The Potential Significance of the EMILIN3 Gene in Augmenting the Aggressiveness of Low-Grade Gliomas is Noteworthy.

Purpose: Low-grade gliomas (LGG) are common brain tumors with high mortality rates. Cancer cell invasion is a significant factor in tumor metastasis. Novel biomarkers are urgently needed to predict LGG prognosis effectively. Methods: The data for LGG were obtained from the Bioinformatics database. A consensus clustering analysis was performed to identify molecular subtypes linked with invasion in LGG. Differential expression analysis was performed to identify differentially expressed genes (DEGs) between the identified clusters. Enrichment analyses were then conducted to explore the function for DEGs. Prognostic signatures were placed, and their predictive power was assessed. Furthermore, the invasion-related prognostic signature was validated using the CGGA dataset. Subsequently, clinical specimens were procured in order to validate the expression levels of the distinct genes examined in this research, and to further explore the impact of these genes on the glioma cell line LN229 and HS-683. Results: Two invasion-related molecular subtypes of LGG were identified, and we sifted 163 DEGs between them. The enrichment analyses indicated that DEGs are mainly related to pattern specification process. Subsequently, 10 signature genes (IGF2BP2, SRY, CHI3L1, IGF2BP3, MEOX2, ABCC3, HOXC4, OTP, METTL7B, and EMILIN3) were sifted out to construct a risk model. Besides, the survival (OS) in the high-risk group was lower. The performance of the risk model was verified. Furthermore, a highly reliable nomogram was generated. Cellular experiments revealed the ability to promote cell viability, value-addedness, migratory ability, invasive ability, and colony-forming ability of the glioma cell line LN229 and HS-683. The qRT-PCR analysis of clinical glioma samples showed that these 10 genes were expressed at higher levels in high-grade gliomas than in low-grade gliomas, suggesting that these genes are associated with poor prognosis of gliomas. Conclusion: Our study sifted out ten invasion-related biomarkers of LGG, providing a reference for treatments and prognostic prediction in LGG.

HPV oncogenes expressed from only one of multiple integrated HPV DNA copies drive clonal cell expansion in cervical cancer.

The integration of HPV DNA into human chromosomes plays a pivotal role in the onset of papillomavirus-related cancers. HPV DNA integration often occurs by linearizing the viral DNA in the E1/E2 region, resulting in the loss of a critical viral early polyadenylation signal (PAS), which is essential for the polyadenylation of the E6E7 bicistronic transcripts and for the expression of the viral E6 and E7 oncogenes. Here, we provide compelling evidence that, despite the presence of numerous integrated viral DNA copies, virus-host fusion transcripts originate from only a single integrated HPV DNA in HPV16 and HPV18 cervical cancers and cervical cancer-derived cell lines. The host genomic elements neighboring the integrated HPV DNA are critical for the efficient expression of the viral oncogenes that leads to clonal cell expansion. The fusion RNAs that are produced use a host RNA polyadenylation signal downstream of the integration site, and almost all involve splicing to host sequences. In cell culture, siRNAs specifically targeting the host portion of the virus-host fusion transcripts effectively silenced viral E6 and E7 expression. This, in turn, inhibited cell growth and promoted cell senescence in HPV16+ CaSki and HPV18+ HeLa cells. Showing that HPV E6 and E7 expression from a single integration site is instrumental in clonal cell expansion sheds new light on the mechanisms of HPV-induced carcinogenesis and could be used for the development of precision medicine tailored to combat HPV-related malignancies. IMPORTANCE: Persistent oncogenic HPV infections lead to viral DNA integration into the human genome and the development of cervical, anogenital, and oropharyngeal cancers. The expression of the viral E6 and E7 oncogenes plays a key role in cell transformation and tumorigenesis. However, how E6 and E7 could be expressed from the integrated viral DNA which often lacks a viral polyadenylation signal in the cancer cells remains unknown. By analyzing the integrated HPV DNA sites and expressed HPV RNAs in cervical cancer tissues and cell lines, we show that HPV oncogenes are expressed from only one of multiple chromosomal HPV DNA integrated copies. A host polyadenylation signal downstream of the integrated viral DNA is used for polyadenylation and stabilization of the virus-host chimeric RNAs, making the oncogenic transcripts targetable by siRNAs. This observation provides further understanding of the tumorigenic mechanism of HPV integration and suggests possible therapeutic strategies for the development of precision medicine for HPV cancers.

