Cell cycle changes mediated by the p53/miR-34c axis are involved in the malignant transformation of human bronchial epithelial cells by benzo[a]pyrene

Characterization of aberrant microRNA (miRNA) expression during carcinogen-induced cell transformation will lead to a better understanding of the role of miRNAs in cancer development. In this investigation, we evaluated changes in p53 function and its downstream target miRNAs in benzo[a]pyrene (BaP)-induced transformation of human bronchial epithelial (HBE) cells. Chronic exposure to BaP induced malignant transformation of cells, in which there were increased levels of mutant p53 (mt-p53) and reduced expression of wild-type p53 (wt-p53) and phosphorylated p53 (p-p53). With acute (12 h) exposure to BaP, p-p53 was increased, and with increasing time of exposure (24 h), the increase in p-p53 at a concentration of 1 μM BaP was followed by a decline with increasing concentrations; wt-p53 and mt-p53 did not change. With prolonged exposure (48 h), p-p53 and wt-p53 decreased, but mt-p53 increased. At different exposure times, the levels of miR-34c were consistent with p-p53. Over-expression of miR-34c resulted in inhibition of the BaP-induced G1-to-S transition and diminished up-regulation of cyclin D. Further, up-regulation of miR-34c or silencing of cylin D prevented BaP-induced malignant transformation. Thus, changes in the cell cycle mediated by the p53/miR-34c axis are involved in the transformation cells induced by BaP.     

The role of miR-506 in transformed 16HBE cells induced by anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide

Growing evidence indicates that the alteration of microRNA (miRNA) expression in tumors that is induced by chemical carcinogens plays an important role in tumor development and progression. However, the mechanism underlying miRNA involvement in lung carcinogenesis induced by anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti-BPDE) remains unclear. In our study, we used the malignant transformation of human bronchial epithelial cells (16HBE-T) induced by anti-BPDE to explore the mechanisms of human lung carcinogenesis. We found that expression of miR-506 was reduced in 16HBE-T transformed malignant human bronchial epithelial cells compared with 16HBE normal human bronchial epithelial cells. Restoration of miR-506 in 16HBE-T cells led to a decrease in cell proliferation, G0/G1 phase cell cycle arrest, as well as significantly suppressed anchorage-dependent growth in vitro and tumor growth inhibition in a nude mouse xenograft model. In addition, we provided novel evidence regarding the role miR-506 potentially plays in negatively regulating the protein and mRNA expression level of N-Ras in cancer cells. Together, these findings revealed that miR-506 acts as an anti-oncogenic miRNA (anti-oncomir) in malignantly transformed cells. The identification of tumor suppressive miRNAs could provide new insight into the molecular mechanisms of chemical carcinogenesis.     

Mechanisms of migration and invasion promoted by NSCLC cells-derived exosomes

OBJECTIVE To explore the function and mechanism of exosomes from non-small cell lung cancer(NSCLC) in promoting the invasion and metastasis of lung cancer cel s. METHODS The exosomes were isolated and identified by differential centrifugation, nanosight,transmission electron microscope and Western blotting.Observation of the exosome uptake was applied by immune fluorescent confocal experiments. Scratch test was used to detect the influence of exosomes to tumor cells migration ability and transwell assay were carried out to investigate the effect of exosomes on migration and invasion. Total RNAs from exosomes were used for miRNA library preparation and sequencing. RT-Q-PCR was employed to detect the miRNA levels. Western blotting was adopted to detect the protein expression levels and dual luciferase reporter assay was used to verify the predict target site of miRNA. RESULTS The extracellular vesicles in the supernatant of NSCLC cells were isolated by the differential centrifugation and confirmed as exosomes by nanosight, transmission electron microscope and Western blotting. Exosomes from NSCLC can promote migration and invasion of non-small cell lung cancer cells showing concentration-and time-dependent manner. By analyzing the differential expression profile of exosomes miRNA of lung epithelial cells and lung cancer cell A549,30 miRNAs were screened out. Then, hsa-miR-34c-3p was selected in combination with clinical specimen detection. The expression level of miR-34c-3p in exosomes from lung cancer was lower than that of the normal one.CONCLUSION Exosomes from NSCLC cells can be uptaken by cells and promote cell migration and invasion in dose-dependent manner. The expression level of miR-34c-3p in exosomes from NSCLC is significantly lower than normal tissues. The level of miR-34c-3p was further declined after incubating with exosomes. Exosomes from NSCLC cells can deliver miR-34c-3p to recepted cells and up-regulate the protein level of integrin α2β1.     

