Bivalent activity of super-enhancer RNA LINC02454 controls 3D chromatin structure and regulates glioma sensitivity to temozolomide.

Glioma cell sensitivity to temozolomide (TMZ) is critical for effective treatment and correlates with patient survival, although mechanisms underlying this activity are unclear. Here, we reveal a new mechanism used by glioma cells to modulate TMZ sensitivity via regulation of SORBS2 and DDR1 genes by super-enhancer RNA LINC02454. We report that LINC02454 activity increases glioma cell TMZ sensitivity by maintaining long-range chromatin interactions between SORBS2 and the LINC02454 enhancer. By contrast, LINC02454 activity also decreased glioma cell TMZ sensitivity by promoting DDR1 expression. Our study suggests a bivalent function for super-enhancer RNA LINC02454 in regulating glioma cell sensitivity to TMZ.

Three-Dimensional Gene Regulation Network in Glioblastoma Ferroptosis.

Ferroptosis is an iron-dependent form of cell death, which is reported to be associated with glioma progression and drug sensitivity. Targeting ferroptosis is a potential therapeutic approach for glioma. However, the molecular mechanism of glioma cell ferroptosis is not clear. In this study, we profile the change of 3D chromatin structure in glioblastoma ferroptosis by using HiChIP and study the 3D gene regulation network in glioblastoma ferroptosis. A combination of an analysis of HiChIP and RNA-seq data suggests that change of chromatin loops mediated by 3D chromatin structure regulates gene expressions in glioblastoma ferroptosis. Genes that are regulated by 3D chromatin structures include genes that were reported to function in ferroptosis, like HDM2 and TXNRD1. We propose a new regulatory mechanism governing glioblastoma cell ferroptosis by 3D chromatin structure.

Systematic fine-mapping and functional studies of prostate cancer risk variants.

To date, genome-wide association studies (GWAS) have revealed over 200 genetic risk loci associated with prostate cancer; yet, true disease-causing variants remain elusive. Identification of causal variants and their targets from association signals is complicated by high linkage disequilibrium and limited availability of functional genomics data for specific tissue/cell types. Here, we integrated statistical fine-mapping and functional annotation from prostate-specific epigenomic profiles, 3D genome features, and quantitative trait loci data to distinguish causal variants from associations and identify target genes. Our fine-mapping analysis yielded 3,395 likely causal variants, and multiscale functional annotation linked them to 487 target genes. We prioritized rs10486567 as a genome-wide top-ranked SNP and predicted HOTTIP as its target. Deletion of the rs10486567-associated enhancer in prostate cancer cells decreased their capacity for invasive migration. HOTTIP overexpression in enhancer-KO cell lines rescued defective invasive migration. Furthermore, we found that rs10486567 regulates HOTTIP through allele-specific long-range chromatin interaction.

KLF4 inhibits early neural differentiation of ESCs by coordinating specific 3D chromatin structure.

Neural differentiation of embryonic stem cells (ESCs) requires precisely orchestrated gene regulation, a process governed in part by changes in 3D chromatin structure. How these changes regulate gene expression in this context remains unclear. In this study, we observed enrichment of the transcription factor KLF4 at some poised or closed enhancers at TSS-linked regions of genes associated with neural differentiation. Combination analysis of ChIP, HiChIP and RNA-seq data indicated that KLF4 loss in ESCs induced changes in 3D chromatin structure, including increased chromatin interaction loops between neural differentiation-associated genes and active enhancers, leading to upregulated expression of neural differentiation-associated genes and therefore early neural differentiation. This study suggests KLF4 inhibits early neural differentiation by regulation of 3D chromatin structure, which is a new mechanism of early neural differentiation.

Paeoniflorin drives the immunomodulatory effects of mesenchymal stem cells by regulating Th1/Th2 cytokines in oral lichen planus.