KSHV promotes oncogenic FOS to inhibit nuclease AEN and transactivate RGS2 for AKT phosphorylation.

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 is a lytic RNA-binding protein. We applied BCBL-1 cells in lytic KSHV infection and performed UV cross-linking immunoprecipitation (CLIP) followed by RNA-seq of the CLIPed RNA fragments (CLIP-seq). We identified ORF57-bound transcripts from 544 host protein-coding genes. By comparing with the RNA-seq profiles from BCBL-1 cells with latent and lytic KSHV infection and from HEK293T cells with and without ORF57 expression, we identified FOS and CITED2 RNAs being two common ORF57-specific RNA targets. FOS dimerizes with JUN as a transcription factor AP-1 involved in cell proliferation, differentiation, and transformation. Knockout of the ORF57 gene from the KSHV genome led BAC16-iSLK cells incapable of FOS expression in KSHV lytic infection. The dysfunctional KSHV genome in FOS expression could be rescued by Lenti-ORF57 virus infection. ORF57 protein does not regulate FOS translation but binds to the 13-nt RNA motif near the FOS RNA 5' end and prolongs FOS mRNA half-life 7.7 times longer than it is in the absence of ORF57. This binding of ORF57 to FOS RNA is competitive to the binding of a host nuclease AEN (also referred to as ISG20L1). KSHV infection inhibits the expression of AEN, but not exosomal RNA helicase MTR4. FOS expression mediated by ORF57 inhibits AEN transcription, but transactivates RGS2, a regulator of G-protein coupled receptors. FOS binds a conserved AP-1 site in the RGS2 promoter and enhances RGS2 expression to phosphorylate AKT. Altogether, we have discovered that KSHV ORF57 specifically binds and stabilizes FOS RNA to increase FOS expression, thereby disturbing host gene expression and inducing pathogenesis during KSHV lytic infection.

miR­149­3p suppresses the proliferation and metastasis of glioma cells by targeting the CBX2/Wnt/ß­catenin pathway.

The present study aimed to investigate the role of miR-149-3p/chromobox 2 (CBX2)/Wnt/ß-catenin pathway in the proliferation and metastasis of glioma cells. The expression and clinical significance of miR-149-3p and CBX2 were analyzed using data from public databases. Cell Counting Kit-8 and colony formation assays were performed to measure cell proliferation. Transwell assays were used to assess cell invasion. The results showed that miR-149-3p was downregulated and CBX2 was upregulated in glioma, and that the downregulated expression of miR-149-3p promoted the proliferation and invasion of glioma cells. In addition, downregulated expression of CBX2 suppressed the proliferation and invasion of glioma cells. Dual-luciferase assay indicated that CBX2 is a target gene of miR149-3p. The possible molecular mechanism of CBX2 was probed by western blotting, which showed that it may further affect the Wnt/ß-catenin pathway. These present findings demonstrated that miR-149-3p may function as a tumor suppressor miRNA by directly regulating CBX2 and serve important roles in the malignancy of glioma.

Anonymous Pattern Molecular Fingerprint and its Applications on Property Identification.

