Search

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.

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.

Circular (circ) RNAs are differentially expressed in gastric cancer (GC) and participate in the biological growth of tumor cells. Given that, investigations were performed to unravel the function of circ_0000654 in GC. GC tissue and normal tissue specimens were obtained, in which circ_0000654, microRNA (miR)-149-5p, and inhibin-beta A (INHBA) levels were examined. GC cell line (BGC-823) was transfected to alter circ_0000654 and miR-149-5p expression, thereby observing cell malignancy. Stably-transfected BGC-823 cells were injected into nude mice to observe tumor growth in vivo . The interaction circ_0000654, miR-149-5p, and INHBA was validated. circ_0000654 and INHBA were up-regulated but miR-149-5p was down-regulated in GC. circ_0000654 absorbed miR-149-5p to target INHBA. Silencing circ_0000654inhibited the progress of GC cell biology. Oppositely, restoring circ_0000654 enhanced the growth of GC cells. Inhibiting miR-149-5p rescued down-regulated circ_0000654-induced anti-tumor effect on GC. circ_0000654 silence or miR-149-5p overexpression limited the growth of GC tumors in vivo . Obviously, circ_0000654 facilitates the growth of GC cells through absorbing miR-149-5p to up-regulate INHBA.

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: Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. Chemotherapy, the treatment of choice in non-operable cases, achieves a dismal success rate, raising the need for new therapeutic options. In about 25% of NSCLC, the activating mutations of the KRAS oncogene define a subclass that cannot benefit from tyrosine kinase inhibitors (TKIs). The tumor suppressor miR-16 is downregulated in many human cancers, including NSCLC. The main objectives of this study were to evaluate miR-16 treatment to restore the TKI sensitivity and compare its efficacy to MEK inhibitors in KRAS -mutated NSCLC., Methods: We performed in vitro and in vivo studies to investigate whether miR-16 could be exploited to overcome TKI resistance in KRAS-mutated NSCLC. We had three goals: first, to identify the KRAS downstream effectors targeted by mir-16, second, to study the effects of miR-16 restoration on TKI resistance in KRAS-mutated NSCLC both in vitro and in vivo, and finally, to compare miR-16 and the MEK inhibitor selumetinib in reducing KRAS-mutated NSCLC growth in vitro and in vivo., Results: We demonstrated that miR-16 directly targets the three KRAS downstream effectors MAPK3 , MAP2K1 , and CRAF in NSCLC, restoring the sensitivity to erlotinib in KRAS -mutated NSCLC both in vitro and in vivo. We also provided evidence that the miR-16-erlotinib regimen is more effective than the selumetinib-erlotinib combination in KRAS -mutated NSCLC., Conclusions: Our findings support the biological preclinical rationale for using miR-16 in combination with erlotinib in the treatment of NSCLC with KRAS -activating mutations.

The complicity of human RAS proteins in cancer is a well-documented fact, both due to the mutational hyperactivation of these GTPases and the overexpression of the genes encoding these proteins. Thus, it can be easily assumed that the study of RAS genes at the transcriptional and post-transcriptional level is of the utmost importance. Although previous research has shed some light on the basic mechanisms by which GTPases are involved in tumorigenesis, limited information is known regarding the transcriptional profile of the genes encoding these proteins. The present study highlights for the first time the wide spectrum of the mRNAs generated by the three most significant RAS genes ( KRAS , NRAS and HRAS ), providing an in-depth analysis of the splicing events and exon/intron boundaries. The implementation of a versatile, targeted nanopore-sequencing approach led to the identification of 39 novel RAS mRNA transcript variants and to the elucidation of their expression profiles in a broad panel of human cell lines. Although the present work unveiled multiple hidden aspects of the RAS gene family, further study is required to unravel the biological function of all the novel alternative transcript variants, as well as the putative protein isoforms.

