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Mitochondria-localized sirtuin 4 (SIRT4) is associated with malignant phenotypes in colorectal cancer (CRC). However, the molecular mechanisms that drive SIRT4-mediated carcinogenesis are unclear. Initially, we confirmed expression of SIRT4 in CRC through public database and in CRC patient tissues using quantitative real-time reverse transcription PCR. We established HCT116 colorectal cells that overexpressed SIRT4 and HT29 cells were transfected with plasmids bearing a small interfering RNA construct to silence SIRT4. Assays to determine the malignant phenotypes (proliferation, invasion and migration) were performed. Xenograft in vivo models were also constructed. A protein interactome network was built using differentially expressed proteins identified using the liquid chromatography/tandem mass spectrophotometry, the findings of which were confirmed using co-immunoprecipitation, western blotting and phenotype rescue experiments. Decreased SIRT4 expression was associated with malignant phenotypes in vitro and in vivo. The ribosomal biogenesis pathway was enriched in the interactome network. SIRT4 suppression activated glutaminase, thereby initiating AKT activation. Our research provided novel insights into the molecular mechanisms underlying CRC, and identified that SIRT4 exerts its antitumor activity in CRC possibly dependent on glutaminase to inhibit proliferation, migration and invasion via the AKT/GSK3β/CyclinD1 pathway., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Circular RNAs (circRNAs), a class of noncoding RNAs generated from pre-mRNAs, participate in regulation of genes. The mechanism for regulation, however, is unknown. Here, to determine if, in human keratinocyte (HaCaT) cells, circular RNAs are involved in arsenite-induced acceleration of the cell cycle, a circRNA microarray was performed to analyze the variability of circRNAs in arsenite-treated HaCaT (As-HaCaT) cells and in arsenite-transformed (T-HaCaT) cells in comparison to control HaCaT cells. Among the circRNAs up-regulated in both As-HaCaT cells and T-HaCaT cells, hsa:circRNA_100284 (circ100284) had the greatest increase and was chosen for further research. The presence of circ100284 was confirmed in HaCaT cells. In these cells, arsenite induced increases of EZH2 and cyclin D1 and accelerated the cell cycle. MicroRNA (miR)-217 suppressed the expression of EZH2 was involved in regulation of the cell cycle. Further, in HaCaT cells exposed to arsenite, EZH2 regulated the cell cycle by binding to the promoter of CCND1, which codes for cyclin D1. Moreover, knockdown of circ100284 with siRNA inhibited the cell cycle acceleration induced by arsenite, but this inhibition was reversed by co-transfection with circ100284 siRNA and by a miR-217 inhibitor. Knockdown of circ100284 with siRNA or transfected with miR-217 mimic inhibited the capacity of T-HaCaT cells for colony formation, invasion, and migration, effects that were reversed by co-transfection with a miR-217 inhibitor or by epigenetic expression of EZH2. These results suggest that, in HaCaT cells, arsenite increases circ100284 levels, which act as a sponge for miR-217 and up-regulate the miR-217 target, EZH2, which, in turn, up-regulates cyclin D1and CDK4, and thus accelerates the cell cycle and leads to malignant transformation. Thus, circ100284, via miR-217 regulation of EZH2, is involved in the arsenite-accelerated cell cycle of human keratinocytes in carcinogenesis. This establishes a previously unknown mechanism between arsenite-induced acceleration of the cell cycle and carcinogenesis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cigarette smoking is the strongest risk factor for the development of lung cancer, the leading cause of cancer-related deaths. However, the molecular mechanisms leading to lung cancer are largely unknown. A long-noncoding RNA (lncRNA), CCAT1, regarded as cancer-associated, has been investigated extensively. Moreover, the molecular mechanisms of lncRNAs in regulation of microRNAs (miRNAs) induced by cigarette smoke remain unclear. In the present investigation, cigarette smoke extract (CSE) caused an altered cell cycle and increased CCAT1 levels and decreased miR-218 levels in human bronchial epithelial (HBE) cells. Depletion of CCAT1 attenuated the CSE-induced decreases of miR-218 levels, suggesting that miR-218 is negatively regulated by CCAT1 in HBE cells exposed to CSE. The CSE-induced increases of BMI1 levels and blocked by CCAT1 siRNA were attenuated by an miR-218 inhibitor. Moreover, in CSE-transformed HBE cells, the CSE-induced cell cycle changes and elevated neoplastic capacity were reversed by CCAT1 siRNA or BMI1 siRNA. This epigenetic silencing of miR-218 by CCAT1 induces an altered cell cycle transition through BMI1 and provides a new mechanism for CSE-induced lung carcinogenesis. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs), which are epigenetic regulators, are involved in human malignancies. Little is known, however, about the molecular mechanisms for lncRNA regulation of genes induced by cigarette smoke. We recently found that, in human bronchial epithelial (HBE) cells, the lncRNA, Hox transcript antisense intergenic RNA (HOTAIR), is associated with changes in the cell cycle caused by cigarette smoke extract (CSE). In the present study, we report that increased expression of HOTAIR and enhancer of zeste homolog 2 (EZH2), and tri-methylation of Lys 27 of histone H3 (H3K27me3), affect cell cycle progression during CSE-induced transformation of HBE cells. Inhibition of HOTAIR and EZH2 by siRNAs attenuated CSE-induced decreases of p21 levels. Further, ChIP assays verified that HOTAIR and EZH2 were needed to maintain the interaction of H3K27me3 with the promoter regions of p21; combined use of a HOTAIR plasmid and EZH2 siRNA supported this observation. Thus, HOTAIR epigenetic silencing of p21 via EZH2-mediated H3K27 trimethylation contributes to changes in the cell cycle induced by CSE. These observations provide further understanding of the regulation of CSE-induced lung carcinogenesis and identify new therapeutic targets. [ABSTRACT FROM AUTHOR]

