Systematic Analysis to Identify the MIR99AHG-has-miR-21-5p-EHD1 CeRNA Regulatory Network as Potential Biomarkers in Lung Cancer.

Lung cancer (LC) remains an extremely lethal disease worldwide, and effective prognostic biomarkers are at top priority. With the rapid development of high-throughput sequencing and bioinformatic analysis methods, the quest to characterize cancer transcriptomes continues to move forward. However, the integrated systematic analysis of lncRNA-miRNA-mRNA regulatory network in LC is lacking. In this study, we collected samples of cancer tissues and adjacent normal tissues from patients with lung cancer and conducted transcriptome and small RNA sequencing to identify differentially expressed genes (DEGs), miRNAs (DEMs), and lncRNAs (DELs). The regulatory roles of miRNAs in LC were explained by functional analysis on DEM-targeted genes. The lncRNA-miRNA pairs, miRNA-mRNA pairs, and lncRNA-mRNA pairs were identified and combined to construct the interplay of lncRNA-miRNA-mRNA. We evaluated the prognostic value of selected lncRNA-miRNA-mRNA by Kaplan-Meier analysis. Finally, we analyzed the expression levels of selected DEM, DELs, and DEGs in lung cancer patients and healthy people to verify our findings. A total of 1492 DEGs, 12 DEMs, and 604 DELs were identified in LC patients. Based on the bioinformatic analysis and the regulatory mechanism of ceRNAs, 3 lncRNAs (GATA2-AS1, LINC00632, MIR99AHG), 1 miRNA (hsa-miR-21-5p) and 5 targeted genes (RECK, TIMP3, EHD1, RASGRP1 and ERG) were figured out first. Through further Kaplan-Meier analysis screening the prognostic value, we finally found the hub subnetwork (MIR99AHG-hsa-miR-21-5p-EHD1) by collating lncRNA-miRNA pairs, miRNA-mRNA pairs and lncRNA-mRNA pairs. As the key of ceRNA regulatory network, the expression of miRNA-21-5p in lung cancer patients was significantly higher than that in healthy people (P < 0.01), and its high expression was significantly associated with poor prognosis (P = 0.0025). Our study successfully constructed a MIR99AHG-hsa-miR-21-5p-EHD1 mutually regulatory network, suggesting the potential efficient biomarkers in LC.

Analysis of Methylome, Transcriptome, and Lipid Metabolites to Understand the Molecular Abnormalities in Polycystic Ovary Syndrome.

Purpose: This study aimed to identify differentially methylated genes (DMGs) and differentially expressed genes (DEGs) to investigate new biomarkers for the diagnosis and treatment of polycystic ovary syndrome (PCOS). Methods: To explore the potential biomarkers of PCOS diagnosis and treatment, we performed methyl-binding domain sequencing (MBD-seq) and RNA sequencing (RNA-seq) on ovarian granulosa cells (GCs) from PCOS patients and healthy controls. MBD-seq was also performed on the ovarian tissue of constructed prenatally androgenized (PNA) mice. Differential methylation and expression analysis were implemented to identify DMGs and DEGs, respectively. The identified gene was further verified by real-time quantitative PCR (RT-qPCR) and methylation-specific PCR (MSP) in clinical samples. Furthermore, ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) was carried out on PCOS patients and healthy controls to identify differential lipid metabolites. Results: Compared to the control group, 13,526 DMGs related to the promoter region and 2429 DEGs were found. The function analysis of DMGs and DEGs showed that they were mainly enriched in glycerophospholipid, ovarian steroidogenesis, and other lipid metabolic pathways. Moreover, 5753 genes in DMGs related to the promoter region were screened in the constructed PNA mice. Integrating the DMGs data from PCOS patients and PNA mice, we identified the following 8 genes: CDC42EP4, ERMN, EZR, PIK3R1, ARHGEF18, NECTIN2, TSC2, and TACSTD2. RT-qPCR and MSP verification results showed that the methylation and expression of TACSTD2 were consistent with sequencing data. Additionally, 15 differential lipid metabolites were shown in the serum of PCOS patients. The differential lipids were involved in glycerophospholipid and glycerolipid metabolism. Conclusion: Using integration of methylome and lipid metabolites profiling we identified 8 potential epigenetic markers and 15 potential lipid metabolite markers for PCOS. Our results suggest that aberrant DNA methylation and lipid metabolite disorders may provide novel insights into the diagnosis and etiology of PCOS.

