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Hsa_circ_0071589 can exacerbate the malignant behavior of colorectal cancer (CRC) cells. However, its function in stemness and oxaliplatin (OXP) resistance of CRC cells remains unclear. To assess the function of hsa_circ_0071589 in stemness and OXP resistance of CRC cells. Western blotting and qRT-PCR were applied to assess protein and mRNA levels. The association between hsa_circ_0071589, miR-133b and SOX13 was explored via a correlation analysis. Sphere formation was used to assess cell stemness. Meanwhile, the viability of CRC cells and OXP-resistant CRC cells was evaluated with the MTT assay. Cell stemness marker (CD133) levels and apoptosis of CRC cells and OXP-resistant CRC cells were tested using flow cytometry. The ALDH level was investigated using the related detection kit. In addition, the association between hsa_circ_0071589 and miR-133b and SOX13 was investigated using the RIP and dual luciferase assay. Finally, in vivo experiments were performed to detect the function of hsa_circ_0071589 in CRC, and the levels of SOX13, Ki67, and CD44 in mice were evaluated via immunohistochemistry staining. The expression of hsa_circ_0071589 and SOX13 was upregulated in CRC, whereas the expression of miR-133b was downregulated. Hsa_circ_0071589 knockdown significantly inhibited CRC stemness via the mediation of miR-133b. Moreover, hsa_circ_0071589 silencing significantly sensitized CRC cells to OXP by upregulating miR-133b. SOX13 was the direct target of miR-133b, and miR-133b could attenuate stemness and OXP resistance in CRC cells by targeting SOX13. Notably, hsa_circ_0071589 knockdown inhibited tumor growth and decreased OXP resistance in mice with CRC. Hsa_circ_0071589 aggravates stemness and OXP resistance by sponging miR-133b to indirectly target SOX13 in CRC. Thus, our study might present a novel treatment strategy against OXP-resistant CRC., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The critical roles of lncRNAs in drug resistance of malignancies have been widely recognized. This investigation aims to study the function of lncRNA PCAT6 in the resistance of non-small cell lung cancer (NSCLC) to gefitinib. In our study, we demonstrated that prostate cancer-associated transcript 6 (PCAT6) was upregulated in gefitinib-resistant NSCLC. PCAT6 knockdown inhibited gefitinib resistance of NSCLC, as indicated by decreased IC 50 value, proliferation, and metastasis, and increased cell apoptosis. Besides, PCAT6 could directly target miR-326 in gefitinib-resistant NSCLC cells and augment NSCLC resistance to gefitinib by serving as ceRNA of miR-326. Furthermore, interferon-alpha receptor 2 (IFNAR2) was validated as a downstream target of miR-326 and miR-326 reduced resistance to gefitinib by inhibiting IFNAR2 expression. Our investigation identified that PCAT6 enhanced gefitinib resistance of NSCLC via miR-326/IFNAR2 axis, which might offer a new therapeutic strategy against gefitinib resistance of NSCLC patients.

Literatures have demonstrated that propofol can improve the efficacy of cisplatin, and miR-195 is implicated in the underlying mechanism concerning the anticancer effects of propofol. However, correlation between propofol and miR-195 has been little studied. This study clarified that propofol enhanced the inhibitory effect of cisplatin in liver cancer cells via miR-195-5p. 50 samples of liver cancer and para-cancer tissues in patients were collected and the difference in the expression of miR-195-5p was then analyzed. The liver cancer cells treated with gradient concentrations of cisplatin (3, 6.25, 12.5, 25, 50 μg/mL) and propofol (2, 5, 10 μg/mL) were tested for drug toxicity using CCK-8 assay. Next, following the transfection, the effects of propofol, cisplatin and miR-195-5p on the functions of liver cancer cells and the expressions of related proteins were analyzed by clone formation, flow cytometry and western blot. The downstream target genes of miR-195-5p were predicted by bio-informatics analysis and verified by dual-luciferase reporter assay, and their expressions in cancer cell was also calculated. The changes on the expressions of target genes were further detected by qRT-PCR and western blot. MiR-195-5p was lowly-expressed in liver cancer, and the up-regulation of miR-195-5p enhanced the sensitivity of liver cancer cells to cisplatin. Propofol inhibited the viability of liver cancer cells and stimulated the up-regulation of miR-195-5p. Propofol enhanced the lethality of cisplatin to liver cancer cells and reversed the repressive effects of miR-195-5p inhibitor on the efficacy of cisplatin. CCNE1 was the downstream target gene of miR-195-5p and its expression was up-regulated by miR-195-5p inhibitor in cisplatin-treated liver cancer cells. Collectively, propofol enhances the lethality of cisplatin to liver cancer cells by up-regulating miR-195-5p., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and is one of the leading causes of cancer-related deaths worldwide. Regorafenib, a multi-kinase inhibitor, is used as a second-line treatment for advanced HCC. Here, we aimed to investigate the mechanism of the antitumor effect of regorafenib on HCC and evaluate altered microRNA (miRNA) expression. Cell proliferation was examined in six HCC cell lines (HuH-7, HepG2, HLF, PLC/PRF/5, Hep3B, and Li-7) using the Cell Counting Kit-8 assay. Xenografted mouse models were used to assess the effects of regorafenib in vivo. Cell cycle analysis, western blotting analysis, and miRNA expression analysis were performed to identify the antitumor inhibitory potential of regorafenib on HCC cells. Regorafenib suppressed proliferation in HuH-7 cell and induced G0/G1 cell cycle arrest and cyclin D1 downregulation in regorafenib-sensitive cells. During miRNA analysis, miRNA molecules associated with the antitumor effect of regorafenib were found. Regorafenib suppresses cell proliferation and tumor growth in HCC by decreasing cyclin D1 via alterations in intracellular and exosomal miRNAs in HCC.

Exposure to high doses of anticancer drugs can induce the emergence of a subpopulation of weakly proliferative and drug-tolerant cells. Drug tolerance can reduce the benefits obtained from canonical treatment and reduce the survival rate of patients. Regulation of SRY-related HMG box transcription factor 4 (SOX4) has been proved to affect drug sensitivity. The current study aimed to explore the role of SOX4 in drug resistance of colorectal cancer (CRC) cells as well as the related molecular mechanisms. Expression patterns of SOX4, microRNA-17 (miR-17), and CYLD in both CRC tissues and cells were determined with their relationship analyzed by bioinformatics analysis, dual-luciferase reporter gene assay, and ChIP. Loss- and gain-function assays were performed to ascertain the effect of SOX4, miR-17, and CYLD on biological cellular processes and drug resistance to 5-FU. SOX4 and miR-17 were found to be highly expressed while CYLD was poorly expressed in CRC tissues and cells. Silencing of SOX4 resulted in the suppression of cellular proliferation, invasion, migration as well as a reduction in CRC drug resistance. Mechanically, CYLD was specifically targeted by miR-17, while SOX4 upregulated the expression of miR-17. Functionally, SOX4 triggered drug resistance of CRC cells to 5-FU through the miR-17/CYLD axis. Taken together, the key findings of the present study provides evidence suggesting that SOX4 elevates miR-17 to decrease CYLD, thus inducing chemotherapy resistance of CRC cells., (© 2021 Wiley Periodicals LLC.)

Gastric cancer (GC) remains one of the most prevalent types of malignancies worldwide, and also one of the most reported lethal tumor-related diseases. Circular RNAs (circRNAs) have been certified to be trapped in multiple aspects of GC pathogenesis. Yet, the mechanism of this regulation is mostly undefined. This research is designed to discover the vital circRNA-microRNA (miRNA)-messenger RNA (mRNA) regulatory network in GC. Expression profiles with diverse levels including circRNAs, miRNAs, and mRNAs were all determined using microarray public datasets from Gene Expression Ominous (GEO). The differential circRNAs expressions were recognized against the published robust rank aggregation algorithm. Besides, a circRNA-based competitive endogenous RNA (ceRNA) interaction network was visualized via Cytoscape software (version 3.8.0). Functional and pathway enrichment analysis associated with differentially expressed targeted mRNAs were conducted using Cytoscape and an online bioinformatics database. Furthermore, an interconnected protein-protein interaction association network which consisted of 51 mRNAs was predicted, and hub genes were screened using STRING and CytoHubba. Then, several hub genes were chosen to explore their expression associated with survival rate and clinical stage in GEPIA and Kaplan-Meier Plotter databases. Finally, a carefully designed circRNA-related ceRNA regulatory subnetwork including four circRNAs, six miRNAs, and eight key hub genes was structured using the online bioinformatics tool.