Lichen planus (LP) is a chronic inflammatory disease. Oral lichen planus (OLP) mainly appears as oral mucosal reticular or ulcerative lesions with an unknown etiology. We aimed to explore the immunomodulatory effect of paeoniflorin (PF) in mesenchymal stem cells (MSCs) and the potential involvement of Th1/Th2 cytokines in OLP. The effects of paeoniflorin on the proliferation and migration of MSCs were detected by Cell Counting Kit-8 (CCK8) and Transwell assays. MSCs were subjected to osteogenic, adipogenic and neurogenic induction followed by Alizarin red, oil red O, real-time PCR and immunofluorescence assays. We found that paeoniflorin promoted the proliferation, migration and multilineage differentiation of MSCs from OLP lesions (OLP-MSCs) in vitro. Paeoniflorin pretreatment increased the inhibitory effect of OLP-MSCs on peripheral blood mononuclear cells. Furthermore, paeoniflorin-pretreated OLP-MSCs simultaneously decreased Th1 cytokine levels and increased Th2 cytokine levels in T lymphocyte cocultures. Finally, paeoniflorin-pretreated OLP-MSCs also promoted the Th1/Th2 balance both in vitro and in the serum of mice that received skin allografts. In conclusion, paeoniflorin enhanced MSC immunomodulation and changed the inflammatory microenvironment via T lymphocytes, suggesting that the improvement of OLP-MSCs is a promising therapeutic approach for OLP.

Automated detection of myopic maculopathy from color fundus photographs using deep convolutional neural networks.

BACKGROUND: Myopic maculopathy (MM) has become a major cause of visual impairment and blindness worldwide, especially in East Asian countries. Deep learning approaches such as deep convolutional neural networks (DCNN) have been successfully applied to identify some common retinal diseases and show great potential for the intelligent analysis of MM. This study aimed to build a reliable approach for automated detection of MM from retinal fundus images using DCNN models. METHODS: A dual-stream DCNN (DCNN-DS) model that perceives features from both original images and corresponding processed images by color histogram distribution optimization method was designed for classification of no MM, tessellated fundus (TF), and pathologic myopia (PM). A total of 36,515 gradable images from four hospitals were used for DCNN model development, and 14,986 gradable images from the other two hospitals for external testing. We also compared the performance of the DCNN-DS model and four ophthalmologists on 3000 randomly sampled fundus images. RESULTS: The DCNN-DS model achieved sensitivities of 93.3% and 91.0%, specificities of 99.6% and 98.7%, areas under the receiver operating characteristic curves (AUC) of 0.998 and 0.994 for detecting PM, whereas sensitivities of 98.8% and 92.8%, specificities of 95.6% and 94.1%, AUCs of 0.986 and 0.970 for detecting TF in two external testing datasets. In the sampled testing dataset, the sensitivities of four ophthalmologists ranged from 88.3% to 95.8% and 81.1% to 89.1%, and the specificities ranged from 95.9% to 99.2% and 77.8% to 97.3% for detecting PM and TF, respectively. Meanwhile, the DCNN-DS model achieved sensitivities of 90.8% and 97.9% and specificities of 99.1% and 94.0% for detecting PM and TF, respectively. CONCLUSIONS: The proposed DCNN-DS approach demonstrated reliable performance with high sensitivity, specificity, and AUC to classify different MM levels on fundus photographs sourced from clinics. It can help identify MM automatically among the large myopic groups and show great potential for real-life applications.

Enhancer architecture-dependent multilayered transcriptional regulation orchestrates RA signaling-induced early lineage differentiation of ESCs.

Signaling pathway-driven target gene transcription is critical for fate determination of embryonic stem cells (ESCs), but enhancer-dependent transcriptional regulation in these processes remains poorly understood. Here, we report enhancer architecture-dependent multilayered transcriptional regulation at the Halr1-Hoxa1 locus that orchestrates retinoic acid (RA) signaling-induced early lineage differentiation of ESCs. We show that both homeobox A1 (Hoxa1) and Hoxa adjacent long non-coding RNA 1 (Halr1) are identified as direct downstream targets of RA signaling and regulated by RARA/RXRA via RA response elements (RAREs). Chromosome conformation capture-based screens indicate that RA signaling promotes enhancer interactions essential for Hoxa1 and Halr1 expression and mesendoderm differentiation of ESCs. Furthermore, the results also show that HOXA1 promotes expression of Halr1 through binding to enhancer; conversely, loss of Halr1 enhances interaction between Hoxa1 chromatin and four distal enhancers but weakens interaction with chromatin inside the HoxA cluster, leading to RA signaling-induced Hoxa1 overactivation and enhanced endoderm differentiation. These findings reveal complex transcriptional regulation involving synergistic regulation by enhancers, transcription factors and lncRNA. This work provides new insight into intrinsic molecular mechanisms underlying ESC fate determination during RA signaling-induced early differentiation.

Regulation Network of Colorectal-Cancer-Specific Enhancers in the Progression of Colorectal Cancer.