MicroRNA biomarkers and their use in evaluating the prognosis of lung cancer.

OBJECTIVE: We aim to use the microRNA (miRNA, micro-ribonucleic acid) data of lung cancer tissues to establish a miRNA biomarker database for lung cancer that can be used for marker screening and analysis of lung cancer prognosis. METHODS: We obtained lung cancer-related data from The Cancer Genome Atlas (TCGA) and analyzed the miRNA expression profiles of lung cancer tissues and normal tissues using bioinformatics techniques to develop a new composite miRNA-based model for the prognostic assessment of lung cancer. The predictive power of this model was verified and evaluated based on grouping of data. We also performed RT-qPCR using lung cancer tissues from patients diagnosed with lung cancer. RESULTS: There was a significant difference between the miRNA expression profiles of lung cancer tissues and normal tissues adjacent to the cancerous lesions. The prognostic survival of patients with lung cancer was closely related to onset age and staging (p = 0.012) but was not related to gender (p = 0.39) and race (p = 0.51). Using three methods of survival model construction, we identified three miRNA composites, namely hsa-mir-21, hsa-mir-141, and has-mir-490, as markers for the prognosis of lung cancer. As confirmed by RT-qPCR, the expressions of hsa-miR-21-5p and hsa-miR-141-5p were upregulated, whereas hsa-miR-490-3p expression was downregulated in lung cancer lesion tissues. CONCLUSION: The three miRNA composites identified, namely hsa-mir-21, hsa-mir-141, and hsa-mir-490, have the potential to serve as novel prognostic biomarkers and therapeutic targets for lung cancer.

miR-29c inhibits metastasis of gastric cancer cells by targeting VEGFA.

Background: Gastric cancer (GC) is characterized by tissue invasion and metastasis, which lead to an aggressive and highly lethal disease. However, the underlying molecular mechanism remains largely unclear. Although multiple miRNAs are known to regulate crucial cellular events during cancer metastasis, their individual roles are still not fully described. Methods: miR-29c overexpressed cell lines were constructed. The wound healing, migration and invasion assays were performed to investigate the effect of miR-29c on metastasis of GC. HUVECs proliferation and tube formation assays were used to test the ability of angiogenesis of miR-29c. The target gene VEGFA was predicted by bioinformatic algorithms and validated by luciferase activity assay. Peritoneal spreading and pulmonary metastasis mice models were applied in vivo. Results: In the current study, we report the results that introduction of exogenous miR-29c inhibits GC cell migration, invasion and angiogenesis. Epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) properties are participated in the miR-29c mediated cell metastasis. Furthermore, by performing tumor metastasis PCR array and luciferase reporter assay, we find that the expression of VEGFA is regulated by miR-29c through direct targeting of its 3'-UTR. In addition, we show that the VEGFA/VEGFR2/ERK pathway is involved in this process. Conclusion: These data taken together reveal the crucial functions of miR-29c-VEGFA/VEGFR2/ERK signaling axis in the metastasis progression of GC via regulating EMT and CSCs properties, which make them potential targets for clinical intervention in GC.

2cChIP-seq and 2cMeDIP-seq: The Carrier-Assisted Methods for Epigenomic Profiling of Small Cell Numbers or Single Cells.

Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) can profile genome-wide epigenetic marks associated with regulatory genomic elements. However, conventional ChIP-seq is challenging when examining limited numbers of cells. Here, we developed a new technique by supplementing carrier materials of both chemically modified mimics with epigenetic marks and dUTP-containing DNA fragments during conventional ChIP procedures (hereafter referred to as 2cChIP-seq), thus dramatically improving immunoprecipitation efficiency and reducing DNA loss of low-input ChIP-seq samples. Using this strategy, we generated high-quality epigenomic profiles of histone modifications or DNA methylation in 10-1000 cells. By introducing Tn5 transposase-assisted fragmentation, 2cChIP-seq reliably captured genomic regions with histone modification at the single-cell level in about 100 cells. Moreover, we characterized the methylome of 100 differentiated female germline stem cells (FGSCs) and observed a particular DNA methylation signature potentially involved in the differentiation of mouse germline stem cells. Hence, we provided a reliable and robust epigenomic profiling approach for small cell numbers and single cells.