The PPARδ gene codes protein that belongs to the peroxisome proliferator-activated receptor (PPAR) family engaged in a variety of biological processes, including carcinogenesis. Specific biological and clinical roles of PPARδ in non-small cell lung cancer (NSCLC) is not fully explained. The association of PPARα with miRNA regulators (e.g. miRNA-17) has been documented, suggesting the existence of a functional relationship of all PPARs with epigenetic regulation. The aim of the study was to determine the PPARδ and miR-17 expression profiles in NSCLC and to assess their diagnostic value in lung carcinogenesis. PPARδ and miR-17 expressions was assessed by qPCR in NSCLC tissue samples (n = 26) and corresponding macroscopically unchanged lung tissue samples adjacent to the primary lesions served as control (n = 26). PPARδ and miR-17 expression were significantly lower in NSCLC than in the control (p = 0.0001 and p = 0.0178; respectively). A receiver operating characteristic (ROC) curve analysis demonstrated the diagnostic potential in discriminating NSCLC from the control with an area under the curve (AUC) of 0.914 for PPARδ and 0.692 for miR-17. Significant increase in PPARδ expression in the control for current smokers vs. former smokers (p = 0.0200) and increase in miR-17 expression in control tissue adjacent to adenocarcinoma subtype (p = 0.0422) were observed. Overexpression of miR-17 was observed at an early stage of lung carcinogenesis, which may suggest that it acts as a putative oncomiR. PPARδ and miR-17 may be markers differentiating tumour tissue from surgical margin and miR-17 may have diagnostic role in NSCLC histotypes differentiation., (© 2021. The Author(s).)

Background: lncRNAs are modulatory factors with critical function in the tumorigenesis pathways, introducing them as promising therapeutic and diagnostic biomarkers for different cancers. This study is thus aimed to evaluate the differences in PVT1 and ZFAS1 gene expression in tumorous tissues as compared with adjacent healthy non-tumorous biopsies of gastric cancer cases., Methods: One hundred two pairs of tumorous and adjacent non-tumorous biopsies of GC cases were sampled. RNA isolation and cDNA production were carried out. The qRT-PCR was performed to evaluate the expression of PVT1 and ZFAS1 genes. Moreover, the associations between PVT1 or ZFAS1 and clinicopathological characteristics as well as the biomarker roles of the lncRNAs were assessed., Results: The PVT1 and ZFAS1 expressions showed a significant increase and decrease in GC samples as compared with non-cancerous tissues, respectively. PVT1 expression was significantly associated with and lymph-node involvement (p = 0.0007). Moreover, ZFAS1 expression demonstrated a significant association with lymph-node involvement (p = 0.0005), and tumor size >5 cm (p = 0.003). The findings of the ROC curve revealed that PVT1 and ZFAS1 may act as a possible biomarker with AUC of 0.71 and 0.79, specificity of 78.43% and 79.41%, and sensitivity of 55.88% and 64.71%., Conclusions: Regarding upregulation of PVT1 and downregulation of ZFAS1 in human GC samples, these genes may respectively act as oncogenic and tumor-suppressive factors in GC cases. Furthermore, PVT1 and ZFAS1 can be considered as possible biomarkers for the detection and treatment of GC cases., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Objective: This study aimed to conduct a genomewide transcriptomic profiling to develop a microRNA (miRNA)-based signature for the identification of peritoneal metastasis (PM) in patients with gastric cancer (GC)., Summary Background Data: Even though PM in patients with GC has long been recognized to associate with poor prognosis, currently there is lack of availability of molecular biomarkers for its robust diagnosis., Methods: We performed a systematic biomarker discovery by analyzing miRNA expression profiles in primary tumors from GC patients with and without PM, and subsequently validated the expression of candidate miRNA biomarkers in 3 independent clinical cohorts of 354 patients with advanced GC., Results: Five miRNAs (miR-30a-5p, -134-5p, -337-3p, -659-3p, and -3917) were identified during the initial discovery phase; three of which (miR-30a-5p, -659-3p, and -3917) were significantly overexpressed in the primary tumors from PM-positive patients in the testing cohort (P = 0.002, 0.04, and 0.007, respectively), and distinguished patients with versus without peritoneal metastasis with the value of area under the curve (AUC) of 0.82. Furthermore, high expression of these miRNAs also associated with poor prognosis (hazard ratio = 2.18, P = 0.04). The efficacy of the combination miRNA signature was subsequently validated in an independent validation cohort (AUC = 0.74). Finally, our miRNA signature when combined together with the macroscopic Borrmann's type score offered a much superior diagnostic in all 3 cohorts (AUC = 0.87, 0.76, and 0.79, respectively)., Conclusions: We have established an miRNA-based signature that have a potential to identify peritoneal metastasis in GC patients., Competing Interests: The authors report no conflicts of interest to disclose., (Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.)

Circular RNAs (circRNAs) are a novel class of widespread noncoding RNAs that regulate gene expression in mammals. Recent studies demonstrate that functional peptides can be encoded by short open reading frames in noncoding RNAs, including circRNAs. However, the role of circRNAs in various physiological and pathological states, such as cancer, is not well understood. In this study, through deep RNA sequencing on human endometrial cancer (EC) samples and their paired adjacent normal tissues, we uncovered that the circRNA hsa-circ-0000437 is significantly reduced in EC compared with matched paracancerous tissue. The hsa-circ-0000437 contains a short open reading frame encoding a functional peptide termed CORO1C-47aa. Overexpression of CORO1C-47aa is capable of inhibiting angiogenesis at the initiation stage by suppressing endothelial cell proliferation, migration, and differentiation through competition with transcription factor TACC3 to bind to ARNT and suppress VEGF. CORO1C-47aa directly bound to ARNT through the PAS-B domain, and blocking the association between ARNT and TACC3, which led to reduced expression of VEGF, ultimately lead to reduced angiogenesis. The antitumor effects of CORO1C-47aa on EC progression suggest that CORO1C-47aa has potential value in anticarcinoma therapies and warrants further investigation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: This work aims to determine the role of circular RNA (circRNA) AGFG1 and related molecular mechanism in esophageal squamous cell carcinoma (ESCC) cells., Methods: CircAGFG1 expression in ESCC cell lines was probed with qRT-PCR. ESCC cells were transfected/cotransfected with si-circAGFG1, pcDNA3.1-circAGFG1, si-Microtubule Associated Protein RP/EB Family Member 2 (MAPRE2), pcDNA3.1-circAGFG1 + miR-4306 mimic or pcDNA3.1-circAGFG1 + si-MAPRE2. The interactions between circAGFG1 and miR-4306 as well as miR-4306 and MAPRE2 were confirmed by dual-luciferase reporter assay. Cell proliferation, migration and invasion were detected by CCK-8, cell scratch and Transwell assays, respectively. Relative RNA expression levels of circAGFG1, miR-4306 and MAPRE2 in ESCC cells were measured by qRT-PCR. The protein level of MAPRE2 in ESCC cells was monitored by Western blot., Results: CircAGFG1 was observably upregulated in ESCC cell lines. Besides, circAGFG1 silencing hindered ESCC cell development in vitro, and these effects were enhanced by miR-4306 overexpression or MAPRE2 silencing. Mechanistic analysis evidenced that circAGFG1 might act as a competitive endogenous RNA of miR-4306 to relieve the repressive effect of miR-4306 on its target MAPRE2., Conclusion: CircAGFG1 facilitates ESCC progression via the miR-4306/MAPRE2 axis, and it may act as a possible biomarker for therapy and diagnosis in ESCC treatment., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Objective: Meningiomas and gliomas are common benign and malignant primary brain tumors, respectively. One of the most prominent features of aggressive malignancies contributing to their progression is their ability to cope with hypoxia. Therefore, glioma tumors are expected to better cope with adverse hypoxic conditions and, consequently, display significantly different expression levels of hypoxia-adaptive genes., Methods: Thirty-three glioma (17 glioblastoma multiforme [GBM], 16 low-grade glioma [LGG]) and 32 meningioma samples were investigated for expression of hypoxia adaptation- related genes by real-time polymerase chain reaction. The same investigation was carried out for GBM, the most malignant form of glioma, versus LGG. The findings were further checked by bioinformatics analysis of expression levels using RNA-seq data. Additional investigations conducted include receiver operating characteristic curve analysis to assess the power for each gene in differential diagnosis of glioma from meningioma., Results: A greater level of hypoxia-inducible factor (HIF) 1α expression in glioma samples compared with meningioma and greater expression levels of Yes-associated protein (YAP) 1 and G-protein-coupled receptor class C group 5 member A (GPRC5A) in meningioma were observed, with P values 0.0005, <0.0001, and <0.0001 for GPRC5A, HIF1α, and YAP1, respectively. Comparison of GBM with LGG also revealed GPRC5A to have significantly greater expression in GBM with P = 0.0381. The calculated area under the curve was 0.7536, 0.8438, and 0.8272 for GPRC5A, HIF1α, and YAP1, respectively, which represented acceptable power for these genes in differential diagnosis of glioma tumor types from meningioma and tumor subtypes GBM from LGG under study., Conclusions: These results imply that these genes can possibly be implicated in brain tumor hypoxia-adaptation response with tumor-specific roles and patterns of expression., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Programmed death ligand 1 (PD-L1) expression on the surface of cancer cells affects the efficacy of anti-PD-1/PD-L1 immune checkpoint therapy. However, the mechanism underlying PD-L1 expression in cancer cells is not fully understood, particularly after ionizing radiation (IR). Here, we examined the impact of high linear energy transfer (LET) carbon-ion irradiation on the expression of PD-L1 in human osteosarcoma U2OS cells. We found that the upregulation of PD-L1 expression after high LET carbon-ion irradiation was greater than that induced by X-rays at the same physical and relative biological effectiveness (RBE) dose, and that the upregulation of PD-L1 induced by high LET carbon-ion irradiation was predominantly dependent on ataxia telangiectasia and Rad3-related (ATR) kinase activity. Moreover, we showed that the downstream signaling, e.g. STAT1 phosphorylation and IRF1 expression, was upregulated to a greater extent after high LET carbon-ion irradiation than X-rays, and that IRF1 upregulation was also ATR dependent. Finally, to visualize PD-L1 molecules on the cell surface in 3D, we applied immunofluorescence-based super-resolution imaging. The three-dimensional structured illumination microscopy (3D-SIM) analyses revealed substantial increases in the number of presented PD-L1 molecules on the cell surface after high LET carbon-ion irradiation compared with X-ray irradiation., (© The Author(s) 2021. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