Aberrant expression of miRNA-192-5p contributes to N,N-dimethylformamide-induced hepatic apoptosis

Excessive exposure to N,N-dimethylformamide (DMF) can lead to occupational liver poisoning in workers; however, the underlying mechanism is not fully clarified. The importance of microRNAs (miRNAs) in chemical-induced hepatotoxicity has been demonstrated. To determine whether miRNAs are also involved in DMF-induced hepatotoxicity, we systematically analyzed the miRNA expression profiles in DMF-treated (75 and 150 mm ) HL-7702 liver cells and controls by high-throughput sequencing. Among the altered miRNAs, miR-192-5p was the most significantly upregulated in HL-7702 cells after DMF exposure and was involved in DMF-mediated cell apoptosis. By contrast, suppression of miR-192-5p in HL-7702 cells attenuated the apoptosis induced by DMF. Furthermore, the anti-apoptotic gene (NIN1/RPN12 binding protein 1 homolog [NOB1]) was predicted to be a potential miR-192-5p target according to bioinformatics analysis. The direct interaction between miR-192-5p and NOB1 was confirmed by the dual-luciferase activity assay in HEK293FT cells. Overexpression of miR-192-5p efficiently reduced NOB1 mRNA and protein expression in HL-7702 cells. Alteration in NOB1 expression influenced DMF-induced hepatotoxicity by affecting hepatic apoptosis. In addition, the inverse correlation between miR-192-5p expression levels and NOB1 expression was further confirmed in DMF-exposed mouse liver tissue samples. These observations demonstrated that promotion of apoptosis from the suppression of NOB1 by miR-192-5p overexpression was responsible for the DMF-induced hepatotoxicity. This work provides the molecular mechanism at the miRNA level for hepatic apoptosis induced by DMF.     

MicroRNA-147b suppresses the proliferation and invasion of non-small-cell lung cancer cells through downregulation of Wnt/β-catenin signalling via targeting of RPS15A

Deregulation of microRNAs (miRNAs) leads to malignant growth and aggressive invasion during cancer occurrence and progression. miR-147b has emerged as one of the cancer-related miRNAs that are dysregulated in multiple cancers. Yet, the relevance of miR-147b in non-small-cell lung cancer (NSCLC) remains unclear. In the present study, we aimed to report the biological function and signalling pathways mediated by miR-147b in NSCLC. Our results demonstrate that miR-147b expression is significantly downregulated in NSCLC tissues and cell lines. Overexpression of miR-147b decreased the proliferative ability, colony-forming capability, and invasive potential of NSCLC cells. Notably, our study identified ribosomal protein S15A (RPS15A), an oncogene in NSCLC, as a target gene of miR-147b. Our results showed that miR-147b negatively modulates RPS15A expression in NSCLC cells. An inverse correlation between miR-147b and RPS15A was evidenced in NSCLC specimens. Moreover, miR-147b overexpression downregulated the activation of Wnt/β-catenin signalling via targeting of RPS15A. Overexpression of RPS15A partially reversed the miR-147b-mediated antitumour effect in NSCLC cells. Collectively, these findings reveal that miR-147b restricts the proliferation and invasion of NSCLC cells by inhibiting RPS15A-induced Wnt/β-catenin signalling and suggest that the miR-147b/RPS15A/Wnt/β-catenin axis is an important regulatory mechanism for malignant progression of NSCLC.     

miR-21 regulates N-methyl-N-nitro-N′-nitrosoguanidine-induced gastric tumorigenesis by targeting FASLG and BTG2

MicroRNAs (miRNAs) are recently discovered regulators of gene expression and are important in the regulation of many cellular events. Evidence collected to date shows that miRNAs are altered after exposure to environmental toxicants. However, the role that miR-21 plays in the gastric tumorigenesis induced by environmental carcinogens remains largely unknown. The aim of this study was to characterize the regulatory role of miR-21 in the carcinogenic processes following exposure to the N-nitroso carcinogen N-methyl-N-nitro-N′-nitrosoguanidine (MNNG). We found a progressive dose- and time-dependent increase in miR-21 expression following treatment with MNNG. Dysregulated miR-21 affected both cell growth in GES-1 cells and the gastric tumorigenesis induced with MNNG. These data demonstrate the involvement of miR-21 in the malignant transformation and tumorigenesis activated by MNNG. We also established that the Fas ligand (FASLG) and B-cell translocation gene 2 (BTG2), regulated by miR-21, contribute to the transformation induced by MNNG in GES-1 cells. This is the first study to show that miR-21 is involved in chemical carcinogenesis in vivo and in vitro. The regulation by miR-21 of the gastric carcinogenesis induced by MNNG highlights the functional roles of miRNAs in chemical carcinogenesis, and offers a new explanation of the mechanisms underlying chemical carcinogenesis.     