Increased homocysteine regulated by androgen activates autophagy by suppressing the mammalian target of rapamycin pathway in the granulosa cells of polycystic ovary syndrome mice.

The purpose of this study was to explore the potential molecular mechanisms of excess homocysteine in relation to autophagic activity in the ovarian tissue of polycystic ovarian syndrome (PCOS) with hyperandrogenism.A PCOS model was constructed using ICR mice. ELISA was used to detect the Hcy levels in the serum and ovarian tissues of PCOS model. The expression level of key enzymes (Methionine synthase and Betaine-homocysteine methyltransferase, MTR and BHMT) in homocysteine metabolism and autophagy-related proteins were detected in ovarian tissues and mouse granulosa cells (mGCs) that were treated with homocysteine, androgen, autophagy inhibitors or BHMT-expressing plasmid by western blot and immunohistochemistry. Electron microscope experiments were used to evaluate autophagosomes in Hcy-treated mGCs. The prenatally androgenized (PNA) PCOS mouse model showed hyperhomocysteinemia and hyperandrogenism. Homocysteine levels displayed a significant increase, while its metabolic enzymes levels were significantly decreased in ovarian tissues of PCOS mice and dihydrotestosterone (DHT)-stimulated mGCs. The LC3II and Beclin1 expression levels were increased and the P62 and p-mTOR levels were decreased in vivo in ovarian tissue from the PCOS mice. The in vitro data were similarly with the in vivo by stimulation of mGCs with DHT or homocysteine. These effects could be diminished by the autophagy inhibitor (MHY1485), androgen receptor antagonists (ARN509) or BHMT-expressing plasmid. Androgen increases homocysteine concentration by downregulating the key enzymes in homocysteine metabolism. And then Hcy promotes GCs autophagy via the mTOR signal pathway.

A Sight of the Diagnostic Value of Aberrant Cell-Free DNA Methylation in Lung Cancer.

BACKGROUND: Lung cancer is one of the most commonly diagnosed cancer worldwide. As one of the liquid biopsy analytes, alternations in cell-free DNA (cfDNA) methylation could function as promising biomarkers for lung cancer detection. METHODS: In this study, differential methylation analysis was performed to identify candidate markers, and lasso regression with 10-fold cross-validation (CV) was used to establish the diagnostic marker panel. The performance of the binary classifier was evaluated using the receiver operating characteristic (ROC) curve and the precision-recall (PR) curve. RESULTS: We identified 4072 differentially methylated regions (DMRs) based on cfDNA methylation data, and then a 10-DMR marker panel was established. The panel achieved an area under the ROC curve (AUROC) of 0.922 and an area under the PR curve (AUPR) of 0.899 in a cfDNA cohort containing 29 lung cancer and 74 normal samples, showing outstanding performance. Besides, the cfDNA-derived markers also performed well in primary tissue datasets, which were more robust than the tissue-derived markers. CONCLUSION: Our study suggested that the 10-DMR marker panel attained high accuracy and robustness and may function as a novel and promising target for lung cancer detection.

Pump-free microfluidic magnetic levitation approach for density-based cell characterization.

Magnetic levitation (MagLev) provides a simple but promising method for density-based analysis and detection down to the individual cell level. However, each existing MagLev configuration for the single-cell density measurement, mainly consisting of a capillary (∼50 mm) placed between two magnets, yields a fairly low sample utilization because of no knowledge about the sample cells in the regions other than the limited microscope vision. Moreover, the quantitative analysis may be affected due to the unclearly defined measurement area, which is specifically associated with the uneven magnetization of magnets, cell size, degree of aggregation. In this work, we explore a pump-free microfluidic magnetic levitation approach for density-based cell characterization, enabling sensitive and effective cellular density measurement on small sample volumes. The microfluidic MagLev comprises a pump-free microfluidic chip placed between two ring magnets with like poles facing. With no external pumps, connectors or control facility, much smaller amounts of fluids (∼4 µL) could be driven automatically in the entire microchannel in 16 s. Based on the pump-free mechanism, unique density signatures of cells from different lineages (ARPE-19, HCT116, HeLa, HT1080, Huh7) are characterized by monitoring the levitation profiles. Furthermore, variation in density of A549 lung cancer cells subjected to a drug treatment are observed in our platform, allowing evaluation of the efficacy of the drug treatment at the individual cell level. Thereby, the proposed pump-free microfluidic MagLev platform, a low-cost, fully automatic and portable design for label-free density-based cell characterization, provides a universal detection tool that operates efficiently within small-volume environments.