RNAscope®, has emerged as an important in-situ hybridisation method to validate mRNA expression within single cells whilst preserving tissue morphology in histological samples. The aim of this research was to compare the utility of various open-source and commercial image analysis methods, to quantify mRNA transcripts identified by RNAscope within formalin fixed paraffin embedded (FFPE) histological samples and cell monolayer preparations. Examination of MLH1 expression from 10 histological FFPE colorectal cancer specimens using four image analysis tools (Colour Deconvolution, SpotStudio, WEKA and the LEICA RNA-ISH algorithm) showed the WEKA tool as having the greatest level of agreement with manual quantification. Comparing image analysis methods to qRT-PCR for quantifying MLH1, GFI1 and TNFRSF11A expression within two colorectal cell lines results suggest that these image analysis methods perform at a similar level to qRT-PCR. Furthermore, we describe the strengths and limitations for each image analysis method when used in combination with RNAscope assays. Our study concludes that there are several freely available and commercial image analysis tools that enable reliable RNA in situ expression analysis, however operators need to consider factors, such as expected expression levels of target genes, software usability and functionality., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Breast cancer (BC) is the most commonly diagnosed malignancy and the leading cause of cancer-related mortality among females. Over 90 % of the cases of death in BC patients are attributed to tumor cell metastasis. Therefore, it is urgently needed to investigate the molecular mechanisms of BC metastasis. The expression of miRNA in BC was evaluated by qRT-PCR and bioinformatics analysis. Clone formation, EdU assays, and subcutaneous xenograft model were used to test the growth of BC cells. Wound healing, transwell assays, and lung-metastasis model were used to explore the effect of miR-934 knockdown on cell metastasis. The miR-934 targets in BC were identified through bioinformatics analysis and luciferase reporter assays. The expression of protein was tested by western blot. The binding of mRNA and RNA-binding-protein was verified using RIP assays. miR-934 expression was significantly elevated in BC tissues, especially in those with lymph node metastasis and associated with poor patient prognosis. Experiments in vitro and in vivo showed that that upregulated miR-934 was not necessarily required for the growth of BC cells. However, miR-934 knockdown significantly inhibited the migration and invasion abilities of BC cells. Moreover, PTEN as identified as the direct target of miR-934 in BC, and miR-934 could promote BC cell metastasis by regulation of PTEN and epithelial-mesenchymal transition (EMT). Our results suggested that targeting miR-934 may be a practical treatment for BC cell metastasis., (Copyright © 2021. Published by Elsevier Ltd.)

Background: Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancers. The drug resistance of NSCLC has clinically increased. This study aimed to screen miRNAs associated with NSCLC using bioinformatics analysis. We hope that the screened miRNA can provide a research direction for the subsequent treatment of NSCLC., Methods: We screened out the common miRNAs after compared the NSCLC-related genes in the TCGA database and GEO database. Selected miRNA was performed ROC analysis, survival analysis, and enrichment analysis (GO term and KEGG pathway)., Results: A total of 21 miRNAs were screened in the two databases. And they were all highly expressed in normal and low in cancerous tissues. Hsa-mir-30a was selected by ROC analysis and survival analysis. Enrichment analysis showed that the function of hsa-mir-30a is mainly related to cell cycle regulation and drug metabolism., Conclusion: Our study found that hsa-mir-30a was differentially expressed in NSCLC, and it mainly affected NSCLC by regulating the cell cycle and drug metabolism., Competing Interests: The authors have declared that no competing interests exist.

Emerging evidence indicates that the abnormal expression of miRNAs involves in the evolution and progression of various human complex diseases. Identifying disease-related miRNAs as new biomarkers can promote the development of disease pathology and clinical medicine. However, designing biological experiments to validate disease-related miRNAs is usually time-consuming and expensive. Therefore, it is urgent to design effective computational methods for predicting potential miRNA-disease associations. Inspired by the great progress of graph neural networks in link prediction, we propose a novel graph auto-encoder model, named GAEMDA, to identify the potential miRNA-disease associations in an end-to-end manner. More specifically, the GAEMDA model applies a graph neural networks-based encoder, which contains aggregator function and multi-layer perceptron for aggregating nodes' neighborhood information, to generate the low-dimensional embeddings of miRNA and disease nodes and realize the effective fusion of heterogeneous information. Then, the embeddings of miRNA and disease nodes are fed into a bilinear decoder to identify the potential links between miRNA and disease nodes. The experimental results indicate that GAEMDA achieves the average area under the curve of $93.56\pm 0.44\%$ under 5-fold cross-validation. Besides, we further carried out case studies on colon neoplasms, esophageal neoplasms and kidney neoplasms. As a result, 48 of the top 50 predicted miRNAs associated with these diseases are confirmed by the database of differentially expressed miRNAs in human cancers and microRNA deregulation in human disease database, respectively. The satisfactory prediction performance suggests that GAEMDA model could serve as a reliable tool to guide the following researches on the regulatory role of miRNAs. Besides, the source codes are available at https://github.com/chimianbuhetang/GAEMDA., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Perineural invasion (PNI) is frequently associated with aggressive clinical behaviour in head and neck cutaneous squamous cell carcinoma (HNcSCC) leading to local recurrence and treatment failure. This study evaluates the gene expression profiles of HNcSCC with PNI using a differential expression analysis approach and constructs a tailored gene panel for sensitivity and specificity analysis. 45 cases of HNcSCC were stratified into three groups (Extensive, Focal and Non PNI) based on predefined clinicopathological criteria. Here we show HNcSCC with extensive PNI demonstrates significant up- and down-regulation of 144 genes associated with extracellular matrix interactions, epithelial to mesenchymal transition, cell adhesion, cellular motility, angiogenesis, and cellular differentiation. Gene expression of focal and non PNI cohorts were indistinguishable and were combined for further analyses. There is clinicopathological correlation between gene expression analysis findings and disease behaviour and a tailored panel of 10 genes was able to identify extensive PNI with 96% sensitivity and 95% specificity.