Objective: To investigate the characteristics of the tumor immune microenvironment in patients with gastrointestinal stromal tumor (GIST) and identify cancer stem-like properties of GIST to screen potential druggable molecular targets., Methods: The gene expression data of 60 patients with GIST was retrieved from the Array Express database. CIBERSORT was applied to calculate the level of immune infiltration. ssGSEA and ESTIMATE were used to calculate the cancer stemness index and tissue purity. The Connectivity Map (CMAP) database was implemented to screen targeted drugs based on cancer stem-like properties of GIST., Result: There was a difference in the level of immune infiltration between the metastasis and non-metastasis GIST groups. The low level of T-cell infiltration was correlated with high tumor purity and tumor stemness index, and the correlation coefficients were -0.87 and -0.61 (p < 0.001), respectively. Furthermore, there was a positive correlation between cancer stemness index and cell purity (p < 0.001). The cancer stemness index in the metastasis group was higher than that in the non-metastasis group (p = 0.0017). After adjusting for tumor purity, there was no significant correlation between T-cell infiltration and cancer stemness index (p = 0.086). Through the pharmacological mechanism of topoisomerase inhibitors, six molecular complexes may be the targets of GIST treatment., Conclusion: Immune infiltration in GIST patients is related to cancer stem-like properties, and the correlation relies on tumor purity. Cancer stemness index can be used as a new predictive biomarker of tumor metastasis and targets of drug therapy for GIST patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Ren, Wu, Zeng, Chen, Han, Lin, Zhang, He and Li.)

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.)

Long non-coding RNAs (lncRNAs) in immune cells play critical roles in tumor cell-immune cell interactions. This study aimed to characterize the landscape of tumor-infiltrating immune-related lncRNAs (Ti-lncRNAs) and reveal their correlations with prognoses and immunotherapy response in head and neck squamous cell carcinoma (HNSCC). We developed a computational model to identify Ti-lncRNAs in HNSCC and analyzed their associations with clinicopathological features, molecular alterations, and immunotherapy response. A signature of nine Ti-lncRNAs demonstrated an independent prognostic factor for both overall survival and disease-free survival among the cohorts from Fudan University Shanghai Cancer Center, The Cancer Genome Atlas, GSE41613, and GSE42743. The Ti-lncRNA signature scores in immune cells showed significant associations with TP53 mutation, CDKN2A mutation, and hypoxia. Inferior signature scores were enriched in patients with high levels of PDCD1 and CTLA4 and high expanded immune gene signature (IGS) scores, who displayed good response to PD-1 blockade in HNSCC. Consistently, superior clinical response emerged in melanoma patients with low signature scores undergoing anti-PD-1 therapy. Moreover, the Ti-lncRNA signature was a prognostic factor independent of PDCD1, CTLA4, and the expanded IGS score. In conclusion, tumor-infiltrating immune profiling identified a prognostic Ti-lncRNA signature indicative of clinical response to PD-1 blockade in HNSCC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer LT declared a shared affiliation with the authors to the handling editor at the time of the review., (Copyright © 2021 Ma, Jiang, Luo, Liao, Xu, Wang, Dong, Ji and Wang.)

B7 homolog 3 (B7-H3) is a recently found superfamily B7 molecule and therefore has significant involvement in immunological regulation. However, the relationships of B7-H3 expression with the tumor microenvironment (TME), response to immunotherapy, and prognosis in head and neck squamous cell carcinoma (HNSCC) are still unknown. In the present analysis, we determined B7-H3 as a novel biomarker that predicts the prognosis and response to immunotherapy in HNSCC. B7-H3 expression is enhanced in HNSCC compared to normal sample and is stably expressed in HNSCC cell line. Besides, high B7-H3 expression is correlated with a dismal prognosis and resistance to immunotherapy and contributes to an immunosuppressive microenvironment. Moreover, single-cell RNA sequencing (scRNA-seq) analysis shows that B7-H3 is mainly expressed in the stromal as well as malignant cells. In conclusion, the study provides insight in understanding the prognostic value of B7-H3 in HNSCC and highlights its involvement in promoting the immunosuppressive microenvironment, which presents an attractive strategy for antibody-based immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lin, Xu, Gao, Zhang, Sun, Qiu, Huang, Kong and Lu.)

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.)