Targeting miR-375 in gastric cancer

INTRODUCTION: Gastric cancer remains a major cancer burden in the world, with a poor 5-year survival rate. It is necessary to develop new effective therapeutic strategies to improve the long-term clinical outcome. MicroRNA (miRNA), a class of small non-coding RNA, has been identified as a key regulator of gene expression, and is implicated in the pathogenesis of gastric cancer. AREAS COVERED: This review summarizes the role of miRNAs in gastric carcinogenesis, with an emphasis on the expression and function of miR-375 in gastric cancer and beyond. It also discusses the opportunities and challenges of miR-375 as a potential therapeutic target for gastric cancer. The genes targeted by miR-375, including JAK2 and 3'-phosphoinositide dependent protein kinase-1 (PDK1), are also candidates for gastric cancer therapy. EXPERT OPINION: Although radical surgery and rational chemotherapy are still the main treatment for gastric cancer, targeting miRNAs, in combination with other conventional therapies, may serve as a promising therapy strategy to improve the clinical outcome.     

Altered miRNA expression in aniline-mediated cell cycle progression in rat spleen

Aniline exposure is associated with toxicity to the spleen, however, early molecular events in aniline-induced cell cycle progression in the spleen remain unknown. MicroRNAs (miRNAs) have been implicated in tumor development by modulating key cell cycle regulators and controlling cell proliferation. This study was, therefore, undertaken on the expression of miRNAs, regulation of cyclins and cyclin-dependent kinases (CDKs) in an experimental condition that precedes a tumorigenic response. Male SD rats were treated with aniline (1?mmol/kg/day by gavage) for 7?days, and expression of miRNAs, cyclins and CDKs in rat spleens were analyzed. Microarray and/or qPCR analyses showed that aniline exposure led to significantly decreased miRNA expression of let-7a, miR-24, miR-34c, miR-100, miR-125b, and greatly increased miR-181a. The aberrant expression of miRNAs was associated with significantly increased protein expression of cyclins A, B1, D3 and E. Furthermore, remarkably enhanced expression of CDKs like CDK1, CDK2, CDK4, CDK6, especially p-CDK1 and p-CDK2 as well as alternations in the expression of pRB, p27, and CDC25A in the spleens of aniline-treated rats was also observed. The data suggest that aniline exposure leads to aberrant expression of miRNAs in the spleen which could be important in the regulation of cell cycle proteins. Our findings, thus, provide new insight into the role of miRNAs in cell cycle progression, which may contribute to aniline-induced tumorigenic response in the spleen.     

Genomic instability and mouse microRNAs

Tumor progression is the continual selection of variant subpopulations of malignant cells that have acquired increasing levels of genetic instability (Nowell Science 1976, 194, 23–28). This instability is manifested as chromosomal aneuploidy or translocations, viral integration or somatic mutations that typically affect the expression of a gene (oncogene) that is especially damaging to the proper function of a cell. With the recent discovery of non-coding RNAs such as microRNAs (miRNAs), the concept that a target of genetic instability must be a protein-encoding gene is no longer tenable. Over the years, we have conducted several studies comparing the location of miRNA genes to positions of genetic instability, principally retroviral integration sites and chromosomal translocations in the mouse as a means of identifying miRNAs of importance in carcinogenesis. In this current study, we have used the most recent annotation of the mouse miRome (miRBase, release 16.0), and several datasets reporting the sites of integration of different retroviral vectors in a variety of mouse strains and mouse models of cancer, including for the first time a model that shows a propensity to form solid tumors, as a means to further identify or define, candidate oncogenic miRNAs. Several miRNA genes and miRNA gene clusters stand out as interesting new candidate oncogenes due to their close proximity to common retroviral integration sites including miR-29a/b/c and miR106a~363. We also discussed some recently identified miRNAs including miR-1965, miR-1900, miR-1945, miR-1931, miR-1894, and miR-1936 that are close to common retroviral integration sites and are therefore likely to have some role in cell homeostasis.     

MiR-199a-5p and miR-375 affect colon cancer cell sensitivity to cetuximab by targeting PHLPP1

Objectives: We aimed to analyze the differentially-expressed miRNAs in colon cancer cells in order to identify novel potential biomarkers involved in cancer cell resistance.

Design and methods: We investigated the miRNA expression profile of GEO human colon carcinoma cells, sensitive to the EGFR inhibitor Cetuximab (CTX) and their CTX-resistant counterpart (GEO CR) by using a miRNA chip.

Results: We found 27 upregulated and 10 downregulated miRNAs in GEO CR compared with GEO cells with a fold change ≥ 2. Among the upregulated miRNAs, we focused on miR-199a-5p and miR-375. We report that their enforced expression promotes CTX resistance, whereas their silencing sensitizes to the same drug. The ability of miR-199a-5p and miR-375 to target PHLPP1 (PH domain and leucine-rich repeat protein phosphatase 1), a tumor suppressor that negatively regulates the AKT pathway, accounts, at least in part, for their drug-resistance activity. Indeed, restoration of PHLPP1 increases sensitivity of the GEO cells to CTX and reverts the resistance-promoting effect of miR-199a-5p and miR-375.