Background : Brain arteriovenous malformations (BAVMs) and cerebral cavernous malformations (CCMs) are rare developmental anomalies of the intracranial vasculature, with an irregular tendency to rupture, and as of yet incompletely deciphered pathophysiology. Because of their variety in location, morphology, and size, as well as unpredictable natural history, they represent a management challenge. MicroRNAs (miRNAs) are strands of non-coding RNA of around 20 nucleotides that are able to modulate the expression of target genes by binding completely or partially to their respective complementary sequences. Recent breakthroughs have been made on elucidating their contribution to BAVM and CCM occurrence, growth, and evolution; however, there are still countless gaps in our understanding of the mechanisms involved. Methods : We have searched the Medline (PubMed; PubMed Central) database for pertinent articles on miRNAs and their putative implications in BAVMs and CCMs. To this purpose, we employed various permutations of the terms and idioms: 'arteriovenous malformation', 'AVM', and 'BAVM', or 'cavernous malformation', 'cavernoma', and 'cavernous angioma' on the one hand; and 'microRNA', 'miRNA', and 'miR' on the other. Using cross-reference search; we then investigated additional articles concerning the individual miRNAs identified in other cerebral diseases. Results : Seven miRNAs were discovered to play a role in BAVMs, three of which were downregulated (miR-18a, miR-137, and miR-195*) and four upregulated (miR-7-5p, miR-199a-5p, miR-200b-3p, and let-7b-3p). Similarly, eight miRNAs were identified in CCM in humans and experimental animal models, two being upregulated (miR-27a and mmu-miR-3472a), and six downregulated (miR-125a, miR-361-5p, miR-370-3p, miR-181a-2-3p, miR-95-3p, and let-7b-3p). Conclusions : The following literature review endeavored to address the recent discoveries related to the various implications of miRNAs in the formation and growth of BAVMs and CCMs. Additionally, by presenting other cerebral pathologies correlated with these miRNAs, it aimed to emphasize the potential directions of upcoming research and biological therapies.

Diffuse large B cell lymphoma (DLBCL) comprises distinct entities due to its heterogeneity. The currently used international prognostic index for DLBCL prognosis prediction is only based on clinical factors and cannot reflect the molecular mechanisms underlying its progression. Here, we aimed to establish a long noncoding RNAs (lncRNA)-based signature for DLBCL prognosis prediction. The data were retrieved from the Gene Expression Omnibus and The Cancer Genome Atlas database. After identifying the differentially expressed lncRNAs (DELs), univariate COX regression, LASSO regression, and stepwise regression analysis were performed to construct a 6-lncRNA risk score system. Kaplan-Meier survival presented that the high-risk group had a significantly poorer overall survival. Based on the risk score and clinical characters, a nomogram was established, which had better predictive accuracy than each factor alone. Finally, weighted gene co-expression network analysis showed that these lncRNAs might regulate immune response, metabolism process, and signal transduction to influence the outcome. Conclusively, our model and nomogram could be reliable prognostic tools for DLBCL patients., (© 2021 Wiley Periodicals LLC.)

The continuous rise in relapse rate and mortality for multiple myeloma (MM) demands an effective treatment option. The microRNAs are emerging nowadays for their promising therapeutic potential. Earlier, we reported involvement of Versican (VCAN) in myeloma pathogenesis which could be inhibited by miR-144 and miR-199 in stroma. However, there is dearth of literature showcasing the direct effect of these miRs in association with VCAN in MM. Expression of miR-144 and miR-199 was determined in myeloma cell lines (RPMI8226 & U266). These miRs were inhibited by small oligos to elucidate changes in expression of VCAN along with variation in parameters such as proliferation, apoptosis, migration and invasion in vitro. Moreover, effect on certain downstream signaling cascades was also evaluated. Lastly, interaction of miRs with VCAN was assessed by reporter luciferase assay. microRNAs expression were found significantly elevated in myeloma cells in comparison to stromal levels reported previously. The antagomirs-mediated inhibition of miR-144 and miR-199 significantly induced VCAN expression in myeloma cells along with alteration in myeloma-associated parameters in favor of myeloma pathogenesis with downstream activation of FAK/STAT3 signaling. Interestingly, miR-144 found to have direct binding with VCAN 3' UTR while miR-199 possess different mechanism. The inhibition of miR-144 and miR-199 contributed in myeloma progression via upregulation of VCAN in vitro affirming the translational significance of VCAN and associated microRNAs in MM. These miRs, hence might be employed for targeting VCAN and might emerge as an effective therapy for the better outcome of MM in clinical settings in future.

Despite recent progress, gastric cancer remains one of the most common cancers and has a high mortality rate worldwide. Aberrant DNA methylation pattern and deregulation of noncoding RNA expression appear in the early stages of gastric cancer. Numerous investigations have confirmed their significant role in gastric cancer tumorigenesis and their high potential as diagnostic and prognostic biomarkers. Currently, it is clear that these epigenetic regulators do not work alone but interact with each other, generating a complex network. The aim of our review was to summarize the current knowledge of this interaction in gastric cancer and estimate its clinical potential for the diagnosis, prognosis, and treatment of the disease.

In this paper, we compared the effects of bortezomib on L1210 (S) cells with its effects on P-glycoprotein (P-gp)-positive variant S cells, which expressed P-gp either after selection with vincristine (R cells) or after transfection with a human gene encoding P-gp (T cells). Bortezomib induced the death-related effects in the S, R, and T cells at concentrations not exceeding 10 nM. Bortezomib-induced cell cycle arrest in the G2/M phase was more pronounced in the S cells than in the R or T cells and was related to the expression levels of cyclins, cyclin-dependent kinases, and their inhibitors. We also observed an increase in the level of polyubiquitinated proteins (via K48-linkage) and a decrease in the gene expression of some deubiquitinases after treatment with bortezomib. Resistant cells expressed higher levels of genes encoding 26S proteasome components and the chaperone HSP90, which is involved in 26S proteasome assembly. After 4 h of preincubation, bortezomib induced a more pronounced depression of proteasome activity in S cells than in R or T cells. However, none of these changes alone or in combination sufficiently suppressed the sensitivity of R or T cells to bortezomib, which remained at a level similar to that of S cells.

Emerging evidence has shown that active enhancers are abundantly transcribed, generating long non-coding RNAs, called enhancer RNAs (eRNAs). While putative eRNAs are often observed from RNA sequencing, the roles of most eRNAs remain largely unknown. Previously, we identified putative enhancer regions at the MALAT1 locus that form chromatin-chromatin interactions under hypoxia, and one of these enhancers is located about 30 kb downstream of the NEAT1 gene and -20 kb upstream of the MALAT1 gene (MALAT1-20 kb enhancer). Here, we report that a novel eRNA, named eRNA of the NEAT1-MALAT1-Locus (eNEMAL), is transcribed from the MALAT1-20 kb enhancer and conserved in primates. We found that eNEMAL is upregulated in response to hypoxia in multiple breast cancer cell lines, but not in non-tumorigenic MCF10A cells. Overexpression and knockdown of eNEMAL revealed that alteration of eNEMAL level does not affect MALAT1 expression. Instead, we found that eNEMAL upregulates the long isoform of NEAT1 (NEAT1&#95;2) without increasing the total NEAT1 transcript level in MCF7 breast cancer cells, suggesting that eNEMAL has a repressive effect on the 3'-end polyadenylation process required for generating the short isoform of NEAT1 (NEAT1&#95;1). Altogether, we demonstrated that an eRNA transcribed from a MALAT1 enhancer regulates NEAT1 isoform expression, implicating the MALAT1-20 kb enhancer and its transcript eNEMAL in co-regulation of MALAT1 and NEAT1 in response to hypoxia in breast cancer cells., Competing Interests: The authors have declared that no competing interests exist.

The transformation of chronic lymphocytic leukemia (CLL) to high-grade B-cell lymphoma is known as Richter syndrome (RS), a rare event with dismal prognosis. In this study, we conducted whole-genome sequencing (WGS) of paired circulating CLL (PB-CLL) and RS biopsies (tissue-RS) from 17 patients recruited into a clinical trial (CHOP-O). We found that tissue-RS was enriched for mutations in poor-risk CLL drivers and genes in the DNA damage response (DDR) pathway. In addition, we identified genomic aberrations not previously implicated in RS, including the protein tyrosine phosphatase receptor (PTPRD) and tumor necrosis factor receptor-associated factor 3 (TRAF3). In the noncoding genome, we discovered activation-induced cytidine deaminase-related and unrelated kataegis in tissue-RS affecting regulatory regions of key immune-regulatory genes. These include BTG2, CXCR4, NFATC1, PAX5, NOTCH-1, SLC44A5, FCRL3, SELL, TNIP2, and TRIM13. Furthermore, differences between the global mutation signatures of pairs of PB-CLL and tissue-RS samples implicate DDR as the dominant mechanism driving transformation. Pathway-based clonal deconvolution analysis showed that genes in the MAPK and DDR pathways demonstrate high clonal-expansion probability. Direct comparison of nodal-CLL and tissue-RS pairs from an independent cohort confirmed differential expression of the same pathways by RNA expression profiling. Our integrated analysis of WGS and RNA expression data significantly extends previous targeted approaches, which were limited by the lack of germline samples, and it facilitates the identification of novel genomic correlates implicated in RS transformation, which could be targeted therapeutically. Our results inform the future selection of investigative agents for a UK clinical platform study. This trial was registered at www.clinicaltrials.gov as #NCT03899337., (© 2021 by The American Society of Hematology.)