Radiation therapy is generally effective for treating breast cancers. However, approximately 30% of patients with breast cancer experience occasional post-treatment local and distant metastasis. Low-dose (0.5 Gy) irradiation is a risk factor that promotes the invasiveness of breast cancers. Although an inhibitor of checkpoint kinase 1 (Chk1) suppresses the growth and motility of breast cancer cell lines, no study has investigated the effects of the combined use of a Chk1 inhibitor and radiation on cancer metastasis. Here, we addressed this question by treating the human breast cancer cell line MDA-MB-231 (in vitro) and mouse mammary tumor cell line 4 T1 (in vitro and in vivo) with γ-irradiation and the Chk1 inhibitor PD407824. Low-dose γ-irradiation promoted invasiveness, which was suppressed by PD407824. Comprehensive gene expression analysis revealed that low-dose γ-irradiation upregulated the mRNA and protein levels of S100A4, the both of which were downregulated by PD407824. We conclude that PD407824 suppresses the expression of S100A4. As the result, γ-irradiation-induced cell invasiveness were inhibited., (© The Author(s) 2021. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

Altered metabolism is an emerging hallmark of cancer. Cancer cells preferentially utilize glycolysis for energy production, termed "aerobic glycolysis." In this study, we performed a comprehensive analysis of the glycolytic activity in head and neck squamous cell carcinoma (HNSCC) using data obtained from The Cancer Genome Atlas database. We first divided 520 patients with HNSCC into four groups based on the mRNA expression of 16 glycolysis-related genes. The upregulated glycolytic activity positively correlated with human papillomavirus-negative tumor type, advanced T factor, and unfavorable prognosis. The gene set enrichment analysis revealed upregulation of several hallmark pathways, including interferon-alpha response, myc targets, unfolded protein response, transforming growth factor-β signaling, cholesterol homeostasis, and interleukin 6-Janus kinase-signal transducer and activator of transcription 3 signaling, in the glycolysis-upregulated groups. Immune cell enrichment analysis revealed decreased infiltration of T cells, dendritic cells, and B cells in the glycolysis-upregulated groups, suggesting impaired tumor antigen presentation, T cell activation, and antibody production in the TME. Moreover, the expression profile of immune-related genes indicated increased immune evasion in the glycolysis-upregulated tumors. Collectively, these findings suggest that transcriptome analysis of glycolytic activity of tumors has the potential as a biomarker for tumor progression and immunological status in patients with HNSCC., (© 2021. The Author(s).)

Lineage tracing in mice indicates that LGR5 is an adult stem cell marker in multiple organs, such as the intestine, stomach, hair follicles, ovary, and mammary glands. Despite many studies exploring the presence of LGR5 cells in human tissues, little is known about its expression profile in either human mammary tissue or pathological lesions. In this study we aim to investigate LGR5 expression in normal, benign, and malignant lesions of the human breast using RNA in situ hybridization. LGR5 expression has not been observed in normal lactiferous ducts and terminal duct lobular units, whereas LGR5-positive cells have been specifically observed in the basal myoepithelium of ducts in the regenerative tissues, ductal carcinoma in situ, and in ducts surrounded by invasive cancer cells. These findings suggest LGR5 marks facultative stem cells that are involved in post injury regeneration instead of homeostatic stem cells. LGR5 positivity was found in 3% (9 of 278 cases) of invasive breast cancers (BC), and it showed positive associations with higher histologic grades (P = 0.001) and T stages (P < 0.001), while having negative correlations with estrogen receptor (P < 0.001) and progesterone receptor (P < 0.001) expression. Remarkably, all LGR5-positive BC, except one, belong to triple-negative BC (TNBC), representing 24% (9 of 38 cases) of all of them. LGR5 histoscores have no correlations with EGFR, CK5/6, Ki-67, or P53 expression. Additionally, no β-catenin nuclear localization was observed in LGR5-positive BC, indicating that canonical Wnt pathway activation is less likely involved in LGR5 expression in BC. Our results demonstrate that LGR5 expression is induced in regenerative conditions in the myoepithelium of human mammary ducts and that its expression is only observed in TNBC subtype among all invasive BC. Further studies regarding the functional and prognostic impact of LGR5 in TNBC are warranted., (© 2021. The Author(s).)