Objective neuromodulation basis for intrafascicular artificial somatosensation through carbon nanotube yarn electrodes.

BACKGROUND: Intrafascicular electrical stimulation has been extensively adopted to achieve sensory feedback for limb amputees. Axon-like carbon nanotube yarn (CNTy) electrodes with both promising flexibility and spatial selectivity index (SSI) can be fascinating alternatives to generate artificial somatosensation. NEW METHOD: Here we systematically disclose objective neuromodulation basis for artificial somatosensation through intrafascicular CNTy electrodes. CNTy electrodes with different exposed lengths were utilized for electrically stimulating tibial nerves in twelve rats. Somatosensory evoked potentials (SEPs) were recorded synchronously using an epidural thirty-channel electrode array. Spatiotemporal characteristics of SEPs were analyzed as current pulse amplitude (PA), pulse width (PW) and pulse frequency (PF) varied. RESULTS: The current thresholds at 1 Hz exhibit the lowest means when compared with those at 4 and 8 Hz for most CNTy electrodes (20/28). For all the electrodes, amplitudes of SEPs and activated areas of perceptive fields increase with PWs and PAs rising, and decrease remarkably with PFs from 1 to 8 Hz. Latencies of P1 and N1 of SEP peaks gradually reduced with PWs and PAs advancing. Considering high SSIs, relatively stable current thresholds, wider variation ranges of sensory magnitudes and optimal stability of perceptive fields, the L-200 µm electrodes are preferable for neuromodulation with PFs of 1 - 8 Hz, PWs of 100 - 800 µs and PAs of 2 - 64 µA. COMPARISON WITH EXISTING METHODS: New-type CNTy electrodes possess both promising flexibility and SSI when compared with other neural interfaces. We systematically explore objective neuromodulation basis for artificial somatosensation through CNTy electrodes for the first time. CONCLUSIONS: Significantly higher SSIs, lower current and charge thresholds exist for CNTy electrodes in comparison with other peripheral-nerve interfaces. This study can, for the first time, lay a solid neuromodulation foundation for CNTy electrodes to achieve fine sensory feedback.

Aberrant miRNA-mRNA regulatory network in polycystic ovary syndrome is associated with markers of insulin sensitivity and inflammation.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common multifactorial metabolic and endocrine disorder in women of reproductive age. Increasingly, evidence indicates that the microRNA (miRNA)-mRNA axis contributes to the development of PCOS. METHODS: To investigate the molecular mechanisms through which miRNA-mRNA expression affects hyperandrogenism, ovarian tissue samples from prenatally androgenized (PNA) mice were subjected to miRNA-seq and RNA-seq analysis. Analyses were performed to identify differentially expressed microRNAs (DEmiRs) and differentially expressed genes (DEGs). In combination with our previous data obtained from clinical samples, we have performed an integrated miRNA-mRNA analysis of PNA mice and granulosa cells (GCs) from patients with PCOS. The changes in expression were validated by RT-qPCR in more mouse models and clinical samples. RESULTS: In total, 3,432 genes and 16 miRNAs were differentially expressed in PNA mice compared with the control mice. We investigated the regulation pattern of miRNAs-mRNAs and observed a total of 12 miRNA-mRNA pairs involved in negative regulation. Functional analysis concentrated on insulin resistance, the T cell receptor signaling pathway, and other inflammation-related pathways. Verification of these results by RT-qPCR showed that the expression of miR-106-5p and miR-155-5p in clinical GCs was consistent with that in PNA mice. After predicting the target genes of miR-106-5p and miR-155-5p and performing negative regulation analysis, six target genes were obtained in GCs. The integration analysis showed that the network of miR-106-5p/miR-155-5p targets was mostly concentrated in pathway related to insulin resistance and inflammation. In addition, the upregulation of the inflammatory genes Il18/IL18 and Socs3/SOCS3 was validated in the PNA mice and GCs from patients compared with the appropriate control sample. The in vitro experiments showed that the regulatory relationship observed may be related to the direct stimulation of DHT. CONCLUSIONS: Our results showed that the miRNA-mRNA regulatory network in PCOS was associated with markers of insulin sensitivity and inflammation. Our study provides a new genetic basis and novel insight regarding the pathogenesis of PCOS.