Molecular heterogeneities and complex microenvironments bring great challenges for cancer diagnosis and treatment. Recent advances in single-cell RNA-sequencing (scRNA-seq) technology make it possible to study cancer cell heterogeneities and microenvironments at single-cell transcriptomic level. Here, we develop an R package named scCancer, which focuses on processing and analyzing scRNA-seq data for cancer research. Except basic data processing steps, this package takes several special considerations for cancer-specific features. Firstly, the package introduced comprehensive quality control metrics. Secondly, it used a data-driven machine learning algorithm to accurately identify major cancer microenvironment cell populations. Thirdly, it estimated a malignancy score to classify malignant (cancerous) and non-malignant cells. Then, it analyzed intra-tumor heterogeneities by key cellular phenotypes (such as cell cycle and stemness), gene signatures and cell-cell interactions. Besides, it provided multi-sample data integration analysis with different batch-effect correction strategies. Finally, user-friendly graphic reports were generated for all the analyses. By testing on 56 samples with 433 405 cells in total, we demonstrated its good performance. The package is available at: http://lifeome.net/software/sccancer/., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The NanoString RNA counting assay for formalin-fixed paraffin embedded samples is unique in its sensitivity, technical reproducibility and robustness for analysis of clinical and archival samples. While commercial normalization methods are provided by NanoString, they are not optimal for all settings, particularly when samples exhibit strong technical or biological variation or where housekeeping genes have variable performance across the cohort. Here, we develop and evaluate a more comprehensive normalization procedure for NanoString data with steps for quality control, selection of housekeeping targets, normalization and iterative data visualization and biological validation. The approach was evaluated using a large cohort ($N=\kern0.5em 1649$) from the Carolina Breast Cancer Study, two cohorts of moderate sample size ($N=359$ and$130$) and a small published dataset ($N=12$). The iterative process developed here eliminates technical variation (e.g. from different study phases or sites) more reliably than the three other methods, including NanoString's commercial package, without diminishing biological variation, especially in long-term longitudinal multiphase or multisite cohorts. We also find that probe sets validated for nCounter, such as the PAM50 gene signature, are impervious to batch issues. This work emphasizes that systematic quality control, normalization and visualization of NanoString nCounter data are an imperative component of study design that influences results in downstream analyses., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Multiple functions of CD38 need exploring to expand clinical application of anti-CD38 antibodies in multiple myeloma (MM). We investigated membrane dynamics of MM cells and subsequent events when CD38 is targeted by therapeutic antibodies. Human MM cells (BF01) were co-cultured in vitro with therapeutic antibody (or control immunoglobulin G) and analysed using gene expression profiling. Microvesicles from antibody-exposed cells were analysed for differential gene and microRNA (miRNA) expression, and for phenotypic characterisation. Exposure of BF01 cells to anti-CD38 antibody resulted in CD38 membrane redistribution, upregulation of metabolism-related genes and downregulation of genes involved in cell cycle processes. Microvesicles derived from antibody-exposed cells showed increased CD73 and CD39 expression, presence of programmed death-ligand 1 and significant up-/down-modulation of miRNAs. Microvesicles accumulated around immunoglobulin Fc receptor-positive (FcR + ) cells. Upon internalisation, natural killer cells displayed significantly increased expression of genes related to activation and immune response, and downregulation of genes involved in the cell cycle. Cells may use microvesicles to transmit signals distally as part of a survival strategy. Microvesicles are equipped on their surface with enzymatic machinery leading to production of tolerogenic adenosine. Further, they are internalised in FcR + cells with significant functional modifications. These observations have relevance for improving anti-CD38 therapeutic antibodies through targeting this mechanism and its sequelae., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

Incidence and mortality rates of cancer continue to increase greatly despite the improved diagnostic and therapeutic methods. Based on GLOBOCAN estimates, the numbers of new cancer cases reported in 2018 were ~18.1 million, while the numbers of cancer mortalities were ~9.6 million. It remains difficult to diagnose most cancer patients at early stages. Although cancer therapy market is rapidly evolving, the effectiveness of therapy is still inadequate. Therefore, exploring new biomarkers for diagnosis, prognosis and treatment is essential for cancer management. Long non-coding RNAs (lncRNAs) are unique regulatory molecules that control several cellular processes and are implicated in diverse human diseases including cancer. LncRNAs could serve as potential biomarkers for cancer patients to aid diagnosis and determine prognosis. In addition, numerous lncRNAs have proved their ability to predict response to cancer treatment. FAM83H antisense RNA 1 (FAM83H-AS1) is among those highly dysregulated lncRNAs in cancer. FAM83H-AS1 was demonstrated to participate in the progression of different malignancies and also shown to play a vital role in diagnosis, prognosis and treatment. Here, we analyse recent studies concerning the oncogenic role and molecular mechanisms of lncRNA FAM83H-AS1 in the following cancer types: bladder, breast, lung, hepatocellular, colorectal, gastric, pancreatic, ovarian, cervical cancer as well as glioma., (© 2020 John Wiley & Sons Ltd.)

The aim of this study was to determine the expression of long-chain non-coding RNA (lncRNA) colon cancer-associated transcript 2 (CCAT2) in children with neuroblastoma and its effect on cancer cell growth. A polymerase chain reaction assay was carried out to quantify lncRNA CCAT2 miRNA in neuroblastoma cells, corresponding paracancerous cells, SH-SY5Y and SK-N-SH cells, and human umbilical vein endothelial cells (HUVEC), and two groups of children with different lncRNA CCAT2 expression were compared in clinical pathological parameters and prognosis. CCAT2-NC and si-CCAT2 were transfected into SH-SY5Y cells, separately. Then a 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay was carried out to analyze the cell proliferation, migration, and invasion ability, a flow cytometry to detect cell apoptosis, and a Western blotting (WB) assay to quantify p53 and Bcl-2 proteins. lncRNA CCAT2 expression in cancer tissues of children with neuroblastoma was notably higher than that in corresponding paracancerous tissues (P < 0.05), and children with different tissue differentiation, tumor staging, and lymph node metastasis (LNM) showed notably different lncRNA CCAT2 expression (P < 0.05). In addition, children with neuroblastoma in the high lncRNA CCAT2 expression group showed lower 3-year survival rate than those in the low expression group (P < 0.05). Multivariate analysis revealed that tissue differentiation, tumor-node-metastasis staging, LNM, and lncRNA CCAT2 expression were all independent risk factors affecting the prognosis of children with neuroblastoma (all P < 0.05). Compared with HUVEC cells, SH-SY5Y and SK-N-SH cells showed notably up-regulated lncRNA CCAT2, and the expression of it in SH-SY5Y was higher than that in SK-N-SH cells (P < 0.05). Compared with the CCAT2-NC group, the si-CCAT2 group presented notably down-regulated CCAT2 (P < 0.05). Moreover, according to the MTT assay, the si-CCAT2 group showed notably weakened cell viability and proliferation than the CCAT2-NC group (both P < 0.05), and SH-SY5Y cells in the former group were less active than those in the latter group in terms of migration and invasion. The cell apoptosis rate of SH-SY5Y cells in the si-CCAT2 was higher than that in the CCAT2-NC. The results suggested that knock down of lncRNA CCAT2 could improve the apoptosis activity of neuroblastoma cells in children. According to the WB assay, the si-CCAT2 group showed notably higher p53 expression and notably lower Bcl-2 protein expression than the CCAT2-NC group (both P < 0.05). LncRNA CCAT2 can inhibit the proliferation of neuroblastoma cells and promote their apoptosis, which provides a basis for the treatment of neuroblastoma.