Ameloblastomas are odontogenic tumors that are rare in people but have a relatively high prevalence in dogs. Because canine acanthomatous ameloblastomas (CAA) have clinicopathologic and molecular features in common with human ameloblastomas (AM), spontaneous CAA can serve as a useful translational model of disease. However, the molecular basis of CAA and how it compares to AM are incompletely understood. In this study, we compared the global genomic expression profile of CAA with AM and evaluated its dental origin by using a bulk RNA-seq approach. For these studies, healthy gingiva and canine oral squamous cell carcinoma served as controls. We found that aberrant RAS signaling, and activation of the epithelial-to-mesenchymal transition cellular program are involved in the pathogenesis of CAA, and that CAA is enriched with genes known to be upregulated in AM including those expressed during the early stages of tooth development, suggesting a high level of molecular homology. These results support the model that domestic dogs with spontaneous CAA have potential for pre-clinical assessment of targeted therapeutic modalities against AM., (© 2021. The Author(s).)

Mutations in genes encoding RNA splicing factors were discovered nearly 10 years ago and are now understood to be among the most recurrent genetic abnormalities in patients with all forms of myeloid neoplasms and several types of lymphoproliferative disorders, as well as subjects with clonal hematopoiesis. These discoveries implicate aberrant RNA splicing, the process by which precursor RNA is converted into mature messenger RNA, in the development of clonal hematopoietic conditions. Both the protein and the RNA components of the splicing machinery are affected by mutations at highly specific residues, and a number of these mutations alter splicing in a manner distinct from loss of function. Importantly, cells bearing these mutations have now been shown to generate mRNA species with novel aberrant sequences, some of which may be critical to disease pathogenesis and/or novel targets for therapy. These findings have opened new avenues of research to understand biological pathways disrupted by altered splicing. In parallel, multiple studies have revealed that cells bearing change-of-function mutation in splicing factors are preferentially sensitized to any further genetic or chemical perturbations of the splicing machinery. These discoveries are now being pursued in several early-phase clinical trials using molecules with diverse mechanisms of action. Here, we review the molecular effects of splicing factor mutations on splicing, the mechanisms by which these mutations drive clonal transformation of hematopoietic cells, and the development of new therapeutics targeting these genetic subsets of hematopoietic malignancies., (© 2021 by The American Society of Hematology.)

The main types of thyroid neoplasms, follicular adenoma (FA), follicular thyroid carcinoma (FTC), classical and follicular variants of papillary carcinoma (clPTC and fvPTC), and anaplastic thyroid carcinoma (ATC), differ in prognosis, progression rate and metastatic behaviour. Specific patterns of lncRNAs involved in the development of clinical and morphological features can be presumed. LncRNA landscapes within distinct benign and malignant histological variants of thyroid neoplasms were not investigated. The aim of the study was to discover long noncoding RNA landscapes common and specific to major benign and malignant histological subtypes of thyroid neoplasms. LncRNA expression in FA, FTC, fvPTC, clPTC and ATC was analysed with comprehensive microarray and RNA-Seq datasets. Putative biological functions were evaluated via enrichment analysis of coexpressed coding genes. In the results, lncRNAs common and specific to FTC, clPTC, fvPTC, and ATC were identified. The discovered lncRNAs are putatively involved in L1CAM interactions, namely, pre-mRNA processing (lncRNAs specific to FTC); PCP/CE and WNT pathways (lncRNAs specific to fvPTC); extracellular matrix organization (lncRNAs specific to clPTC); and the cell cycle (lncRNAs specific to ATC). Known oncogenic and suppressor lncRNAs (RMST, CRNDE, SLC26A4-AS1, NR2F1-AS1, and LINC00511) were aberrantly expressed in thyroid carcinomas. These findings enhance the understanding of lncRNAs in the development of subtype-specific features in thyroid cancer., (© 2021. The Author(s).)