Conclusion: This study proposes miR-199a-5p and miR-375 as contributors to CTX resistance in colon cancer and suggests a novel approach based on miRNAs as tools for the therapy of this tumor.     

Genomic instability and mouse microRNAs

Tumor progression is the continual selection of variant subpopulations of malignant cells that have acquired increasing levels of genetic instability (Nowell Science 1976, 194, 23-28). This instability is manifested as chromosomal aneuploidy or translocations, viral integration or somatic mutations that typically affect the expression of a gene (oncogene) that is especially damaging to the proper function of a cell. With the recent discovery of non-coding RNAs such as microRNAs (miRNAs), the concept that a target of genetic instability must be a protein-encoding gene is no longer tenable. Over the years, we have conducted several studies comparing the location of miRNA genes to positions of genetic instability, principally retroviral integration sites and chromosomal translocations in the mouse as a means of identifying miRNAs of importance in carcinogenesis. In this current study, we have used the most recent annotation of the mouse miRome (miRBase, release 16.0), and several datasets reporting the sites of integration of different retroviral vectors in a variety of mouse strains and mouse models of cancer, including for the first time a model that shows a propensity to form solid tumors, as a means to further identify or define, candidate oncogenic miRNAs. Several miRNA genes and miRNA gene clusters stand out as interesting new candidate oncogenes due to their close proximity to common retroviral integration sites including miR-29a/b/c and miR106a~363. We also discussed some recently identified miRNAs including miR-1965, miR-1900, miR-1945, miR-1931, miR-1894, and miR-1936 that are close to common retroviral integration sites and are therefore likely to have some role in cell homeostasis.     

MicroRNA delivery by cationic lipoplexes for lung cancer therapy

Lung cancer is the leading cause of cancer deaths in western countries and carries a poor overall five year survival rate. Several studies demonstrate that microRNAs (miRNAs or miRs) are actively involved in tumor development by serving as tumor suppressors, oncogenes or both. In lung cancer, miRNAs may serve as both diagnostic and prognostic biomarkers as well as regulate in vitro and in vivo tumor progression. However, miRNA-based therapy is faced with several challenges including lack of tissue specificity, lack of optimal delivery systems, poor cellular uptake and risk of systemic toxicity. Here, we report a cationic lipid based miRNA delivery system to address some of these challenges. Among many lung cancer related miRNAs, miR-133b, a tumor suppressor, was selected as a therapeutic target because it directly targets the prosurvival gene MCL-1 thus regulating cell survival and sensitivity of lung cancer cells to chemotherapeutic agents. The efficacy of pre-miR-133b containing lipoplexes was evaluated in A549 non-small cell lung cancer (NSCLC) cells. Compared with siPORT NeoFX transfection agent, lipoplexes delivered pre-miR-133b in a more efficient manner with ~2.3-fold increase in mature miR-133b expression and ~1.8-fold difference in MCL-1 protein downregulation in vitro. In the in vivo biodistribution study, lipoplexes achieved ~30% accumulation in lung tissue, which was ~50-fold higher than siPORT NeoFX transfection agent. Mice treated with pre-miR-133b containing lipoplexes had mature miR-133b expression in lung ~52-fold higher than untreated mice. Our results demonstrated that cationic lipoplexes are a promising carrier system for the development of miRNA-based therapeutics in lung cancer treatment.     

MicroRNAs: small but potent oncogenes or tumor suppressors

MicroRNAs (miRNAs) are small, non-coding RNAs that modulate the expression of target mRNA. Many miRNAs are known to be up- or downregulated in a variety of cancers, suggesting a role for miRNAs in tumorigenesis. The correlation between the expression of miRNAs and their effects on target oncogenes, on tumorigenesis and on the proliferation of cancer cells is beginning to gain experimental evidence. For example, the miRNA (miR)-17-92 cluster has been characterized as an oncogene, while let-7 represses Ras and miR-15a/-16-1 represses Bcl-2, thereby acting as tumor suppressors. Thus, an oncogenic or tumor suppressive miRNA may have potential as a therapeutic target to control cancers. This review will discuss the relationship between miRNAs and tumorigenesis, and the potential for modulating miRNAs for the treatment of cancer.     

鸦胆亭上调miR-29a-3p抑制非小细胞肺癌H1299细胞增殖、迁移与侵袭

本研究主要探讨鸦胆亭(bruceantin,BCT)对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞的增殖、侵袭与迁移的抑制作用及其机制.采用MTT法检测BCT对NSCLC细胞株的细胞毒作用;采用集落形成、划痕和Transwell实验分别检测细胞的增殖、迁移与侵袭能力;Wester...