Enhancers regulate multiple genes via higher-order chromatin structures, and they further affect cancer progression. Epigenetic changes in cancer cells activate several cancer-specific enhancers that are silenced in normal cells. These cancer-specific enhancers are potential therapeutic targets of cancer. However, the functions and regulation networks of colorectal-cancer-specific enhancers are still unknown. In this study, we profile colorectal-cancer-specific enhancers and reveal their regulation network through the analysis of HiChIP data that were derived from a colorectal cancer cell line and Hi-C and RNA-seq data that were derived from tissue samples by in silico analysis and in vitro experiments. Enhancer-promoter loops in colorectal cancer cells containing colorectal-cancer-specific enhancers are involved in more than 50% of the topological associated domains (TADs) changed in colorectal cancer cells compared to normal colon cells. In addition, colorectal-cancer-specific enhancers interact with 152 genes that are significantly and highly expressed in colorectal cancer cells. These colorectal-cancer-specific enhancer target genes include ITGB4, RECQL4, MSLN, and GDF15. We propose that the regulation network of colorectal-cancer-specific enhancers plays an important role in the progression of colorectal cancer.

Bergamottin Induces DNA Damage and Inhibits Malignant Progression in Melanoma by Modulating miR-145/Cyclin D1 Axis.

BACKGROUND: Melanoma is a prevalent skin cancer with the high rate of metastasis and mortality, affecting the increasing number of people worldwide. Bergamottin (BGM) is a natural furanocoumarin derived from grapefruits and presents the potential anti-cancer activity in several tumor models. However, the role of BGM in the development of melanoma remains unclear. Here, we aimed to explore the effect of BGM on the DNA damage and progression of melanoma. METHODS: The effect of BGM on the melanoma progression was analyzed by CCK-8 assays, colony formation assays, transwell assays, Annexin V-FITC Apoptosis Detection Kit, cell-cycle analysis, in vivo tumorigenicity analysis. The mechanism investigation was performed using luciferase reporter gene assays, qPCR assays, and Western blot analysis. RESULTS: We identified that BGM repressed cell proliferation, migration, and invasion of melanoma cells. BGM induced cell cycle arrest at the G0/G1 phase and enhanced apoptosis of melanoma cells. The DNA damage in the melanoma cells was stimulated by the BGM treatment. Meanwhile, BGM was able to up-regulate the expression of miR-145 and miR-145 targeted Cyclin D1 in the melanoma cells. Furthermore, BGM inhibited the progression of melanoma by targeting miR-145/Cyclin D1 axis in vitro. BGM attenuated the tumor growth of melanoma in vivo. CONCLUSION: Thus, we conclude that BGM induces DNA damage and inhibits tumor progression in melanoma by modulating the miR-145/Cyclin D1 axis. Our finding provides new insights into the mechanism by which BGM modulates the development of melanoma. BGM may be applied as a potential anti-tumor candidate for the clinical treatment of melanoma.

Two Enhancers Regulate HoxB Genes Expression During Retinoic Acid-Induced Early Embryonic Stem Cells Differentiation Through Long-Range Chromatin Interactions.

Homeobox B cluster (HoxB) genes play important roles in retinoic acid (RA)-induced early embryonic stem cells (ESCs) differentiation. Knowledge of regulation network of HoxB is important to further unveil the mechanism of ESCs differentiation. In this study, we identified two enhancers that were activated by RA treatment and 4C data showed long-range interactions between HoxB genes and the two enhancers. CRISPR/Cas9-mediated individual or compound deletion of the two enhancers significantly inhibits HoxB gene expression, and transcriptome analysis revealed that RA-induced early ESCs differentiation was blocked in the enhancer KO cells. We propose new mechanism by which two enhancers regulate HoxB gene expression by different regulation modes during RA-induced early ESCs differentiation through long-range chromatin interactions.

CTCF-binding element regulates ESC differentiation via orchestrating long-range chromatin interaction between enhancers and HoxA.