Thyroid cancer is the most common endocrine malignancy, and the characterization of the genetic alterations in coding-genes that drive thyroid cancer are well consolidated in MAPK signaling. In the context of non-coding RNAs, microRNAs (miRNAs) are small non-coding RNAs that, when deregulated, cooperate to promote tumorigenesis by targeting mRNAs, many of which are proto-oncogenes and tumor suppressors. In thyroid cancer, miR-146b-5p is the most overexpressed miRNA associated with tumor aggressiveness and progression, while the antisense blocking of miR-146b-5p results in anti-tumoral effect. Therefore, inactivating miR-146b has been considered as a promising strategy in thyroid cancer therapy. Here, we applied the CRISPR/Cas9n editing system to target the MIR146B gene in an aggressive anaplastic thyroid cancer (ATC) cell line. For that, we designed two single-guide RNAs cloned into plasmids to direct Cas9 nickase (Cas9n) to the genomic region of the pre-mir-146b structure to target miR-146b - 5p and miR-146b - 3p sequences. In this plasmidial strategy, we cotransfected pSp-Cas9n- miR-146b -GuideA-puromycin and pSp-Cas9n- miR-146b -GuideB-GFP plasmids in KTC2 cells and selected the puromycin resistant + GFP positive clones (KTC2-Cl). As a result, we observed that the ATC cell line KTC2-Cl1 showed a 60% decrease in the expression of miR-146b-5p compared to the control, also showing reduced cell viability, migration, colony formation, and blockage of tumor development in immunocompromised mice. The analysis of the MIR146B edited sequence shows a 5 nt deletion in the miR-146b-5p region and a 1 nt deletion in the miR-146b-3p region in KTC2-Cl1. Thus, we developed an effective CRISPR/Cas9n system to edit the MIR146B miRNA gene and reduce miR-146b-5p expression which constitutes a potential molecular tool for the investigation of miRNAs function in thyroid cancer.

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.)

Cancer is a complex disease involving alterations of multiple processes, with both genetic and epigenetic features contributing as core factors to the disease. In recent years, it has become evident that non-coding RNAs (ncRNAs), an epigenetic factor, play a key role in the initiation and progression of cancer. MicroRNAs, the most studied non-coding RNAs subtype, are key controllers in a myriad of cellular processes, including proliferation, differentiation, and apoptosis. Furthermore, the expression of miRNAs is controlled, concomitantly, by other epigenetic factors, such as DNA methylation and histone modifications, resulting in aberrant patterns of expression upon the occurrence of cancer. In this sense, aberrant miRNA landscape evaluation has emerged as a promising strategy for cancer management. In this review, we have focused on the regulation (biogenesis, processing, and dysregulation) of miRNAs and their role as modulators of the epigenetic machinery. We have also highlighted their potential clinical value, such as validated diagnostic and prognostic biomarkers, and their relevant role as chromatin modifiers in cancer therapy.

Hypoxia and hyperthermia, which can be induced by high environmental temperature or strenuous exercise, are two common stressors that affect intestinal epithelial integrity and lead to multiple clinical symptoms. In this study, we developed an in-vitro intestinal monolayer model using two human colonic epithelial cell lines, Caco-2 and HT-29, co-cultured in Transwell inserts, and investigated the effects of heat treatment and/or hypoxia on the epithelial barrier function. The monolayer with a ratio of 9:1 (Caco-2:HT-29) showed high trans-epithelial electrical resistance (TEER), low Lucifer Yellow permeability and high mucin production. Hyperthermia and/or hypoxia exposure (2 h) triggered heat shock and oxidative stress responses. HSP-70 and HSF-1 protein levels were up-regulated by hyperthermia, which were further enhanced when hyperthermia was combined with hypoxia. Increased HIF-1α protein expression and Nrf2 nuclear translocation was only caused by hypoxia. Hyperthermia and/or hypoxia exposure disrupted the established monolayer by increasing paracellular permeability, decreasing ZO-1, claudin-3 and occludin protein/mRNA expression, while enhancing E-cadherin protein expression. Tight junction protein distribution in the monolayer was also modulated by the hyperthermia and/or hypoxia exposure. In addition, transcription levels of mucin genes, MUC-2 and MUC-5AC, were increased after 2 h of hyperthermia and/or hypoxia exposure. In conclusion, this Caco-2/HT-29 cell model is valid and effective for studying detrimental effects of hyperthermia and/or hypoxia on intestinal barrier function and related heat shock and oxidative stress pathways and can be used to investigate possible interventions to reverse hyperthermia and/or hypoxia-induced intestinal epithelial injury.

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.