Suppression of CPSF6 Enhances Apoptosis Through Alternative Polyadenylation-Mediated Shortening of the VHL 3'UTR in Gastric Cancer Cells.

Alternative polyadenylation (APA) is an important RNA post-transcriptional process, which can generate diverse mRNA isoforms. Increasing evidence shows that APA is involved in cell self-renewal, development, immunity, and cancer. CPSF6 is one of the core proteins of CFIm complex and can modulate the APA process. Although it has been reported to play oncogenic roles in cancer, the underlying mechanisms remain unclear. The aim of the present study was to characterize CPSF6 in human gastric cancer (GC). We observed that CPSF6 was upregulated in GC. Knockdown of CPSF6 inhibited proliferation and enhanced apoptosis of GC cells both in vitro and in vivo. Global APA site profiling analysis revealed that knockdown of CPSF6 induced widespread 3'UTR shortening of genes in GC cells, including VHL. We also found CPSF6 negatively regulated the expression of VHL through APA and VHL short-3'UTR isoform enhanced apoptosis and inhibited cell growth in GC cells. Our data suggested that CPSF6-induced cell proliferation and inhibition of apoptosis were mediated by the preferential usage of poly(A) in VHL. Our data provide insights into the function of CPSF6 and may imply potential therapeutic targets against GC.

Integrated Transcriptomic and Epigenetic Study of PCOS: Impact of Map3k1 and Map1lc3a Promoter Methylation on Autophagy.

Polycystic ovary syndrome (PCOS) is a prevalent heterogeneous endocrine and metabolic disorder in women of reproductive age. Epigenetic mechanisms contribute to the development of PCOS. Nevertheless, the role of DNA methylation in the development of PCOS remains unclear. To investigate the molecular mechanisms underlying the hyperandrogenic phenotype of PCOS, dihydrotestosterone (DHT)-induced prenatally androgenized (PNA) mice were used to mimic this phenotype. Ovarian samples from PNA and control mice were subjected to methyl-CpG-binding domain (MBD)-seq and RNA-seq, and validation was conducted using methylation-specific polymerase chain reaction (MSP) and quantitative real-time PCR (RT-qPCR). Immunohistochemical analysis (using anti-LC3II antibody) and transmission electron microscopy were conducted using ovarian tissue sections (which included granulosa cells) from PNA and control mice. There were 857 genes with differentially methylated promoter regions and 3,317 differentially expressed genes (DEGs) in the PNA mice compared to the control mice. Downregulation of Dnmt1 (which encodes DNA methyltransferase 1), accompanied by global hypomethylation, was observed in the PNA mice compared to the control mice. The promoter regions of Map3k1 (which encodes MEKK1) and Map1lc3a (which encodes LC3II) were hypomethylated, accompanied by upregulation of Map3k1 and Map1lc3a mRNA expression. The autophagy profiling results showed that LC3II protein expression and autophagosomes were significantly increased in the granulosa cells of PNA mice. Additionally, the mRNA expression of genes related to the mitogen-activated protein kinase (MAPK)/p53 pathway (Mapk14, Mapkapk3, and Trp53) and the autophagy-related gene Becn1 were significantly increased. DHT could change the DNA methylation and transcription level of Map3k1 and lead to an activation of autophagy in granulosa cells. These observations indicated that the change in autophagy may be driven by MAPK/p53 pathway activation, which may have been caused by DHT-induced transcriptional, and the methylation level changed of the key upstream gene Map3k1. Our study provides a novel genetic basis and new insights regarding the pathogenesis of PCOS.

CPSF6 links alternative polyadenylation to metabolism adaption in hepatocellular carcinoma progression.

