Qianlei Yang

Epigenetic silencing of miR-218 by the lncRNA CCAT1, acting via BMI1, promotes an altered cell cycle transition in the malignant transformation of HBE cells induced by cigarette smoke extract

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.

NF-kB-regulated exosomal miR-155 promotes the inflammation associated with arsenite carcinogenesis

In the cancer microenvironment, extracellular communication allows various types of cells to coordinate and execute biological functions. Emerging evidence indicates that exosomes, as mediators of cell communication, are involved in tumor progression. Little is known, however, about the mechanism by which exosomal miRNAs regulate inflammatory infiltration in arsenite-induced liver cancer. The present research aimed to determine if miRNAs secreted from arsenite-transformed human hepatic epithelial (L-02) cells are transferred into normal L-02 and THLE-3 cells, which are functionally active in the recipient cells. The results show that the exosomes from arsenite-transformed L-02 cells enhance miR-155 expression and the pro-inflammatory properties of normal L-02 and THLE-3 cells. Transformed cells transfer miR-155 into normal L-02 cells via exosomes. The inhibition of NF-κB by siRNA and inhibitor, which reduces miR-155 levels in exosomes derived from transformed L-02 cells, blocks inflammation. Arsenite-transformed cells secrete exosomes to enhance inflammation, but the inhibition of the synthesis of exosomes fails to stimulate inflammation. miR-155 is involved in exosome-mediated intercellular communication between neoplastic and normal liver cells. In addition, miR-155, IL-6, and IL-8 were over-expressed in the serum of arsenite exposure group. And there was a positive correlation between miR-155 and IL-6 or IL-8 levels. Further, exosomal miR-155 was up-regulated in the serum of arsenite exposure group. Thus, these results show that exosomes derived from transformed L-02 cells transfer miR-155 to surrounding cells, which induces pro-inflammatory activity of normal liver cells. The findings support the concept that exosomal miRNAs are involved in cell-cell communication during carcinogenesis induced by environmental chemicals.

MicroRNA-21 activation of ERK signaling via PTEN is involved in arsenite-induced autophagy in human hepatic L-02 cells.

Autophagy, an evolutionarily conserved cellular process, has diverse physiological and pathological roles in biological functions. Whether autophagy is induced by arsenite, a well-established human carcinogen, and the molecular mechanisms involved, remain to be established. Further, microRNAs (miRNAs) act as regulators in various cancers, but how miRNAs regulate autophagy remains largely unexplored. We have found that, in human hepatic epithelial (L-02) cells, arsenite increases levels of autophagy-related proteins in a concentration- and time-dependent manner and elevates the number of autophagic vacuoles (AVs). Arsenite also activates the ERK pathway in a dose- and time-dependent manner. In L-02 cells exposed to arsenite, microRNA-21 (miRNA-21) is over-expressed, and its target proteins, PTEN, PDCD4, and Spry1, are decreased. Moreover, inhibition of miR-21 increases levels of PTEN, and reduces levels of Beclin 1 and LC3 II/I, indicating that miR-21 is involved in arsenite-induced autophagy. In addition, ectopic expression of PTEN blocks the effect of miR-21 on the arsenite-induced autophagy and decreases p-ERK levels. Also, ERK promotes the autophagy induced by arsenite. In sum, upon exposure of cells to arsenite, over-expression of miR-21 activates ERK through PTEN, factors that participate in arsenite-induced autophagy. This link, mediated through miRNAs, establishes a mechanism for the development of autophagy that is associated with arsenic toxicity. Such information contributes to an understanding of the liver toxicity caused by arsenite.

Circ100284, via miR-217 regulation of EZH2, is involved in the arsenite-accelerated cell cycle of human keratinocytes in carcinogenesis

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.