Tumor progression and metastatic dissemination are driven by cell-intrinsic and biomechanical cues that favor the growth of life-threatening secondary tumors. We recently identified pro-metastatic vascular regions with blood flow profiles that are permissive for the arrest of circulating tumor cells. We have further established that such flow profiles also control endothelial remodeling, which favors extravasation of arrested CTCs. Yet, how shear forces control endothelial remodeling is unknown. In the present work, we aimed at dissecting the cellular and molecular mechanisms driving blood flow-dependent endothelial remodeling. Transcriptomic analysis of endothelial cells revealed that blood flow enhanced VEGFR signaling, among others. Using a combination of in vitro microfluidics and intravital imaging in zebrafish embryos, we now demonstrate that the early flow-driven endothelial response can be prevented upon specific inhibition of VEGFR tyrosine kinase and subsequent signaling. Inhibitory targeting of VEGFRs reduced endothelial remodeling and subsequent metastatic extravasation. These results confirm the importance of VEGFR-dependent endothelial remodeling as a driving force of CTC extravasation and metastatic dissemination. Furthermore, the present work suggests that therapies targeting endothelial remodeling might be a relevant clinical strategy in order to impede metastatic progression.

miR-365 is a microRNA that regulates transcription and has been demonstrated to promote oncogenesis and metastasis in some cancers while suppressing these effects in others. Virtually no information is known about the presence or function of miR-365 in oral cancers. Based upon this information, the primary goal of this project was to evaluate the expression of miR-365 in existing oral cancer cell lines. Five commercially available oral cancer cell lines (SCC4, SCC9, SCC15, SCC25, and CAL27) were obtained and cultured. RNA was then screened by PCR using primers specific for miR-365, as well as matrix metalloproteinase (MMP-2) and a downstream cancer stem cell regulator (NKX2.1), and structural and metabolic standards (beta actin, GAPDH). miR-365 was detected among these oral cancers, and some cells also expressed NKX2.1 and MMP-2, which correlated with miR-365 levels. The relative expression of miR-365, NKX2.1, and MMP-2 RNA was higher than expected. Transfection of miR-365 resulted in a significant increase in proliferation, which was not observed in the negative controls. These data appear to confirm miR-365 expression in oral cancers, which may also be correlated with MMP-2 and NKX2.1 expression. Moreover, the fastest growing oral cancers with the highest viability produced the most miR-365. In addition, miR-365 transfected into cells significantly increased growth, even in normal cells. More research is needed to elucidate the pathways responsible for these observations.

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.

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.)

Aberrant B-cell receptor/NF-κB signaling is a hallmark feature of B-cell non-Hodgkin lymphomas, especially in diffuse large B-cell lymphoma (DLBCL). Recurrent mutations in this cascade, for example, in CD79B, CARD11, or NFKBIZ, and also in the Toll-like receptor pathway transducer MyD88, all deregulate NF-κB, but their differential impact on lymphoma development and biology remains to be determined. Here, we functionally investigate primary mouse lymphomas that formed in recipient mice of Eµ-myc transgenic hematopoietic stem cells stably transduced with naturally occurring NF-κB mutants. Although most mutants supported Myc-driven lymphoma formation through repressed apoptosis, CARD11- or MyD88-mutant lymphoma cells selectively presented with a macrophage-activating secretion profile, which, in turn, strongly enforced transforming growth factor β (TGF-β)-mediated senescence in the lymphoma cell compartment. However, MyD88- or CARD11-mutant Eµ-myc lymphomas exhibited high-level expression of the immune-checkpoint mediator programmed cell death ligand 1 (PD-L1), thus preventing their efficient clearance by adaptive host immunity. Conversely, these mutant-specific dependencies were therapeutically exploitable by anti-programmed cell death 1 checkpoint blockade, leading to direct T-cell-mediated lysis of predominantly but not exclusively senescent lymphoma cells. Importantly, mouse-based mutant MyD88- and CARD11-derived signatures marked DLBCL subgroups exhibiting mirroring phenotypes with respect to the triad of senescence induction, macrophage attraction, and evasion of cytotoxic T-cell immunity. Complementing genomic subclassification approaches, our functional, cross-species investigation unveils pathogenic principles and therapeutic vulnerabilities applicable to and testable in human DLBCL subsets that may inform future personalized treatment strategies., (© 2021 by The American Society of Hematology.)