Objectives: Recently, immunotherapy and microRNA have shown much more promises in oncology research, inspiring new hope for a cure for various malignancies. Specifically, the function and mechanisms of action of pembrolizumab have been investigated in many cancers, but not in laryngeal squamous cell carcinoma. The present study thus focused on the effect of hsa-miR-128a on pembrolizumab in laryngeal cancer cells as well as tried to elucidate the mechanisms that may mediate this effect., Methods: Hep2 and AMC-HN8 cell lines were utilized to create stable cell lines that overexpressing hsa-miR-128a. Using the immunotherapy assay, the contribution of hsa-miR-128a to pembrolizumab sensitivity was evaluated. By performing the dual luciferase assay and quantitative real-time polymerase chain reaction, the possible mechanisms of hsa-miR-128a were identified., Results: Hsa-miR-128a was overexpressed in laryngeal cancer cell lines successfully. The immunotherapy assay revealed that upregulating hsa-miR-128a augmented the effect of pembrolizumab. Moreover, hsa-miR-128a targeted BMI-1 and might played a role in the p53 pathway., Conclusion: Hsa-miR-128a boosted the effect of pembrolizumab on laryngeal cancer cells, perhaps via the p53 pathway. Therefore, hsa-miR-128a might be a novel target in laryngeal cancer treatment., Competing Interests: The authors declare that there is no competing interest relevant to the publication of this paper., (Copyright © 2021 Hui Chen et al.)

Technological advances in molecular profiling have enabled the comprehensive identification of common regions of gene amplification on chromosomes (amplicons) in muscle invasive bladder cancer (MIBC). One such region is 8q22.2, which is largely unexplored in MIBC and could harbor genes with potential for outcome prediction or targeted therapy. To investigate the prognostic role of 8q22.2 and to compare different amplicon definitions, an in-silico analysis of 357 patients from The Cancer Genome Atlas, who underwent radical cystectomy for MIBC, was performed. Amplicons were generated using the GISTIC2.0 algorithm for copy number alterations (DNA&#95;Amplicon) and z-score normalization for mRNA gene overexpression (RNA&#95;Amplicon). Kaplan-Meier survival analysis, univariable, and multivariable Cox proportional hazard ratios were used to relate amplicons, genes, and clinical parameters to overall (OS) and disease-free survival (DFS). Analyses of the biological functions of 8q22.2 genes and genomic events in MIBC were performed to identify potential targets. Genes with prognostic significance from the in silico analysis were validated using RT-qPCR of MIBC tumor samples (n = 46). High 8q22.2 mRNA expression (RNA-AMP) was associated with lymph node metastases. Furthermore, 8q22.2 DNA and RNA amplified patients were more likely to show a luminal subtype (DNA&#95;Amplicon&#95;core: p = 0.029; RNA&#95;Amplicon&#95;core: p = 0.01). Overexpression of the 8q22.2 gene OSR2 predicted shortened DFS in univariable (HR [CI] 1.97 [1.2; 3.22]; p = 0.01) and multivariable in silico analysis (HR [CI] 1.91 [1.15; 3.16]; p = 0.01) and decreased OS (HR [CI] 6.25 [1.37; 28.38]; p = 0.0177) in RT-qPCR data analysis. Alterations in different levels of the 8q22.2 region are associated with manifestation of different clinical characteristics in MIBC. An in-depth comprehensive molecular characterization of genomic regions involved in cancer should include multiple genetic levels, such as DNA copy number alterations and mRNA gene expression, and could lead to a better molecular understanding. In this study, OSR2 is identified as a potential biomarker for survival prognosis., Competing Interests: R. M. Wirtz is the CEO and employee of STRATIFYER Molecular Pathology GmbH. The XTRAKT FFPE Kit for RNA Isolation was developed by STRATIFYER and is commercially available on the German market. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Objective: Cholesteatoma is a clinically heterogeneous disease, with some patients showing spontaneous regression, while others experiencing an aggressive, lethal disease. Cholesteatoma in children can be divided into two types: congenital and acquired. Identifying good prognostic markers is needed to help select patients who will require immediate surgical intervention. Matrix metalloproteinase-2 (MMP2) was previously reported to play an important role in cholesteatoma progression, by promoting bone destruction and keratinocyte infiltration. Herein, we analyzed MMP2 mRNA expression level in cholesteatoma using RNA-in situ hybridization in formalin-fixed, paraffin-embedded (FFPE) tissue samples., Methods: Sixty patients with cholesteatoma under 15 years old, who underwent their primary surgery at Aichi Medical University's Otolaryngology Department, were analyzed for MMP2 expression level, using RNA-in situ hybridization., Results: There were no significant differences in MMP2 mRNA expression level between congenital cholesteatoma and acquired cholesteatomas. In congenital cholesteatoma, higher MMP2 signals were observed in the open type than in the closed type ( p < 0.001). In acquired cholesteatoma, higher MMP2 signals were observed in the pars tensa than in the pars flaccida ( p < 0.001). MMP2 mRNA expression level was almost exclusively found in the fibroblasts or in the inflammatory cells in the stroma, but not in the epithelium., Conclusion: Our study reveals that MMP2 mRNA expression level is strongly associated with the subtypes of cholesteatoma. The findings suggest that the level of expression of MMP2 mRNA may be related to the pathogenesis and aggressive features of cholesteatoma., Competing Interests: The authors have no significant relationships with or financial interests in any commercial companies pertaining to this article., (Copyright © 2021 Taichi Kan et al.)

In this study, we identified long non-coding RNAs (lncRNAs) associated with DNA methylation in lung adenocarcinoma (LUAD) using clinical and methylation/expression data from 184 qualified LUAD tissue samples and 21 normal lung-tissue samples from The Cancer Genome Atlas (TCGA). We identified 1865 differentially expressed genes that correlated negatively with the methylation profiles of normal lung tissues, never-smoker LUAD tissues and smoker LUAD tissues, while 1079 differentially expressed lncRNAs were identified using the same criteria. These transcripts were integrated using ingenuity pathway analysis to determine significant pathways directly related to cancer, suggesting that lncRNAs play a crucial role in carcinogenesis. When comparing normal lung tissues and smoker LUAD tissues, 86 candidate genes were identified, including six lncRNAs. Of the 43 candidate genes revealed by comparing never-smoker LUAD tissues and smoker LUAD tissues, 13 were also different when compared to normal lung tissues. We then investigated the expression of these genes using the Gene Expression of Normal and Tumor Tissues (GENT) and Methylation and Expression Database of Normal and Tumor Tissues (MENT) databases. We observed an inverse correlation between the expression of 13 genes in normal lung tissues and smoker LUAD tissues, and the expression of five genes between the never-smoker and smoker LUAD tissues. These findings were further validated in clinical specimens using bisulfite sequencing, revealing that AGR2, AURKB, FOXP3, and HMGA1 displayed borderline differences in methylation. Finally, we explored the functional connections between DNA methylation, lncRNAs, and gene expression to identify possible targets that may contribute toward the pathogenesis of cigarette smoking-associated LUAD. Together, our findings suggested that differentially expressed lncRNAs and their target transcripts could serve as potential biomarkers for LUAD., Competing Interests: The authors have declared that no competing interests exist.

Chemotherapeutic insensitivity is a major obstacle for effective treatment of hepatocellular carcinoma (HCC). Recently, new evidence showed that microRNAs (miRNAs) are closely related to drug sensitivity. This study aimed to investigate the relationship between miR-138 expression and cisplatin sensitivity of HCC cells by regulation of EZH2. CCK-8, EdU, and western blotting are determining the cell viability, proliferation, EZH2, and EMT-related protein expression. It was found that compared with normal samples, miR-138 expression was lower in cancer tissue; it was also downregulated in HCC cells. Transfected with miR-138 mimic increased sensitivity of HCC cells to cisplatin. Mechanistically, Luciferase Reporter analysis verified the interaction between miR-138 and target gene EZH2. Inhibition of EZH2 enhanced cisplatin sensitivity and transfection with EZH2 mimic mirrored the function of miR-138 in cisplatin sensitivity. Furthermore, the role of miR-138 on reversed cisplatin-induced epithelial-mesenchymal transition (EMT) was attenuated when combined with EZH2 plasmid. In conclusion, all data from this study illustrate that miR-138 may as a tumor suppressor provides a potential treatment method to treating HCC., Competing Interests: We declare that we have no conflicts of interest., (Copyright © 2021 Taohui Zeng et al.)