Molecular fingerprints are significant cheminformatics tools to map molecules into vectorial space according to their characteristics in diverse functional groups, atom sequences, and other topological structures. In this paper, we investigate a novel molecular fingerprint Anonymous-FP that possesses abundant perception about the underlying interactions shaped in small, medium, and large-scale atom chains. In detail, the possible atom chains from each molecule are sampled and extended as anonymous atom chains using an anonymous encoding manner. After that, the molecular fingerprint Anonymous-FP is embedded into vectorial space in virtue of the Natural Language Processing technique PV-DBOW. Anonymous-FP is studied on molecular property identification via molecule classification experiments on a series of molecule databases and has shown valuable advantages such as less dependence on prior knowledge, rich information content, full structural significance, and high experimental performance. During the experimental verification, the scale of the atom chain or its anonymous pattern is found significant to the overall representation ability of Anonymous-FP. Generally, the typical scale r = 8 could enhance the molecule classification performance, and specifically, Anonymous-FP gains the classification accuracy to above 93% on all NCI datasets.

Characteristics of glioblastomas and immune microenvironment in a Chinese family with Lynch syndrome and concurrent porokeratosis.

Background: Lynch syndrome (LS)-associated glioblastoma (GBM) is rare in clinical practice, and simultaneous occurrence with cutaneous porokeratosis is even rarer. In this study, we analyzed the clinicopathological and genetic characteristics of LS-associated GBMs and concurrent porokeratosis, as well as evaluated the tumor immune microenvironment (TIME) of LS-associated GBMs. Methods: Immunohistochemical staining was used to confirm the histopathological diagnosis, assess MMR and PD-1/PD-L1 status, and identify immune cell subsets. FISH was used to detect amplification of EGFR and PDGFRA, and deletion of 1p/19q and CDKN2A. Targeted NGS assay analyzed somatic variants, MSI, and TMB status, while whole-exome sequencing and Sanger sequencing were carried out to analyze the germline mutations. Results: In the LS family, three members (I:1, II:1 and II:4) were affected by GBM. GBMs with loss of MSH2 and MSH6 expression displayed giant and multinucleated bizarre cells, along with mutations in ARID1A, TP53, ATM, and NF1 genes. All GBMs had TMB-H but not MSI-H. CD8+ T cells and CD163+ macrophages were abundant in each GBM tissue. The primary and recurrent GBMs of II:1 showed mesenchymal characteristics with high PD-L1 expression. The family members harbored a novel heterozygous germline mutation in MSH2 and FDPS genes, confirming the diagnosis of LS and disseminated superficial actinic porokeratosis. Conclusion: LS-associated GBM exhibits heterogeneity in clinicopathologic and molecular genetic features, as well as a suppressive TIME. The presence of MMR deficiency and TMB-H may serve as predictive factors for the response to immune checkpoint inhibitor therapy in GBMs. The identification of LS-associated GBM can provide significant benefits to both patients and their family members, including accurate diagnosis, genetic counseling, and appropriate screening or surveillance protocols. Our study serves as a reminder to clinicians and pathologists to consider the possibility of concurrent genetic syndromes in individuals or families.

Oncogenic SRSF3 in health and diseases.

Serine/arginine rich splicing factor 3 (SRSF3) is an important multi-functional splicing factor, and has attracted increasing attentions in the past thirty years. The importance of SRSF3 is evidenced by its impressively conserved protein sequences in all animals and alternative exon 4 which represents an autoregulatory mechanism to maintain its proper cellular expression level. New functions of SRSF3 have been continuously discovered recently, especially its oncogenic function. SRSF3 plays essential roles in many cellular processes by regulating almost all aspects of RNA biogenesis and processing of many target genes, and thus, contributes to tumorigenesis when overexpressed or disregulated. This review updates and highlights the gene, mRNA, and protein structure of SRSF3, the regulatory mechanisms of SRSF3 expression, and the characteristics of SRSF3 targets and binding sequences that contribute to SRSF3's diverse molecular and cellular functions in tumorigenesis and human diseases.

Integration and viral oncogene expression of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma and gastric cancer.