MicroRNAs (miRNAs) play crucial roles in multifarious biological processes associated with human diseases. Identifying potential miRNA-disease associations contributes to understanding the molecular mechanisms of miRNA-related diseases. Most of the existing computational methods mainly focus on predicting whether a miRNA-disease association exists or not. However, the roles of miRNAs in diseases are prominently diverged, for instance, Genetic variants of miRNA (mir-15) may affect the expression level of miRNAs leading to B cell chronic lymphocytic leukemia, while circulating miRNAs (including mir-1246, mir-1307-3p, etc.) have potentials to detecting breast cancer in the early stage. In this paper, we aim to predict multi-type miRNA-disease associations instead of taking them as binary. To this end, we innovatively represent miRNA-disease-type triples as a tensor and introduce tensor decomposition methods to solve the prediction task. Experimental results on two widely-adopted miRNA-disease datasets: HMDD v2.0 and HMDD v3.2 show that tensor decomposition methods improve a recent baseline in a large scale (up to $38\%$ in Top-1F1). We then propose a novel method, Tensor Decomposition with Relational Constraints (TDRC), which incorporates biological features as relational constraints to further the existing tensor decomposition methods. Compared with two existing tensor decomposition methods, TDRC can produce better performance while being more efficient., (© 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.)

Axicabtagene ciloleucel (axi-cel) is a chimeric antigen receptor (CAR) T-cell therapy for relapsed or refractory large B-cell lymphoma (LBCL). This study evaluated whether immune dysregulation, present before CAR T-cell therapy, was associated with treatment failure. Tumor expression of interferon (IFN) signaling, high blood levels of monocytic myeloid-derived suppressor cells (M-MDSCs), and high blood interleukin-6 and ferritin levels were each associated with a lack of durable response. Similar to other cancers, we found that in LBCL tumors, IFN signaling is associated with the expression of multiple checkpoint ligands, including programmed cell death-ligand 1, and these were higher in patients who lacked durable responses to CAR-T therapy. Moreover, tumor IFN signaling and blood M-MDSCs associated with decreased axi-cel expansion. Finally, patients with high tumor burden had higher immune dysregulation with increased serum inflammatory markers and tumor IFN signaling. These data support that immune dysregulation in LBCL promotes axi-cel resistance via multiple mechanistic programs: insufficient axi-cel expansion associated with both circulating M-MDSC and tumor IFN signaling, which also gives rise to expression of immune checkpoint ligands., (© 2021 by The American Society of Hematology.)

Molecular classification of acute myeloid leukemia (AML) aids prognostic stratification and clinical management. Our aim in this study is to identify transcriptome-wide mRNAs that are specific to each of the molecular subtypes of AML. We analyzed RNA-sequencing data of 955 AML samples from three cohorts, including the BeatAML project, the Cancer Genome Atlas, and a cohort of Swedish patients to provide a comprehensive transcriptome-wide view of subtype-specific mRNA expression. We identified 729 subtype-specific mRNAs, discovered in the BeatAML project and validated in the other two cohorts. Using unique proteomics data, we also validated the presence of subtype-specific mRNAs at the protein level, yielding a rich collection of potential protein-based biomarkers for the AML community. To enable the exploration of subtype-specific mRNA expression by the broader scientific community, we provide an interactive resource to the public., (© 2021 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.)