Proper expression of Homeobox A cluster genes (HoxA) is essential for embryonic stem cell (ESC) differentiation and individual development. However, mechanisms controlling precise spatiotemporal expression of HoxA during early ESC differentiation remain poorly understood. Herein, we identified a functional CTCF-binding element (CBE+47) closest to the 3'-end of HoxA within the same topologically associated domain (TAD) in ESC. CRISPR-Cas9-mediated deletion of CBE+47 significantly upregulated HoxA expression and enhanced early ESC differentiation induced by retinoic acid (RA) relative to wild-type cells. Mechanistic analysis by chromosome conformation capture assay (Capture-C) revealed that CBE+47 deletion decreased interactions between adjacent enhancers, enabling formation of a relatively loose enhancer-enhancer interaction complex (EEIC), which overall increased interactions between that EEIC and central regions of HoxA chromatin. These findings indicate that CBE+47 organizes chromatin interactions between its adjacent enhancers and HoxA. Furthermore, deletion of those adjacent enhancers synergistically inhibited HoxA activation, suggesting that these enhancers serve as an EEIC required for RA-induced HoxA activation. Collectively, these results provide new insight into RA-induced HoxA expression during early ESC differentiation, also highlight precise regulatory roles of the CTCF-binding element in orchestrating high-order chromatin structure.

Branched-chain amino acid aminotransferase-1 regulates self-renewal and pluripotency of mouse embryonic stem cells through Ras signaling.

The developmental plasticity of embryonic stem cells (ESCs) is mainly controlled by well-characterized transcription factors, but additional factors, especially those related to metabolism that modulate this intrinsic program remain elusive. Here, using whole transcriptome analysis, we identified branched-chain amino acid aminotransferase-1(Bcat1) as highly-expressed in mouse ESCs and dramatically down-regulated upon differentiation. Bcat1 deletion impaired pluripotency and self-renewal in mouse ESCs, while Bcat1 overexpression resulted in robust ESC self-renewal and inhibition of differentiation. Whole genome bisulfite sequencing (WGBS) analysis showed that Bcat1 deletion altered whole genome methylation levels and hence gene expression in multiple pathways. Specifically, Bcat1 deletion increased expression of RAS protein activator like 1(Rasal1), leading to inactivation of Ras-Erk/MAPK signaling, while Rasal1 inhibition rescued defects seen in Bcat1 deleted cells. In summary, we demonstrate that Bcat1 is essential for mouse ESC self-renewal and pluripotency and that this effect is mediated by DNA methylation and the Ras signaling pathway.

HOTAIRM1, an enhancer lncRNA, promotes glioma proliferation by regulating long-range chromatin interactions within HOXA cluster genes.

The long noncoding RNA HOTAIRM1 reportedly plays important roles in acute myeloid leukemia, gastric cancer and colorectal cancer. Here, we analyzed potential function of HOTAIRM1 in glioma and asked whether it participates in long-range chromatin interactions. We monitored expression of HOTAIRM1 in glioma tissues and correlated levels with patient survival using the TCGA dataset. HOTAIRM1 was highly expressed in glioma tissue, with high levels associated with shortened patient survival time. We then suppressed HOTAIRM1 activity in the human glioblastoma U251 line using CRISPR-cas9 to knock in a truncating polyA fragment. Reporter analysis of these and control cells confirmed that the HOTAIRM1 locus serves as an active enhancer. We then performed Capture-C analysis to identify target genes of that locus and applied RNA antisense purification to assess chromatin interactions between the HOTAIRM1 locus and HOXA cluster genes. HOTAIRM1 knockdown in glioma cells decreased proliferation and reduced expression of HOXA cluster genes. HOTAIRM1 regulates long-range interactions between the HOTAIRM1 locus and HOXA genes. Our work suggests a new mechanism by which HOTAIRM1 regulates glioma progression by regulating high-order chromatin structure and could suggest novel therapeutic targets to treat an intractable cancer.

A HOTAIR regulatory element modulates glioma cell sensitivity to temozolomide through long-range regulation of multiple target genes.

Temozolomide (TMZ) is a frequently used chemotherapy for glioma; however, chemoresistance is a major problem limiting its effectiveness. Thus, knowledge of mechanisms underlying this outcome could improve patient prognosis. Here, we report that deletion of a regulatory element in the HOTAIR locus increases glioma cell sensitivity to TMZ and alters transcription of multiple genes. Analysis of a combination of RNA-seq, Capture Hi-C, and patient survival data suggests that CALCOCO1 and ZC3H10 are target genes repressed by the HOTAIR regulatory element and that both function in regulating glioma cell sensitivity to TMZ. Rescue experiments and 3C data confirmed this hypothesis. We propose a new regulatory mechanism governing glioma cell TMZ sensitivity.

The prostate cancer risk variant rs55958994 regulates multiple gene expression through extreme long-range chromatin interaction to control tumor progression.