Persistent high-risk human papillomavirus (HR-HPV) infections cause cervical cancer and a fraction of head and neck cancer. To investigate whether HR-HPV infection might be also involved in the development of gastric cancer (GC), we developed a platform utilizing a rolling circle amplification (RCA)-based nested L1 polymerase chain reaction with Sanger sequencing to genotype the HPV DNA in cancer tissues of 361 GC and 89 oropharyngeal squamous cell carcinomas (OPSCC). HPV transcriptional activity was determined by E6/E7 mRNA expression and a 3' rapid amplification of cDNA ends was performed to identify HPV integration and expression of virus-host fusion transcripts. Ten of 361 GC, 2 of 89 OPSCC, and 1 of 22 normal adjacent tissues were HPV L1 DNA-positive. Five of the 10 HPV-positive GC were genotyped as HPV16 by sequencing and 1 of 2 GC with RCA/nested HPV16 E6/E7 DNA detection exhibited HPV16 E6/E7 mRNA. Two OPSCC displayed HPV16 L1 DNA and E6/E7 mRNA, of which 1 OPSCC tissue showed virus-host RNA fusion transcripts from an intron region of KIAA0825 gene. Together, our data reveal viral oncogene expression and/or integration in GC and OPSCC and a possible etiology role of HPV infections in gastric carcinogenesis.

Using radiomics based on multicenter magnetic resonance images to predict isocitrate dehydrogenase mutation status of gliomas.

Background: Isocitrate dehydrogenase (IDH) mutation status is an important biomarker for the treatment strategy selection and prognosis evaluation of glioma. The purpose of this study is to predict the IDH mutation status of gliomas based on multicenter magnetic resonance (MR) images using radiomic models, which were composed from the selected radiomics features and logistic regression (LR), support vector machine (SVM), and LR least absolute shrinkage and selection operator (LASSO) classifiers. Methods: We retrospectively reviewed the medical records of 205 patients with gliomas. We enrolled 78 patients from Shandong Provincial Hospital from January 2018 to December 2019 as testing sets and 127 patients from The Cancer Genome Atlas (TCGA) as training sets. Preoperative MR images were stratified according to their IDH status, and the participants formed a consecutive and random series. Four MR modalities, including T1C, T2, T1 fluid-attenuated inversion recovery (FLAIR), and T2 FLAIR, were used for analysis. Five-fold cross-validation was adopted to train the models, and the models' performances were verified through the testing set. Tumor volumes of interest (VOI) were delineated on the 4 MR modalities. A total of 428 radiomics features were extracted. Two feature selection algorithms, Pearson correlation coefficient (PCC) and recursive feature elimination (RFE), were used to select radiomics features. These features were fed into 3 machine learning classifiers, which were LR, SVM, and LR LASSO, to construct prediction models. The accuracy (ACC), sensitivity (SEN), specificity (SPEC), and area under the curve (AUC) were applied to measure the predictive performance of the radiomics models. Results: The LR (SVM and LR LASSO) classifier predicted IDH mutation status with an average testing set ACC of 80.77% (80.64% and 80.41%), a SEN of 73.68% (84.21% and 89.47%), a SPEC of 87.50% (67.50% and 62.50%), and an AUC of 0.8572 (0.8217 and 0.8164). Conclusions: The radiomics models based on MR modalities demonstrated the potential to be used as tools across different data sets for the noninvasive prediction of the IDH mutation status in glioma.

Modeling HPV-Associated Disease and Cancer Using the Cottontail Rabbit Papillomavirus.

Approximately 5% of all human cancers are attributable to human papillomavirus (HPV) infections. HPV-associated diseases and cancers remain a substantial public health and economic burden worldwide despite the availability of prophylactic HPV vaccines. Current diagnosis and treatments for HPV-associated diseases and cancers are predominantly based on cell/tissue morphological examination and/or testing for the presence of high-risk HPV types. There is a lack of robust targets/markers to improve the accuracy of diagnosis and treatments. Several naturally occurring animal papillomavirus models have been established as surrogates to study HPV pathogenesis. Among them, the Cottontail rabbit papillomavirus (CRPV) model has become known as the gold standard. This model has played a pivotal role in the successful development of vaccines now available to prevent HPV infections. Over the past eighty years, the CRPV model has been widely applied to study HPV carcinogenesis. Taking advantage of a large panel of functional mutant CRPV genomes with distinct, reproducible, and predictable phenotypes, we have gained a deeper understanding of viral-host interaction during tumor progression. In recent years, the application of genome-wide RNA-seq analysis to the CRPV model has allowed us to learn and validate changes that parallel those reported in HPV-associated cancers. In addition, we have established a selection of gene-modified rabbit lines to facilitate mechanistic studies and the development of novel therapeutic strategies. In the current review, we summarize some significant findings that have advanced our understanding of HPV pathogenesis and highlight the implication of the development of novel gene-modified rabbits to future mechanistic studies.