BACKGROUND: Alternative polyadenylation (APA) is an important mechanism of gene expression regulation through generation of RNA isoforms with distinct 3' termini. Increasing evidence has revealed that APA is actively involved in development and disease, including hepatocellular carcinoma (HCC). However, how APA functions in tumor formation and progression remains elusive. In this study, we investigated the role of cleavage factor I (CFIm) subunit CPSF6 in human hepatocellular carcinoma (HCC). METHODS: Expression levels of CPSF6 in clinical tissues and cell lines were determined by qRT-PCR and western blot. Functional assays, including the cell number, MTT, colony formation and transwell, were used to determine the oncogenic role of CPSF6 in HCC. Animal experiments were used to determine the role of CPSF6 in HCC tumorigenicity in vivo. Deep sequencing-based 3 T-seq was used to profile the transcriptome-wide APA sites in both HCC cells and CPSF6 knockdown HCC cells. The function of CPSF6-affected target NQO1 with distinct 3'UTRs was characterized by metabolism assays. RESULTS: We observed CPSF6 was upregulated in HCC and the high expression of CPSF6 was associated with poor prognosis in patients. Overexpression of CPSF6 promoted proliferation, migration and invasion of HCC cells in vitro and in vivo. Transcriptome-wide APA profiling analysis indicated that high expression of CPSF6 promoted the favorable usage of the proximal poly(A) site in the 3'UTR of NQO1. We demonstrated CPSF6-induced tumorigenic activities were mediated by the NQO1 isoform with short 3'UTR. Furthermore, we found that CPSF6 induced metabolic alterations in liver cells through NQO1. CONCLUSION: CPSF6 plays a critical role in HCC progression by upregulating NQO1 expression through APA. These findings provide evidence to demonstrate that APA of NQO1 contributes to HCC progression and may have implications for developing new therapeutic strategy against this disease.

The binding epitope of sintilimab on PD-1 revealed by AbMap.

PD-1 plays an important role as an immune checkpoint. Sintilimab is a newly approved PD-1 antibody for cancer immunotherapy with an unknown binding epitope on PD-1. In this study, to elucidate the molecular mechanism by which sintilimab blocks PD-1 activation, we applied Antibody binding epitope Mapping (AbMap) to identify the binding epitope of sintilimab. An epitope was successfully identified, i.e. SLAPKA, aa 127-132. By constructing a series of point mutations, the dominant residues S127, L128, A129, P130, and A132 of PD-1 were further validated by western blot analysis, biolayer interferometry, and flow cytometry. Structural analysis showed that the epitope is partially within the binding interface of PD-1 and PD-L1, and this epitope also partially overlaps with that of nivolumab and pembrolizumab. These results demonstrate that sintilimab can attenuate PD-1 activation by directly competing with the interaction between PD-1 and PD-L1 through binding with the key residues of the FG loop on PD-1. This study also demonstrates the high efficiency and accuracy of AbMap for determining the binding epitope of therapeutic antibodies.

Methylome and transcriptome profiling revealed epigenetic silencing of LPCAT1 and PCYT1A associated with lipidome alterations in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine diseases of fertile women and a major cause of infertility. The regulatory effects of DNA methylation on gene transcription and downstream lipid metabolism have not been explored in PCOS. In this study, MBD-seq and RNA-seq were performed on ovarian granulosa cells of PCOS patients and controls, and methylation specific PCR and quantitative polymerase chain reaction were used to validate the results. Then lipidomic profiling was conducted on serum of PCOS patients and controls using UPLC-MS. We identified 73 genes with differently methylated promoters and 830 differently expressed genes. The promoter regions of LPCAT1 and PCYT1A were hypermethylated, accompanied by downregulation of their messenger RNA expression, which may be involved in the regulation of PCOS through downstream glycerophospholipid metabolism and phosphatidylcholine synthesis. The lipid profiling results showed significant changes in 21 lipids, which demonstrated the disturbance in glycerophospholipid metabolism and glycerolipid metabolism pathways. Furthermore, the metabolites-genes interaction network was constructed to illustrate the association of aberrant methylome and transcriptome with lipidome alterations in glycerolipid and glycerophospholipid metabolism pathways. Our study suggested that the methylation silencing of LPCAT1 and PCYT1A may promote glycerophospholipids metabolism dysregulation, which provided a novel genetic and lipometabolic basis for the pathogenesis of PCOS.

Antibody Binding Epitope Mapping (AbMap) of Hundred Antibodies in a Single Run.

Antibodies play essential roles in both diagnostics and therapeutics. Epitope mapping is essential to understand how an antibody works and to protect intellectual property. Given the millions of antibodies for which epitope information is lacking, there is a need for high-throughput epitope mapping. To address this, we developed a strategy, Antibody binding epitope Mapping (AbMap), by combining a phage displayed peptide library with next-generation sequencing. Using AbMap, profiles of the peptides bound by 202 antibodies were determined in a single test, and linear epitopes were identified for >50% of the antibodies. Using spike protein (S1 and S2)-enriched antibodies from the convalescent serum of one COVID-19 patient as the input, both linear and potentially conformational epitopes of spike protein specific antibodies were identified. We defined peptide-binding profile of an antibody as the binding capacity (BiC). Conceptually, the BiC could serve as a systematic and functional descriptor of any antibody. Requiring at least one order of magnitude less time and money to map linear epitopes than traditional technologies, AbMap allows for high-throughput epitope mapping and creates many possibilities.