Genome-wide association studies identified single-nucleotide polymorphism (SNP) rs55958994 as a significant variant associated with increased susceptibility to prostate cancer. However, the mechanisms by which this SNP mediates increased risk to cancer are still unknown. In this study, we show that this variant is located in an enhancer active in prostate cancer cells. Deletion of this enhancer from prostate tumor cells resulted in decreased tumor initiation, tumor growth, and invasive migration, as well as a loss of stem-like cells. Using a combination of capture chromosome conformation capture (Capture-C) and RNA sequencing, we identified genes on the same and different chromosomes as targets regulated by the enhancer. Furthermore, we show that expression of individual candidate target genes in an enhancer-deleted cell line rescued different aspects of tumorigenesis. Our data suggest that the rs55958994-associated enhancer affects prostate cancer progression by influencing expression of multiple genes via long-range chromatin interactions.

A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.

Retinoic acid (RA) induces rapid differentiation of embryonic stem cells (ESCs), partly by activating expression of the transcription factor Hoxa1, which regulates downstream target genes that promote ESCs differentiation. However, mechanisms of RA-induced Hoxa1 expression and ESCs early differentiation remain largely unknown. Here, we identify a distal enhancer interacting with the Hoxa1 locus through a long-range chromatin loop. Enhancer deletion significantly inhibited expression of RA-induced Hoxa1 and endoderm master control genes such as Gata4 and Gata6. Transcriptome analysis revealed that RA-induced early ESCs differentiation was blocked in Hoxa1 enhancer knockout cells, suggesting a requirement for the enhancer. Restoration of Hoxa1 expression partly rescued expression levels of ∼40% of genes whose expression changed following enhancer deletion, and ∼18% of promoters of those rescued genes were directly bound by Hoxa1. Our data show that a distal enhancer maintains Hoxa1 expression through long-range chromatin loop and that Hoxa1 directly regulates downstream target genes expression and then orchestrates RA-induced early differentiation of ESCs. This discovery reveals mechanisms of a novel enhancer regulating RA-induced Hoxa genes expression and early ESCs differentiation.

Oral mucosal mesenchymal stem cell­derived exosomes: A potential therapeutic target in oral premalignant lesions.

Emerging evidence indicates that mesenchymal stem cells (MSCs) serve an indispensable role in the tumor microenvironment. However, whether MSCs participate in the development of oral carcinogenesis remains unclear. The present study isolated MSCs from clinical tissues and investigated the differences of MSCs derived from normal oral mucosa (N­MSC), oral leukoplakia with dysplasia (LK­MSC) and oral carcinoma (Ca­MSC). The results revealed that the LK­MSCs exhibited reduced proliferation and migration, compared with the N­MSCs and Ca­MSCs. Furthermore, it was demonstrated that the exosomes secreted by LK­MSCs have significant roles in promoting proliferation, migration and invasion in vitro, which was similar to the Ca­MSC­derived exosomes. The promoting effect was also demonstrated in a 3D coculture model. When the secretion of exosomes was blocked, the promoting effect of LK­MSCs was reversed. Based on a microarray analysis of MSC­derived exosomes, microRNA­8485 (miR­8485) was identified to be ectopically expressed. The exosomal miR­8485 was capable of promoting the proliferation, migration and invasion of tumor cells. Therefore, the present study highlights the significance of MSC­derived exosomes and exosomal miR­8485 in premalignant lesions and carcinogenesis. Intervention with the secretion of MSC­derived­exosomes may be an innovative strategy to retard the carcinogenesis.

Total glucosides of paeony improves the immunomodulatory capacity of MSCs partially via the miR-124/STAT3 pathway in oral lichen planus.

Mesenchymal stem cells (MSCs) have been used clinically and experimentally to relieve severe immune-related diseases due to their immunomodulatory properties, but these are impaired by inflammation. Oral lichen planus (OLP) is a T cell-mediated chronic inflammatory mucosal disease. In the present study, we found MSCs from OLP with higher expression of interleukin (IL)-6, tumour necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-ß) and IL-10 compared with control. Total glucosides of paeony (TGP) significantly improves the immunomodulatory function of MSCs by inhibiting IL-6 and TNF-α expression and increasing TGF-ß and IL-10 expression. Moreover, TGP can downregulate p-STAT3 expression through upregulation of miR-124. The changes of IL-6, TGF-ß and p-STAT3 were further confirmed by overexpression and knockdown of miR-124 in MSCs. Taken together, the immune-regulating function of MSCs can be improved by TGP via the miR-124/STAT3 pathway.