Genome-wide regulation of KSHV RNA splicing by viral RNA-binding protein ORF57.

RNA splicing plays an essential role in the expression of eukaryotic genes. We previously showed that KSHV ORF57 is a viral splicing factor promoting viral lytic gene expression. In this report, we compared the splicing profile of viral RNAs in BCBL-1 cells carrying a wild-type (WT) versus the cells containing an ORF57 knock-out (57KO) KSHV genome during viral lytic infection. Our analyses of viral RNA splice junctions from RNA-seq identified 269 RNA splicing events in the WT and 255 in the 57KO genome, including the splicing events spanning large parts of the viral genome and the production of vIRF4 circRNAs. No circRNA was detectable from the PAN region. We found that the 57KO alters the RNA splicing efficiency of targeted viral RNAs. Two most susceptible RNAs to ORF57 splicing regulation are the K15 RNA with eight exons and seven introns and the bicistronic RNA encoding both viral thymidylate synthase (ORF70) and membrane-associated E3-ubiquitin ligase (K3). ORF57 inhibits splicing of both K15 introns 1 and 2. ORF70/K3 RNA bears two introns, of which the first intron is within the ORF70 coding region as an alternative intron and the second intron in the intergenic region between the ORF70 and K3 as a constitutive intron. In the WT cells expressing ORF57, most ORF70/K3 transcripts retain the first intron to maintain an intact ORF70 coding region. In contrast, in the 57KO cells, the first intron is substantially spliced out. Using a minigene comprising of ORF70/K3 locus, we further confirmed ORF57 regulation of ORF70/K3 RNA splicing, independently of other viral factors. By monitoring protein expression, we showed that ORF57-mediated retention of the first intron leads to the expression of full-length ORF70 protein. The absence of ORF57 promotes the first intron splicing and expression of K3 protein. Altogether, we conclude that ORF57 regulates alternative splicing of ORF70/K3 bicistronic RNA to control K3-mediated immune evasion and ORF70 participation of viral DNA replication in viral lytic infection.

Correction: Yu et al. HPV16 and HPV18 Genome Structure, Expression, and Post-Transcriptional Regulation. Int. J. Mol. Sci. 2022, 23, 4943.

In the published review [...].

HPV16 and HPV18 Genome Structure, Expression, and Post-Transcriptional Regulation.

Human papillomaviruses (HPV) are a group of small non-enveloped DNA viruses whose infection causes benign tumors or cancers. HPV16 and HPV18, the two most common high-risk HPVs, are responsible for ~70% of all HPV-related cervical cancers and head and neck cancers. The expression of the HPV genome is highly dependent on cell differentiation and is strictly regulated at the transcriptional and post-transcriptional levels. Both HPV early and late transcripts differentially expressed in the infected cells are intron-containing bicistronic or polycistronic RNAs bearing more than one open reading frame (ORF), because of usage of alternative viral promoters and two alternative viral RNA polyadenylation signals. Papillomaviruses proficiently engage alternative RNA splicing to express individual ORFs from the bicistronic or polycistronic RNA transcripts. In this review, we discuss the genome structures and the updated transcription maps of HPV16 and HPV18, and the latest research advances in understanding RNA cis-elements, intron branch point sequences, and RNA-binding proteins in the regulation of viral RNA processing. Moreover, we briefly discuss the epigenetic modifications, including DNA methylation and possible APOBEC-mediated genome editing in HPV infections and carcinogenesis.