Background/aim: Germline mutations in PTCH1 or SUFU in the sonic hedgehog (SHH) pathway cause Gorlin's syndrome with increased risk of developing SHH-subgroup medulloblastoma. Gorlin's syndrome precludes the use of radiotherapy (a standard component of treatment) due to the development of multiple basal cell carcinomas. Also, current SHH inhibitors are ineffective against SUFU-mutated medulloblastoma, as they inhibit upstream genes. In this study, we aimed to detect differences in the expression of genes and microRNAs between SUFU- and PTCH1-mutated SHH medulloblastomas which may hint at new treatment directions., Patients and Methods: We sequenced RNA and microRNA from tumors of two patients with germline Gorlin's syndrome - one having PTCH1 mutation and one with SUFU mutation - followed by bioinformatics analysis to detect changes in genes and miRNAs expression in these two tumors. Expression changes were validated using qRT-PCR. Ingenuity pathway analysis was performed in search for targetable pathways., Results: Compared to the PTCH1 tumor, the SUFU tumor demonstrated lower expression of miR-301a-3p and miR-181c-5p, matrix metallopeptidase 11 (MMP11) and OTX2, higher expression of miR-7-5p and corresponding lower expression of its targeted gene, connexin 30 (GJB6). We propose mechanisms to explain the phenotypic differences between the two types of tumors, and understand why PTCH1 and SUFU tumors tend to relapse locally (rather than metastatically as in other medulloblastoma subgroups)., Conclusion: Our results help towards finding new treatable molecular targets for these types of medulloblastomas., (Copyright© 2021, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Recent advances in our understanding of breast cancer have demonstrated that cancer stem-like cells (CSCs, also known as tumor-initiating cell (TICs)) are central for progression and recurrence. CSCs are a small subpopulation of cells present in breast tumors that contribute to growth, metastasis, therapy resistance, and recurrence, leading to poor clinical outcome. Data have shown that cancer cells can gain characteristics of CSCs, or stemness, through alterations in key signaling pathways. The dysregulation of miRNA expression and signaling have been well-documented in cancer, and recent studies have shown that miRNAs are associated with breast cancer initiation, progression, and recurrence through regulating CSC characteristics. More specifically, miRNAs directly target central signaling nodes within pathways that can drive the formation, maintenance, and even inhibition of the CSC population. This review aims to summarize these research findings specifically in the context of breast cancer. This review also discusses miRNAs as biomarkers and promising clinical therapeutics, and presents a comprehensive summary of currently validated targets involved in CSC-specific signaling pathways in breast cancer.

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.

Ovarian cancer (OC) is the eighth most common type of cancer for women worldwide. The current diagnostic and prognostic routine available for OC management either lack specificity or are very costly. Gene expression profiling has shown to be a very effective tool in exploring new molecular markers for patients with OC, although association of such markers with patient survival and clinical outcome is still elusive. Here, we performed gene expression profiling of different subtypes of OC to evaluate its association with patient overall survival (OS) and aggressive forms of the disease. By global mRNA microarray profiling in a total of 196 epithelial OC patients (161 serous, 15 endometrioid, 11 mucinous, and 9 clear cell carcinomas), we found four candidates-HSPA1A, CD99, RAB3A and POM121L9P, which associated with OS and poor clinicopathological features. The overexpression of all combined was correlated with shorter OS and progression-free survival (PFS). Furthermore, the combination of at least two markers were further associated with advanced grade, chemotherapy resistance, and progressive disease. These results indicate that a panel comprised of a few predictors that associates with a more aggressive form of OC may be clinically relevant, presenting a better performance than one marker alone.

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.)

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.

Objective: The study is aimed at investigating the regulatory relationship between miR-145-5p and ABRACL, and has tried at clarifying the mechanisms underlying the proliferation, migration, and invasion of esophageal carcinoma (EC) cells., Methods: Gene expression data related to EC were accessed from TCGA database, and the "edgeR" package was used to screen differentially expressed genes. TargetScan, miRDB, and miRTarBase databases were used to predict potential targets for the target miRNA miR-145-5p. qRT-PCR and Western blot were performed to assess the expression of miR-145-5p and ABRACL in EC cells. Dual-luciferase reporter assay was performed to validate the targeting relationship between miR-145-5p and ABRACL. Functional experiments including CCK-8 assay, Transwell migration, and invasion assays were used to detect the proliferation, migration, and invasion of EC cells., Results: The expression of miR-145-5p was significantly decreased in EC, while ABRACL was remarkably increased. In addition, there was a negative correlation identified between miR-145-5p and ABRACL mRNA. Overexpressing miR-145-5p was able to suppress cell proliferation, migration, and invasion, whereas silencing miR-145-5p posed an opposite effect. In the meantime, ABRACL was identified as a direct target of miR-145-5p by dual-luciferase reporter assay. Furthermore, miR-145-5p could inhibit the expression of ABRACL, in turn inhibiting the proliferation, migration, and invasion of EC cells., Conclusion: miR-145-5p functions on the proliferation, migration, and invasion of EC cells via targeting ABRACL, and it may be a novel therapeutic target in EC treatment., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Shengming Fan et al.)