Feedback circuitry via let-7c between lncRNA CCAT1 and c-Myc is involved in cigarette smoke extract-induced malignant transformation of HBE cells

Cigarette smoking is a primary risk factor for the development of lung cancer, which is regarded as the leading cause of cancer-related deaths. The process of malignant transformation of cells, however, is complex and elusive. The present study investigated the roles of an lncRNA, CCAT1, and a transcriptional factor, c-Myc, in human bronchial epithelial (HBE) cell transformation induced by cigarette smoke extract. With acute and chronic treatment of HBE cells, cigarette smoke extract induced increases of CCAT1 and c-Myc levels and decreases of levels of let-7c, a microRNA. Down-regulation of c-Myc reduced the degree of malignancy and the invasion/migration capacity of HBE cells transformed by cigarette smoke extract. ChIP assays established that c-Myc, increased by cigarette smoke extract, binds to the promoter of CCAT1, activating its transcription. Further, let-7c suppressed the expression of c-Myc through binding to its 3′-UTR. In turn, CCAT1 promoted the accumulation of c-Myc through binding to let-7c and decreasing free let-7c, which influenced the neoplastic capacity of HBE cells transformed by cigarette smoke extract. These results indicate that a positive feedback loop ensures expression of cigarette smoke extract-induced CCAT1 and c-Myc via let-7c, which is involved in cigarette smoke extract-induced malignant transformation of HBE cells. Thus, the present research establishes a new mechanism for the reciprocal regulation between CCAT1 and c-Myc and provides an understanding of cigarette smoke extract-induced lung carcinogenesis.

MicroRNA-191, regulated by HIF-2α, is involved in EMT and acquisition of a stem cell-like phenotype in arsenite-transformed human liver epithelial cells

Inorganic arsenic is widely distributed in the environment, and epidemiologic data show a strong association between arsenic exposure and risk of liver cancer. An understanding of the mechanisms underlying development of liver cancer and metastasis would be useful in reducing the incidence and mortality of liver cancer. MicroRNAs (miRs) act as regulators in liver cancer. Here, we show that acute or chronic exposure of human liver epithelial L-02 cells to arsenite increased expression of miR-191. There were decreased levels of BASP-1 and E-cadherin and increased levels of WT-1 and N-cadherin, indicating that arsenite induced epithelial-mesenchymal transition (EMT). Moreover, arsenite increased EpCAM and CD90 mRNA levels, showing the acquisition of stem cell-like properties by these cells. Suppression of miR-191 resulted in repression of EMT and reduced expression of stem-cell markers. Further, a miR-191 inhibitor blocked spheroid formation and production of side population cells. Luciferase reporter assays indicated that miR-191 was a target of HIF-2α, and inhibition of miR-191 decreased the neoplastic and metastatic properties of arsenite-transformed L-02 cells. Thus, in arsenite-transformed liver epithelial cells, transcriptional activation of the miR-191 promoter by HIF-2α is involved in EMT and in the acquisition of a stem cell-like phenotype.

MicroRNA-191, acting via the IRS-1/Akt signaling pathway, is involved in the hepatic insulin resistance induced by cigarette smoke extract

Cigarette smoke causes insulin resistance, which is associated with type 2 diabetes mellitus (T2DM). However, the mechanism by which this occurs remains poorly understood. Because the involvement of microRNAs (miRNAs) in the development of insulin resistance is largely unknown, we investigated, in hepatocytes, the roles of miR-191 in cigarette smoke extract (CSE)-induced insulin resistance. In L-02 cells, CSE not only decreased glucose uptake and glycogen levels but also reduced levels of insulin receptor substrate-1 (IRS-1) and Akt activation, effects that were blocked by SC79, an activator of Akt. CSE also increased miR-191 levels in L-02 cells. Furthermore, the inhibition of miR-191 blocked the decreases of IRS-1 and p-Akt levels, which antagonized the decreases of glucose uptake and glycogen levels in L-02 cells induced by CSE. These results reveal a mechanism by which miR-191 is involved in CSE-induced hepatic insulin resistance via the IRS-1/Akt signaling pathway, which helps to elucidate the mechanism for cigarette smoke-induced T2DM.