KSHV episome tethering sites on host chromosomes and regulation of latency-lytic switch by CHD4.

Kaposi sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the cell nucleus, but where KSHV episomal genomes are tethered and the mechanisms underlying KSHV lytic reactivation are unclear. Here, we study the nuclear microenvironment of KSHV episomes and show that the KSHV latency-lytic replication switch is regulated via viral long non-coding (lnc)RNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. KSHV episomes localize with CHD4 and ADNP proteins, components of the cellular ChAHP complex. The CHD4 and ADNP proteins occupy the 5'-region of the highly inducible lncRNAs and terminal repeats of the KSHV genome together with latency-associated nuclear antigen (LANA). Viral lncRNA binding competes with CHD4 DNA binding, and KSHV reactivation sequesters CHD4 from the KSHV genome, which is also accompanied by detachment of KSHV episomes from host chromosome docking sites. We propose a model in which robust KSHV lncRNA expression determines the latency-lytic decision by regulating LANA/CHD4 binding to KSHV episomes.

Vitamin K2 Modulates Mitochondrial Dysfunction Induced by 6-Hydroxydopamine in SH-SY5Y Cells via Mitochondrial Quality-Control Loop.

Vitamin K2, a natural fat-soluble vitamin, is a potent neuroprotective molecule, owing to its antioxidant effect, but its mechanism has not been fully elucidated. Therefore, we stimulated SH-SY5Y cells with 6-hydroxydopamine (6-OHDA) in a proper dose-dependent manner, followed by a treatment of vitamin K2. In the presence of 6-OHDA, cell viability was reduced, the mitochondrial membrane potential was decreased, and the accumulation of reactive oxygen species (ROS) was increased. Moreover, the treatment of 6-OHDA promoted mitochondria-mediated apoptosis and abnormal mitochondrial fission and fusion. However, vitamin K2 significantly suppressed 6-OHDA-induced changes. Vitamin K2 played a significant part in apoptosis by upregulating and downregulating Bcl-2 and Bax protein expressions, respectively, which inhibited mitochondrial depolarization, and ROS accumulation to maintain mitochondrial structure and functional stabilities. Additionally, vitamin K2 significantly inhibited the 6-OHDA-induced downregulation of the MFN1/2 level and upregulation of the DRP1 level, respectively, and this enabled cells to maintain the dynamic balance of mitochondrial fusion and fission. Furthermore, vitamin K2 treatments downregulated the expression level of p62 and upregulated the expression level of LC3A in 6-OHDA-treated cells via the PINK1/Parkin signaling pathway, thereby promoting mitophagy. Moreover, it induced mitochondrial biogenesis in 6-OHDA damaged cells by promoting the expression of PGC-1α, NRF1, and TFAM. These indicated that vitamin K2 can release mitochondrial damage, and that this effect is related to the participation of vitamin K2 in the regulation of the mitochondrial quality-control loop, through the maintenance of the mitochondrial quality-control system, and repair mitochondrial dysfunction, thereby alleviating neuronal cell death mediated by mitochondrial damage.

Human Papillomavirus Type 16 Circular RNA Is Barely Detectable for the Claimed Biological Activity.