Long non-coding RNAs (lncRNAs) serve a key role in different types of cancer, including colorectal cancer (CRC). The exact roles and mechanisms underlying lncRNA00963 [long intergenic non‑protein coding RNA 963 (LINC00963)] in CRC are not completely understood. The present study aimed to identify the effects and mechanisms underlying LINC00963 in CRC. Firstly, the LINC00963 expression was detected using reverse transcription‑quantitative PCR and the results demonstrated that LINC00963 expression levels were significantly increased in CRC tissues and cell lines compared with healthy tissues and HpoEpiC cells, respectively. Online database analysis indicated that high levels of LINC00963 were associated with low survival rates. The results of functional experiments, such as CCK‑8 assay, colony formation assay, wound healing assay and Transwell invasion assay, indicated that LINC00963 knockdown significantly inhibited CRC cell proliferation, colony formation, migration and invasion compared with the small interfering RNA (si)‑negative control (NC) group. Furthermore, the luciferase reporter indicated that LINC00963 competitively regulated microRNA (miR)‑10b by targeting fibroblast growth factor 13 (FGF13). Compared with si‑NC, LINC00963 knockdown decreased the expression levels of FGF13, vimentin and N‑cadherin, and increased the expression of E‑cadherin as detecting by western blotting. miR‑10b inhibitors partly attenuated si‑LINC00963‑induced inhibition of CRC cell proliferation, migration and invasion. Collectively, the results of the present study suggested a potential role of the LINC00963/miR-10b/FGF13 axis in the tumorigenesis and progression of CRC, indicating a novel lncRNA-based diagnostic or therapeutic target for CRC.

Inflammatory breast cancer (IBC) as a rare and highly aggressive type of breast cancer displays phenotypic characteristics. To date, the IBC-associated molecular mechanisms are entirely unknown. In addition, there is an urgent need to identify the new biomarkers involved in the diagnosis and therapeutic purposes of IBC. MicroRNAs, a category of short noncoding RNAs, are capable of controlling the post-transcriptional expression of genes and thus can act as diagnostic predictive tools. In this review, we addressed the status of oncogenic and tumor suppressor miRNA-mediated IBC in current studies. Furthermore, based on their targets, their involvement in cancer progression, angiogenesis, metastasis, and apoptosis were determined.

Hepatocellular carcinoma (HCC) poses an increasing threat to humans, due to its poor prognosis. Nuclear‑enriched abundant transcript 1 (NEAT1), a type of long non‑coding (lnc)RNA, has been found to function in a variety of cancer types. However, the role of NEAT1 in HCC is poorly understood. Reverse transcription‑quantitative PCR was used to detect the expression levels of NEAT1, microRNA (miR)‑503 and Smoothened (SMO) mRNA in HCC tissues and cells. MTT and flow cytometry assays were used to investigate cell viability and apoptosis, respectively, while Transwell assays were performed to investigate cell invasion and migration. StarBase and TargetScan were utilized to predict the target sequence between miR‑503 and NEAT1 or SMO, the results of which were verified using a dual‑luciferase reporter assay. The protein expression level of SMO was measured using western blot. The RNA expression level of NEAT1 and SMO was significantly elevated in HCC tissues and cells compared with that in the corresponding healthy tissues and cells, which was contrary to miR‑503 expression level. NEAT1 silencing was found to restrict the viability, migration and invasion of the cells, while simultaneously induced apoptosis in the HCC cell line. Further studies found that miR‑503 expression was negatively correlated with NEAT1 or SMO. It was also confirmed that NEAT1 directly interacted with miR‑503 and miR‑503 could bind to the 3'‑untranslated region of SMO. Furthermore, overexpression of NEAT1 or SMO could reverse the effects of miR‑503‑mediated inhibition on cell viability, invasion, migration and promotion of apoptosis in the HCC cell lines. These results demonstrated that downregulation of NEAT1 impeded the viability, migration, invasion and induced apoptosis through the NEAT1/miR‑503/SMO axis in the HCC cell line.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer with fewer treatment options than other types of invasive breast cancer due to the loss of the estrogen, progesterone receptors and low levels of the HER2 protein, resulting in a poor prognosis for these patients. Here, we found that the expression of the lncRNA, ZFAS1, was significantly downregulated (∼3.0-fold) in blood samples of TNBC patients (n=40) compared to matched healthy controls (n=40). Functionally, silencing of ZFAS1 promoted cell proliferation and colonization of human MDA-MB-231 TNBC cells by inhibiting the expression levels of the cyclin-dependent kinase (CDK) inhibitors p21 (CDKN1A) and p27 (CDKN1B) compared to the scrambled siRNA control cells. Further, we found that downregulation of ZFAS1 led to decreased protein levels of the epithelial markers, E-cadherin, Claudin-1, and Zo-1, with increased protein levels of the mesenchymal markers, Slug and ZEB1. In addition, by utilizing the bioinformatic tools such as RAID v2.0 (RNA Interactome Database Version 2.0), AnnoLnc (Annotate human lncRNA database), and GEPIA (Gene Expression Profiling Interactive Analysis), we identified a strong negative correlation between ZFAS1 and signal transducer and activator of transcription 3 (STAT3) gene expression (R = -0.11, p-value = 0.0002). Further, we observed that decreased ZFAS1 expression significantly (p < 0.05) increased STAT3 and phosphorylated STAT3 (at Ser727 residue) protein levels in TNBC cells. The composite data indicate that ZFAS1 may function as a tumor-suppressor lncRNA with potential as a diagnostic/prognostic marker and may offer a new target for the treatment of TNBC patients., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Alternative splicing (AS), a critical post-transcriptional regulatory mechanism, expands gene expression patterns, thereby leading to increased protein diversity. Indeed, more than 95% of human genes undergo alternative splicing events (ASEs). In this study, we drew an all-around AS profile of thyroid cancer cells based on RNA-seq data. In total, there were 45,150 AS in 10,446 thyroid cancer cell genes derived from 506 patients, suggesting that ASEs is a common process in TC. Moreover, 1819 AS signatures were found to be significantly associated with the overall survival (OS) of TC patients. Kaplan-Meier survival analyses suggested that seven types of ASEs were associated with poor prognosis of TC (P < 0.05). Among them, exon skipping (ES) was the most common, with alternate promoter (AP) and alternate terminator (AT) coming second and third, respectively. Our results indicated that acceptor sites (AA) (AUC: 0.937), alternate donor sites (AD) (AUC: 0.965), AT (AUC: 0.964), ES (AUC: 0.999), mutually exclusive exons (ME) (AUC: 0.999), and retained intron (RI) (AUC: 0.837) exhibited an AUC greater than 0.6. In addition, age and risk score (All) were risk factors for TC patients. We also evaluated whether TC-ASEs are regulated by various splicing factors (SFs). We found that the expression of 90 SFs was associated with 469 ASEs and OS of TC patients. Our findings provide an insight into the role of spliceosomes in TC, which may offer novel perspectives in tumor research.

Numerous studies have confirmed that microRNAs (miRNAs or miRs) have important roles in cancer biogenesis and development including multiple myeloma (MM). MicroRNA‑25‑3p (miR‑25‑3p) has been proven to promote cancer progression, whereas its functions in MM has not yet been reported, at least to the best of our knowledge. Therefore, the present study aimed to investigate the function of miR‑25‑3p in MM and to identify the potential underlying mechanistic pathway. Herein, it was found that miR‑25‑3p expression was significantly increased in MM tissues and cell lines. The upregulation of miR‑25‑3p was closely associated with anemia, renal function impairment international staging system (ISS) staging and Durie‑Salmon (D‑S) staging. A high level of miR‑25‑3p was predictive of a poor prognosis of patients with MM. In vitro , the knockdown of miR‑25‑3p suppressed the proliferation and promoted the apoptosis of RPMI‑8226 and U266 cells, while the overexpression of miR‑25‑3p exerted opposite effects. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a well‑known tumor suppressor, was confirmed as a target of miR‑25‑3p in MM cells. Moreover, it was found that the PTEN expression levels were decreased, and inversely correlated with miR‑25‑3p expression levels in MM tissues. Further analyses revealed that the overexpression of PTEN exerted effects similar to those of miR‑25‑3p knockdown, whereas the knockdown of PTEN partially abolished the effects of miR‑25‑3p inhibitor on MM cells. Accompanied by PTEN induction, miR‑25‑3p promoted PI3K/AKT signaling pathway activation in MM cells. Collectively, these findings demonstrate critical roles for miR‑25‑3p in the pathogenesis of MM, and suggest that miR‑25‑3p may serve as a novel prognostic biomarker and therapeutic target of MM.