CircLRP6 Regulation of ZEB1 via miR-455 Is Involved in the Epithelial-Mesenchymal Transition During Arsenite-Induced Malignant Transformation of Human Keratinocytes

Circular RNAs (circRNAs), a class of noncoding RNAs generated from pre-mRNAs, participate in the regulation of tumorigenesis. The mechanism for regulation, however, is unclear. Here, to determine whether circRNAs are involved in arsenite-induced epithelial-mesenchymal transition (EMT) and malignant transformation in human keratinocyte (HaCaT) cells, the up-regulation of circLRP6 was confirmed in arsenite-transformed HaCaT (T-HaCaT) cells. In HaCaT cells, circLRP6 acted as an microRNA (miR)-455 sponge. For these cells, chronic exposure to arsenite caused an increase of circLRP6 and the transcription factor ZEB1, which induced the EMT. miR-455 suppressed the expression of ZEB1. Further, in T-HaCaT cells, knockdown of circLRP6 with siRNA inhibited ZEB1 expression, but cotransfection with circLRP6 siRNA and an miR-455 inhibitor reversed this inhibition. These results suggest that, in HaCaT cells, arsenite increases circLRP6 levels, which act as a sponge for miR-455 and up-regulate the miR-455 target, ZEB1, which subsequently induces the EMT, thus promoting malignant transformation. Thus, for HaCaT cells chronically exposed to arsenite, circLRP6 via miR-455 regulation of ZEB1 is involved in the EMT during malignant transformation. The results establish a previously unknown mechanism for arsenite-induced carcinogenesis.

NF-κB-regulated miR-155, via repression of QKI, contributes to the acquisition of CSC-like phenotype during the neoplastic transformation of hepatic cells induced by arsenite

Chronic exposure to arsenite can cause various human tumors. For the initiation and recurrence of human liver cancer, the acquisition of CSC‐like properties is essential. In various cancers, microRNAs (miRNAs) act as regulators in induction of CSC‐like properties. Liver cancers over‐express miR‐155, but the mechanism relating miR‐155 and arsenite‐induced liver cancer is unknown. Here, we show that long‐term exposure of L‐02 cells to arsenite increases miR‐155 levels by activation of NF‐κB and leads to the acquisition of CSC‐like properties. In spheroids formed from arsenite‐transformed L‐02 cells, the levels of miR‐155 positively relate to the levels of CD90, EpCAM, and OCT4. Inhibition of miR‐155, by reduction of SOX2 and OCT4, results in suppression of spheroid formation. Luciferase reporter assays indicate that QKI is a target of miR‐155. Inhibition of QKI expression by miR‐155 promotes arsenite‐induced acquisition of CSC‐like properties, whereas QKI over‐expression has the opposite effect. Collectively, the findings demonstrate that miR‐155, driven by NF‐κB, reduces QKI expression and is involved in acquisition of the CSC‐like phenotype during neoplastic transformation of hepatic cells induced by arsenite.

Impaired autophagic flux and p62-mediated EMT are involved in arsenite-induced transformation of L-02 cells

&NA; Autophagy is a catabolic process essential for preserving cellular homeostasis, and the epithelial‐to‐mesenchymal transition (EMT) is involved during tissue development and cancer progression. In arsenite‐treated human hepatic epithelial (L‐02) cells, arsenite reduced the autophagic flux, which caused accumulation of p62, an adaptor and receptor of autophagy. Further, in arsenite‐transformed L‐02 cells, the levels of E‐cadherin were attenuated, but the levels of vimentin, which is expressed in mesenchymal cells, and Snail, a transcription regulator of the EMT, were up‐regulated. Thus, after chronic exposure of L‐02 cells to arsenite, the impaired autophagic flux induced the accumulation of p62, which up‐regulated the expression of Snail, a protein involved in arsenite‐induced EMT of these cells. Knockdown of p62 by siRNA reversed the arsenite‐induced EMT and decreased the capacities of arsenite‐transformed L‐02 cells for colony formation and invasion and migration. Therefore, in arsenite‐induced transformation of L‐02 cells, the accumulation of p62, by impairing autophagic flux, mediates the EMT via Snail. These results provide a previously unknown mechanism underlying arsenic toxicity and carcinogenicity. HighlightsImpaired autophagic flux induces the accumulation of p62 in arsenite‐treated L‐02 cells.The EMT is associated with arsenite‐induced transformation in L‐02 cells.p62 mediates the EMT via Snail in arsenite‐induced transformation of L‐02 cells.