Human papillomavirus type 16 (HPV16) E7 oncoprotein plays an essential role in cervical carcinogenesis and is encoded predominantly by an E6*I mRNA through alternative RNA splicing of a P97 promoter-transcribed bicistronic E6E7 pre-mRNA. Recently, an HPV16 circular RNA, circE7, was detected in two HPV16-positive cervical cancer cell lines, CaSki and SiHa. It was generated through back-splicing of the E6E7 pre-mRNA. The reported findings showed that, because viral E6*I RNA was nuclear, E7 was mainly translated from the cytoplasmic circE7, and knockdown of circE7 in CaSki cells led to reduction of E7 oncoprotein, cell proliferation, and xenograft tumor formation. We have reanalyzed the published data, conducted detailed experiments, and found that the circE7 in CaSki cells is only 0.4 copies per cell, which is ∼1,640-fold lower than E6*I RNA and also barely detectable from two W12 subclone cell lines, 20861 (integrated HPV16) and 20863 (extrachromosomal HPV16) cells derived from a low-grade cervical lesion. We also determined HPV16 E6*I and E6*II RNAs in CaSki cells are mainly cytoplasmic in cell fractionation analyses, as reported in other studies. We further demonstrated that the claimed circE7 functions in the published report have resulted from off-target effects on E6*I RNA by the circE7 small interfering RNAs used in the reported study. IMPORTANCE RNA back-splicing is a rare splicing event accounting for <1% of canonical RNA splicing and, thus, is thought to have little or no biological significance. Recently, circular RNAs (circRNAs) from RNA back-splicing have been found widely in cells and tissues and may have a role in modulating RNA transcription, splicing, and interference and antiviral innate immunity. A recent report claimed that the predominant HPV16 E6*I RNA was nuclear and unable to encode E7. Rather, a viral circE7 was responsible for translating the oncoprotein E7 in CaSki cells, a cervical cancer cell line. However, we found that both HPV16 E6*I and circE7 RNAs in CaSki cells are primarily cytoplasmic and that the copy number of viral E6*I RNA is 656 copies per cell, whereas the viral circE7 is only 0.4 copies per cell. Most importantly, we found that the claimed circE7 function resulted from off-target effect on viral E6*I RNA by the small interfering RNA (siRNA) si-circE7 designed to knock down the back-spliced circE7 RNA.

Protein-RNA Interactome Analysis Reveals Wide Association of Kaposi's Sarcoma-Associated Herpesvirus ORF57 with Host Noncoding RNAs and Polysomes.

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 is an RNA-binding posttranscriptional regulator. We recently applied an affinity-purified anti-ORF57 antibody to conduct ORF57 cross-linking immunoprecipitation (CLIP) in combination with RNA-sequencing (CLIP-seq) and analyzed the genome-wide host RNA transcripts in association with ORF57 in BCBL-1 cells with lytic KSHV infection. Mapping of the CLIP RNA reads to the human genome (GRCh37) revealed that most of the ORF57-associated RNA reads were from rRNAs. The remaining RNA reads mapped to several classes of host noncoding and protein-coding mRNAs. We found that ORF57 binds and regulates expression of a subset of host long noncoding RNAs (lncRNAs), including LINC00324, LINC00355, and LINC00839, which are involved in cell growth. ORF57 binds small nucleolar RNAs (snoRNAs) responsible for 18S and 28S rRNA modifications but does not interact with fibrillarin or NOP58. We validated ORF57 interactions with 67 snoRNAs by ORF57 RNA immunoprecipitation (RIP)-snoRNA array assays. Most of the identified ORF57 rRNA binding sites (BS) overlap the sites binding snoRNAs. We confirmed ORF57-snoRA71B RNA interaction in BCBL-1 cells by ORF57 RIP and Northern blot analyses using a 32P-labeled oligonucleotide probe from the 18S rRNA region complementary to snoRA71B. Using RNA oligonucleotides from the rRNA regions that ORF57 binds for oligonucleotide pulldown-Western blot assays, we selectively verified ORF57 interactions with 5.8S and 18S rRNAs. Polysome profiling revealed that ORF57 associates with both monosomes and polysomes and that its association with polysomes increases PABPC1 binding to polysomes but prevents Ago2 association with polysomes. Our data indicate a functional correlation with ORF57 binding and suppression of Ago2 activities for ORF57 promotion of gene expression. IMPORTANCE As an RNA-binding protein, KSHV ORF57 regulates RNA splicing, stability, and translation and inhibits host innate immunity by blocking the formation of RNA granules in virus-infected cells. In this study, ORF57 was found to interact with many host noncoding RNAs, including lncRNAs, snoRNAs, and rRNAs, to carry out additional unknown functions. ORF57 binds a group of lncRNAs via the RNA motifs identified by ORF57 CLIP-seq to regulate their expression. ORF57 associates with snoRNAs independently of fibrillarin and NOP58 proteins and with rRNA in the regions that commonly bind snoRNAs. Knockdown of fibrillarin expression decreases the expression of snoRNAs and CDK4 but does not affect viral gene expression. More importantly, we found that ORF57 binds translationally active polysomes and enhances PABPC1 but prevents Ago2 association with polysomes. Data provide compelling evidence on how ORF57 in KSHV-infected cells might regulate protein synthesis by blocking Ago2's hostile activities on translation.