Inhalational anaesthetics were previously reported to promote ovarian cancer malignancy, but underlying mechanisms remain unclear. The present study aims to investigate the role of sevoflurane- or desflurane-induced microRNA (miRNA) changes on ovarian cancer cell behaviour. The cultured SKOV3 cells were exposed to 3.6% sevoflurane or 10.3% desflurane for 2 h. Expression of miR-138, -210 and -335 was determined with qRT-PCR. Cell proliferation and migration were assessed with wound healing assay, Ki67 staining and Cell Counting Kit-8 (CCK8) assay with or without mimic miR-138/-210 transfections. The miRNA downstream effector, hypoxia inducible factor-1α (HIF-1α), was also analysed with immunofluorescent staining. Sevoflurane or desflurane exposure to cancer cells enhanced their proliferation and migration. miR-138 expression was suppressed by both sevoflurane and desflurane, while miR-210 expression was suppressed only by sevoflurane. miR-335 expression was not changed by either sevoflurane or desflurane exposure. The administration of mimic miR-138 or -210 reduced the promoting effects of sevoflurane and desflurane on cancer cell proliferation and migration, in line with the HIF-1α expression changes. These data indicated that inhalational agents sevoflurane and desflurane enhanced ovarian cancer cell malignancy via miRNA deactivation and HIF-1α. The translational value of this work needs further study.

MicroRNAs (miRNAs) are attractive therapeutic targets and promising candidates as molecular biomarkers for various therapy-resistant tumors. However, the association between miRNAs and drug resistance in melanoma remains to be elucidated. We used an integrative genomic analysis to comprehensively study the miRNA expression profiles of drug-resistant melanoma patients and cell lines. MicroRNA-181a and -181b (miR181a/b) were identified as the most significantly down-regulated miRNAs in resistant melanoma patients and cell lines. Re-establishment of miR-181a/b expression reverses the resistance of melanoma cells to the BRAF inhibitor dabrafenib. Introduction of miR-181 mimics markedly decreases the expression of TFAM in A375 melanoma cells resistant to BRAF inhibitors. Furthermore, melanoma growth was inhibited in A375 and M14 resistant melanoma cells transfected with miR-181a/b mimics, while miR-181a/b depletion enhanced resistance in sensitive cell lines. Collectively, our study demonstrated that miR-181a/b could reverse the resistance to BRAF inhibitors in dabrafenib resistant melanoma cell lines. In addition, miR-181a and -181b are strongly down-regulated in tumor samples from patients before and after the development of resistance to targeted therapies. Finally, melanoma tissues with high miR-181a and -181b expression presented favorable outcomes in terms of Progression Free Survival, suggesting that miR-181 is a clinically relevant candidate for therapeutic development or biomarker-based therapy selection.

Nasopharyngeal carcinoma (NPC) is a kind of malignancy generated from the nasopharyngeal epithelium. Recently, long noncoding RNA (lncRNA) has been shown to be involved in the regulation of many signaling pathways and is closely associated with carcinogenesis and tumor progression. However, the precise role of lncRNA Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) in NPC is not well understood. Here, we find that OIP5-AS1 is overexpressed in NPC patient specimens and NPC cell lines. Further investigations reveal that knockdown of OIP5-AS1 significantly inhibits the proliferation, migration, and invasion and accelerates the apoptosis of NPC cells in vitro. Consistent with these findings, NPC progression is significantly slowed in mice when OIP5-AS1 is knocked down. Interestingly, there is a functional link between OIP5-AS1 and microRNA-203 (miR-203), a tumor suppressor, in NPC cells. In conclusion, our data demonstrate that OIP5-AS1 plays an important role in the development and progression of NPC by targeting miR-203 and therefore provide a promising target for the treatment of NPC., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2021 Jian Tang et al.)

MET inhibitors have shown activity in non-small-cell lung cancer patients (NSCLC) with MET amplification and exon 14 skipping (METΔex14). However, patient stratification is imperfect, and thus, response rates have varied widely. Here, we studied MET alterations in 474 advanced NSCLC patients by nCounter, an RNA-based technique, together with next-generation sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR), exploring correlation with clinical benefit. Of the 474 samples analyzed, 422 (89%) yielded valid results by nCounter, which identified 13 patients (3%) with METΔex14 and 15 patients (3.5%) with very-high MET mRNA expression. These two subgroups were mutually exclusive, displayed distinct phenotypes and did not generally coexist with other drivers. For METΔex14, 3/8 (37.5%) samples positive by nCounter tested negative by NGS. Regarding patients with very-high MET mRNA, 92% had MET amplification by FISH and/or NGS. However, FISH failed to identify three patients (30%) with very-high MET RNA expression, among which one received MET tyrosine kinase inhibitor treatment deriving clinical benefit. Our results indicate that quantitative mRNA-based techniques can improve the selection of patients for MET-targeted therapies., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Increasing evidence has demonstrated the important roles of long non‑coding (lnc) RNA in non‑small cell lung cancer (NSCLC). lncRNA gastric cancer‑associated transcript 1 (GACAT1) has been reported to play an oncogenic role in different types of cancer; however, the function of GACAT1 in NSCLC remains unclear. The present study found that GACAT1 was overexpressed in NSCLC tissues and was associated with poor outcomes in patients with NSCLC. Functional experiments revealed that GACAT1 downregulation inhibited proliferation, induced apoptosis and cell cycle arrest of 2 NSCLC cell lines. GACAT1 was found to target microRNA(miR)‑422a mechanically and negatively regulated miR‑422a expression. Reduced expression of miR‑422a in NSCLC tissues was inversely correlated with that of GACAT1. Furthermore, YY1 transcription factor (YY1) was identified as a downstream miR‑422a target. Reduced expression of GACAT1 inactivated YY1 by sponging miR‑422a in NSCLC cells. YY1 reintroduction reversed the reduced proliferation of NSCLC cells via GACAT1 knockdown. Taken together, these results revealed the novel role of the GACAT1/miR‑422a pathway in the progression of NSCLC cell lines, providing a possible therapeutic strategy for NSCLC treatment.

Natural products remain a significant source of anticancer chemotherapeutics. The search for targeted drugs for cancer treatment includes consideration of natural products, which may provide new opportunities for antitumor cytotoxicity as single agents or in combination therapy. We examined the association of molecular genomic features in the well-characterized NCI-60 cancer cell line panel with in vitro response to treatment with 1302 small molecules which included natural products, semisynthetic natural product derivatives, and synthetic compounds based on a natural product pharmacophore from the Developmental Therapeutics Program of the US National Cancer Institute's database. These compounds were obtained from a variety of plant, marine, and microbial species. Molecular information utilized for the analysis included expression measures for 23059 annotated transcripts, lncRNAs, and miRNAs, and data on protein-changing single nucleotide variants in 211 cancer-related genes. We found associations of expression of multiple genes including SLFN11, CYP2J2, EPHX1, GPC1, ELF3, and MGMT involved in DNA damage repair, NOTCH family members, ABC and SLC transporters, and both mutations in tyrosine kinases and BRAF V600E with NCI-60 responses to specific categories of natural products. Hierarchical clustering identified groups of natural products, which correlated with a specific mechanism of action. Specifically, several natural product clusters were associated with SLFN11 gene expression, suggesting that potential action of these compounds may involve DNA damage. The associations between gene expression or genome alterations of functionally relevant genes with the response of cancer cells to natural products provide new information about potential mechanisms of action of these identified clusters of compounds with potentially similar biological effects. This information will assist in future drug discovery and in design of new targeted cancer chemotherapy agents., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Persistent mortality rates of medulloblastoma (MB) and severe side effects of the current therapies require the definition of the molecular mechanisms that contribute to tumor progression. Using cultured MB cancer stem cells and xenograft tumors generated in mice, we show that low expression of miR-326 and its host gene β-arrestin1 (ARRB1) promotes tumor growth enhancing the E2F1 pro-survival function. Our models revealed that miR-326 and ARRB1 are controlled by a bivalent domain, since the H3K27me3 repressive mark is found at their regulatory region together with the activation-associated H3K4me3 mark. High levels of EZH2, a feature of MB, are responsible for the presence of H3K27me3. Ectopic expression of miR-326 and ARRB1 provides hints into how their low levels regulate E2F1 activity. MiR-326 targets E2F1 mRNA, thereby reducing its protein levels; ARRB1, triggering E2F1 acetylation, reverses its function into pro-apoptotic activity. Similar to miR-326 and ARRB1 overexpression, we also show that EZH2 inhibition restores miR-326/ARRB1 expression, limiting E2F1 pro-proliferative activity. Our results reveal a new regulatory molecular axis critical for MB progression., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)