Lung cancer is regarded as the leading cause of cancer-related deaths, and cigarette smoking is one of the strongest risk factors for the development of lung cancer. However, the mechanisms for cigarette smoke-induced lung carcinogenesis remain unclear. The present study investigated the effects of an miRNA (miR-217) on levels of an lncRNA (MALAT1) and examined the role of these factors in the epithelial–mesenchymal transition (EMT) induced by cigarette smoke extract (CSE) in human bronchial epithelial (HBE) cells. In these cells, CSE caused decreases of miR-217 levels and increases in lncRNA MALAT1 levels. Over-expression of miR-217 with a mimic attenuated the CSE-induced increase of MALAT1 levels, and reduction of miR-217 levels by an inhibitor enhanced expression of MALAT1. Moreover, the CSE-induced increase of MALAT1 expression was blocked by an miR-217 mimic, indicating that miR-217 negatively regulates MALAT1 expression. Knockdown of MALAT1 reversed CSE-induced increases of EZH2 (enhancer of zeste homolog 2) and H3K27me3 levels. In addition to the alteration from epithelial to spindle-like mesenchymal morphology, chronic exposure of HBE cells to CSE increased the levels of EZH2, H3K27me3, vimentin, and N-cadherin and decreased E-cadherin levels, effects that were reversed by MALAT1 siRNA or EZH2 siRNA. The results indicate that miR-217 regulation of EZH2/H3K27me3 via MALAT1 is involved in CSE-induced EMT and malignant transformation of HBE cells. The posttranscriptional silencing of MALAT1 by miR-217 provides a link, through EZH2, between ncRNAs and the EMT and establishes a mechanism for CSE-induced lung carcinogenesis. [ABSTRACT FROM AUTHOR]

Arsenic is well established as a human carcinogen, but the molecular mechanisms leading to arsenic-induced carcinogenesis are complex and elusive. It is not been determined if the epithelial–mesenchymal transition (EMT) contributes to carcinogen-induced malignant transformation and subsequent tumor formation. We have found that, during the neoplastic transformation induced in human bronchial epithelial (HBE) cells by a low concentration (1.0 μM) of arsenite, the cells undergo an EMT and show enhanced invasion and migration. With longer times for transformation of HBE cells, there was increased miR-21 expression. Further, during the transformation of HBE cells, inhibition of miR-21 with an miR-21 inhibitor increased levels of PDCD4, an inhibitor of neoplastic transformation; reduced Twist1, a transcription factor involved in cell differentiation; and inhibited cell invasion and migration. In addition, PDCD4 interacted with Twist1 and inhibited its expression function, which is involved in arsenite-induced EMT. Thus, miR-21, acting on PDCD4, which interacts with Twist1 and represses the expression of Twist1, contributes to the EMT induced by arsenite. These observations add to an understanding of the processes involved in arsenite-induced carcinogenesis. [ABSTRACT FROM AUTHOR]

The incidence of lung diseases, including cancer, caused by cigarette smoke is increasing, but the molecular mechanisms of gene regulation induced by cigarette smoke remain unclear. This report describes a long noncoding RNA (lncRNA) that is induced by cigarette smoke extract (CSE) and experiments utilizing lncRNAs to integrate inflammation with the epithelial-mesenchymal transition (EMT) in human bronchial epithelial (HBE) cells. The present study shows that, induced by CSE, IL-6, a pro-inflammatory cytokine, leads to activation of STAT3, a transcription activator. A ChIP assay determined that the interaction of STAT3 with the promoter regions of HOX transcript antisense RNA (HOTAIR) increased levels of HOTAIR. Blocking of IL-6 with anti-IL-6 antibody, decreasing STAT3, and inhibiting STAT3 activation reduced HOTAIR expression. Moreover, for HBE cells cultured in the presence of HOTAIR siRNA for 24 h, the CSE-induced EMT, formation of cancer stem cells (CSCs), and malignant transformation were reversed. Thus, IL-6, acting on STAT3 signaling, which up-regulates HOTAIR in an autocrine manner, contributes to the EMT and to CSCs induced by CSE. These data define a link between inflammation and EMT, processes involved in the malignant transformation of cells caused by CSE. This link, mediated through lncRNAs, establishes a mechanism for CSE-induced lung carcinogenesis. [ABSTRACT FROM AUTHOR]