Ecdysoneless Protein Regulates Viral and Cellular mRNA Splicing to Promote Cervical Oncogenesis.

High-risk human papillomaviruses (HPV), exemplified by HPV16/18, are causally linked to human cancers of the anogenital tract, skin, and upper aerodigestive tract. Previously, we identified Ecdysoneless (ECD) protein, the human homolog of the Drosophila ecdysoneless gene, as a novel HPV16 E6-interacting protein. Here, we show that ECD, through its C-terminal region, selectively binds to high-risk but not to low-risk HPV E6 proteins. We demonstrate that ECD is overexpressed in cervical and head and neck squamous cell carcinoma (HNSCC) cell lines as well as in tumor tissues. Using The Cancer Genome Atlas dataset, we show that ECD mRNA overexpression predicts shorter survival in patients with cervical and HNSCC. We demonstrate that ECD knockdown in cervical cancer cell lines led to impaired oncogenic behavior, and ECD co-overexpression with E7 immortalized primary human keratinocytes. RNA-sequencing analyses of SiHa cells upon ECD knockdown showed to aberrations in E6/E7 RNA splicing, as well as RNA splicing of several HPV oncogenesis-linked cellular genes, including splicing of components of mRNA splicing machinery itself. Taken together, our results support a novel role of ECD in viral and cellular mRNA splicing to support HPV-driven oncogenesis. IMPLICATIONS: This study links ECD overexpression to poor prognosis and shorter survival in HNSCC and cervical cancers and identifies a critical role of ECD in cervical oncogenesis through regulation of viral and cellular mRNA splicing.

Improved diagnosis of thyroid cancer aided with deep learning applied to sonographic text reports: a retrospective, multi-cohort, diagnostic study.

OBJECTIVE: Large volume radiological text data have been accumulated since the incorporation of electronic health record (EHR) systems in clinical practice. We aimed to determine whether deep natural language processing algorithms could aid radiologists in improving thyroid cancer diagnosis. METHODS: Sonographic EHR data were obtained from the EHR database. Pathological reports were used as the gold standard for diagnosing thyroid cancer. We developed thyroid cancer diagnosis based on natural language processing (THCaDxNLP) to interpret unstructured sonographic text reports for thyroid cancer diagnosis. We used the area under the receiver operating characteristic curve (AUROC) as the primary metric to measure the performance of the THCaDxNLP. We compared the performance of thyroid ultrasound radiologists aided with THCaDxNLP vs. those without THCaDxNLP using 5 independent test sets. RESULTS: We obtained a total number of 788,129 sonographic radiological reports. The number of thyroid sonographic data points was 132,277, 18,400 of which were thyroid cancer patients. Among the 5 test sets, the numbers of patients per set were 439, 186, 82, 343, and 171. THCaDxNLP achieved high performance in identifying thyroid cancer patients (the AUROC ranged from 0.857-0.932). Thyroid ultrasound radiologists aided with THCaDxNLP achieved significantly higher performances than those without THCaDxNLP in terms of accuracy (93.8% vs. 87.2%; one-sided t-test, adjusted P = 0.003), precision (92.5% vs. 86.0%; P = 0.018), and F1 metric (94.2% vs. 86.4%; P = 0.007). CONCLUSIONS: THCaDxNLP achieved a high AUROC for the identification of thyroid cancer, and improved the accuracy, sensitivity, and precision of thyroid ultrasound radiologists. This warrants further investigation of THCaDxNLP in prospective clinical trials.