targeted Silencing of S100A8 Gene by miR-24 to Increase Chemotherapy Sensitivity of Endometrial Carcinoma Cells to Paclitaxel.

BACKGROUND The objective of this study was to determine whether miR-24 can regulate malignant proliferation and chemotherapy sensitivity of EC cells by targeted silencing of the S100 Calcium Binding Protein A8 (S100A8) gene. MATERIAL AND METHODS The expression of miR-24 in EC tissues was detected by quantitative real-time PCR. The proliferation ability and chemotherapy sensitivity were analyzed by MTT assay. Bioinformatics software was used to predict some potential target genes of miR-24. Luciferase activity assay was used to verify the relationship between target genes and miR-24. S100A8 protein expression was detected by Western blot analysis. RESULTS The low expression of miR-24 in EC tissues compared with normal control tissues suggests miR-24 might play a role in tumorigenesis of EC. EC HEC-1A cells were transfected with miR-24 agonist (agomiR-24) to up-regulate the expression of miR-24. Up-regulation of miR-24 inhibited the cell proliferation and advanced the chemotherapy sensitivity to paclitaxel in HEC-1A cells significantly. We used several types of bioinformatic software to predict that miR-24 could specifically combine with the 3' untranslated region (3'UTR) of the S100A8 gene, and this prediction was verified by Western blot and luciferase activities assay. The regulation effects of miR-24 enhancement on cell proliferation and chemotherapy sensitivity were largely reversed by S100A8 up-regulation. CONCLUSIONS miR-24 acts as a tumor-suppressing gene to inhibit malignant proliferation and advance chemotherapy sensitivity to paclitaxel in EC by targeted silencing of the S100A8 gene.

Cisplatin-induced epigenetic activation of miR-34a sensitizes bladder cancer cells to chemotherapy.

BACKGROUND: Accumulating evidence suggests a tumor suppressive role for miR-34a in human carcinogenesis. However, its precise biological role remains largely elusive. This study aimed to reveal the association of the miR-34a expression and its modulation of sensitivity to cisplatin in muscle-invasive bladder cancer (MIBC). METHODS: miR-34a expression in MIBC cell lines and patient tissues was investigated using qPCR. The methylation analysis of miR-34a promoter region was performed by MassARRAY. Synthetic short single or double stranded RNA oligonucleotides and lentiviral vector were used to regulate miR-34a expression in MIBC cells to investigate its function in vitro and in vivo. RESULTS: miR-34a expression was frequently decreased in MIBC tissues and cell lines through promoter hypermethylation while it was epigenetically increased in MIBC cells following cisplatin treatment. Increased miR-34a expression significantly sensitized MIBC cells to cisplatin and inhibited the tumorigenicity and proliferation of cancer cells in vitro and in vivo. Furthermore, we identified CD44 as being targeted by miR-34a in MIBC cells following cisplatin treatment, and increased CD44 expression could efficiently reverse the effect of miR-34a on MIBC cell proliferation, colongenic potential and chemosensitivity. CONCLUSIONS: Cisplatin-based chemotherapy induced demethylation of miR-34a promoter and increased miR-34a expression, which in turn sensitized MIBC cells to cisplatin and decreased the tumorigenicity and proliferation of cancer cells that by reducing the production of CD44.

Epigenetic inactivation of KLF4 is associated with urothelial cancer progression and early recurrence.

PURPOSE: KLF4 is a transcription factor with divergent functions in different malignancies. We analyzed KLF4 expression and DNA methylation, and their clinical relevance and biological function in urothelial cancer. MATERIALS AND METHODS: Immunohistochemistry and Sequenom™ MassARRAY® were done to detect the expression and promoter methylation of KLF4 in urothelial cancer tissues. The association of the recurrence-free survival rate and decreased KLF4 or KLF4 methylation status was analyzed by the Kaplan-Meier method, Cox regression analysis and ROC assay. Lentivirus based KLF4 over expression and dsRNA mediated knockdown were used to detect KLF4 functions in urothelial cancer in vitro and in vivo. RESULTS: KLF4 was down-regulated in urothelial cancer due to promoter hypermethylation. Each correlated with recurrence-free survival in patients with nonmuscle invasive bladder cancer after transurethral resection of bladder cancer, which potentiates them as valuable predictive biomarkers for early recurrence. Moreover, in and ex vivo experiments showed that KLF4 suppressed urothelial cancer cell growth, migration and invasion inhibited the epithelial-to-mesenchymal transition. CONCLUSIONS: KLF4 may function as a tumor suppressor gene in urothelial cancer since down-regulation of KLF4 by promoter hypermethylation would promote cancer progression. In addition, decreased expression of KLF4 or its promoter hypermethylation may have predictive value for early recurrence in patients with nonmuscle invasive bladder cancer.