Identification of epigenetic interactions between microRNA and DNA methylation associated with polycystic ovarian syndrome.

Aberration in microRNA expression or DNA methylation is a causal factor for polycystic ovarian syndrome. However, the epigenetic interactions between miRNA and DNA methylation remain unexplored in PCOS. We conducted a novel integrated analysis of RNA-seq, miRNA-seq, and methylated DNA-binding domain sequencing on ovarian granulosa cells to reveal the epigenetic interactions involved in the pathogenesis of PCOS. We identified 830 genes and 30 miRNAs that were expressed differently in PCOS, and seven miRNAs negatively regulated target mRNA expression. 130 miRNAs' promoters were significantly differently methylated, while 13 were associated with miRNA expression. Furthermore, the hypermethylation of miR-429, miR-141-3p, and miR-126-3p' promoter was found related to miRNA expression suppression and therefore their corresponding genes upregulation, including XIAP, BRD3, MAPK14, and SLC7A5. Our findings provide a novel insight in PCOS. The consequential reversal of genes silencing may participate in PCOS pathogenesis and served as potential molecular targets for PCOS.

An Iron Metabolism-Related SLC22A17 for the Prognostic Value of Gastric Cancer.

PURPOSE: Gastric cancer (GC) is a type of malignant cancer with a poor prognosis. The iron's metabolism plays an important role in the process of GC. The aim of this study was to evaluate the effectiveness of SLC22A17, associated with iron metabolism, in predicting the prognosis of GC patients. MATERIALS AND METHODS: We analyzed genes related to iron metabolism of gastric cancer mRNA-seq data from TCGA database. We identified an iron metabolism-related SLC22A17 as an independent prognostic factor using univariate and multivariate Cox regression analysis. RESULTS: Further research showed that SLC22A17 was related with many pathways involved in the process of gastric cancer, and the expression was associated with diverse cancer-infiltrating immune cells. The expression of SLC22A17 was associated with T (Topography). CONCLUSION: We validated that SLC22A17 associated with iron metabolism could serve as a prognostic biomarker for GC patients.

Post-Transcriptional Dysregulation of microRNA and Alternative Polyadenylation in Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide. microRNAs (miRNAs) repress gene expression by binding to complementary sequences in the 3' untranslated region (3'UTR) of target mRNAs. Alternative polyadenylation (APA) are relevant to the variability of the 3'UTR of mRNA. However, the posttranscriptional dysregulation of miRNAs and APA in CRC are poorly understood. METHOD: In this study, we conducted small RNA sequencing to identify differentially expressed miRNAs (DERs) and their target genes. Function analysis on DER-target genes can explain the regulation roles of miRNAs in CRC. The mutual regulation of miRNAs and APA was analyzed by combining miRNA data to 3'UTR alteration using 3' termini of polyadenylated RNAs sequencing (3T-seq) technique, and this was validated using TCGA gene expression data. RESULTS: Our results showed 64 significant differentially expressed miRNAs (DERs) in CRC patients. Their target genes were related to cell adhesion and transcription regulation and were prevailingly involved in the CRC-related pathway. Integrative analysis of the miRNA and APA profile revealed 16 DERs were correlated with 12 polyadenylation factors, and six of them were significantly differently expressed in CRC. We also found four DERs that lost binding sites due to APA and showed a positive correlation between the miRNA and gene expression. CONCLUSION: Our study found that miRNAs regulated APA by modulating key polyadenylation factors, and several miRNAs lost their suppression on mRNA due to APA. Associating this with gene expression may provide some important clues for a deeper study of posttranscriptional cellular regulation and biomarker research in CRC. Our data provided the first evidence that the interaction between miRNAs and APA associated with gene expression could serve as biomarkers for CRC, suggesting that hsa-miR-133a-3p and MLEC, hsa-miR-145-5p and SET, hsa-miR-1-3p and PPIA, and hsa-miR-378d and YY1 might be novel and potential biomarkers in improving the diagnosis of CRC.