Overexpression of the lung cancer-prognostic miR-146b microRNAs has a minimal and negative effect on the malignant phenotype of A549 lung cancer cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Patnaik Santosh
PROVIDER: S-ECPF-GEOD-29496 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:INTRODUCTION:Expression levels of miR-146b-5p and -3p microRNAs in human non-small cell lung cancer (NSCLC) are associated with recurrence of the disease after surgery. To understand this, the effect of miR-146b overexpression was studied in A549 human lung cancer cells. METHODS:A549 cells, engineered with lentiviruses to overexpress the human pre-miR-146b precursor microRNA, were examined for proliferation, colony formation on plastic surface and in soft agar, migration and invasiveness in cell culture and in vivo in mice, chemosensitivity to cisplatin and doxorubicin, and global gene expression. miR-146b expressions were assessed in microdissected stroma and epithelia of human NSCLC tumors. Association of miR-146b-5p and -3p expression in early stage NSCLC with recurrence was analyzed. PRINCIPAL FINDINGS:A549 pre-miR-146b-overexpressors had 3-8-fold higher levels of both miR-146b microRNAs than control cells. Overexpression did not alter cellular proliferation, chemosensitivity, migration, or invasiveness; affected only 0.3% of the mRNA transcriptome; and, reduced the ability to form colonies in vitro by 25%. In human NSCLC tumors, expression of both miR-146b microRNAs was 7-10-fold higher in stroma than in cancerous epithelia, and higher miR-146b-5p but lower -3p levels were predictive of recurrence. CONCLUSIONS:Only a minimal effect of pre-miR-146b overexpression on the malignant phenotype was seen in A549 cells. This could be because of opposing effects of miR-146b-5p and -3p overexpression as suggested by the conflicting recurrence-predictive values of the two microRNAs, or because miR-146b expression changes in non-cancerous stroma and not cancerous epithelia of tumors are responsible for the prognostic value of miR-146b.
| S-EPMC3138784 | biostudies-other
Project description:Biological effects of overexpression of miR-146b microRNAs in the A549 human lung cancer cell-line was studied. A549 cells were engineered to express the precursor RNA (pre-miR-146b) that generates the miR-146b microRNAs. Control cells were engineered using the same gene expression plasmid (pLemiR, Open Biosystems®) but without the pre-miR-146b insert. The Trans-Lentiviral GIPZ™ packaging system (Open Biosystems®) was used to generate stable transfectant populations of the engineered cells.
2011-05-24 | GSE29496 | GEO
Project description:Biological effects of overexpression of miR-146b microRNAs in the A549 human lung cancer cell-line was studied. A549 cells were engineered to express the precursor RNA (pre-miR-146b) that generates the miR-146b microRNAs. Control cells were engineered using the same gene expression plasmid (pLemiR, Open Biosystems®) but without the pre-miR-146b insert. The Trans-Lentiviral GIPZ™ packaging system (Open Biosystems®) was used to generate stable transfectant populations of the engineered cells. Gene expression was compared between A549 stable transfectants of empty pLemiR plasmid (A549/vec cells) or of the pLemiR plasmid containing an insert for overexpressing human pre-miR-146b (A549/146b cells). Two different cell cultures were used for each cell-line (for biological replicates).
2011-05-24 | E-GEOD-29496 | biostudies-arrayexpress
Project description:Over the last few years, incidental thyroid nodules are being diagnosed with increasing frequency with the use of highly sensitive imaging techniques. The ultrasound thyroid gland examination, followed by the fine-needle aspiration cytology is the standard diagnostic approach. However, in cases of the follicular nature of nodules, cytological diagnosis is not enough. Analysis of miRNAs in the biopsy presents a promising approach. Increasing our knowledge of miRNA's role in follicular carcinogenesis, and development of the appropriate the miRNA analytical technologies are required to implement miRNA-based tests in clinical practice. We used material from follicular thyroid nodes (n.84), grouped in accordance with their invasive properties. The invasion-associated miRNAs expression alterations were assayed. Expression data were confirmed by highly sensitive two-tailed RT-qPCR. Reciprocally dysregulated miRNAs pair concentration ratios were explored as a diagnostic parameter using receiver operation curve (ROC) analysis. A new bioinformatics method (MiRImpact) was applied to evaluate the biological significance of the observed expression alterations. Coupled experimental and computational approaches identified reciprocal dysregulation of miR-146b and miR-451 as important attributes of follicular cell malignant transformation and follicular thyroid cancer progression. Thus, evaluation of combined dysregulation of miRNAs relevant to invasion and metastasis can help to distinguish truly malignant follicular thyroid cancer from indolent follicular adenoma.
| S-EPMC7503510 | biostudies-literature
Project description:Cancer develops by a multistep process during which cells acquire characteristics that allow them to evade apoptosis and proliferate unchecked. Sequential acquisition of genetic alterations drives this process but also causes cellular stress, frequently prompting cells to enter a premalignant period during which they mount a defense against transformation. T cell-specific deletion of the tumor suppressor PTEN in mice induces premalignancy in the thymus and development of CD4(+) T-cell lymphomas in the periphery. Here we sought to identify factors mediating the cellular defense against transformation during the premalignant period. We identified several microRNAs upregulated specifically in premalignant thymocytes, including miR-146a, miR-146b, and the miR-183/96/182 cluster. CD4-driven T cell-specific transgenic overexpression of mir-146a and mir-146b significantly delayed PTEN-deficient lymphomagenesis and delayed c-myc oncogene induction, a key driver of transformation in PTEN-deficient T-cell malignancies. We found that miR-146a and miR-146b targeting of Traf6 attenuates TCR signaling in the thymus and inhibits downstream NF-κB-dependent induction of c-myc. Additionally, c-myc repression in mature CD4 T cells by miR-146b impaired TCR-mediated proliferation. Hence, we have identified 2 miRNAs that are upregulated as part of the cellular response against transformation that, when overrepresented, can effectively inhibit progression to malignancy in the context of PTEN deficiency.
| S-EPMC4073325 | biostudies-literature
Project description:p32 is a multifunctional and multicompartmental protein that has been found upregulated in numerous adenocarcinomas, including colorectal malignancy. High levels of p32 expression have been correlated with poor prognosis in colorectal cancer. However, the functions performed by p32 in colorectal cancer have not been characterized. Here we show that p32 is overexpressed in colorectal cancer cell lines compared to non-malignant colon cells. Colon cancer cells also display higher nuclear levels of p32 than nuclear levels found in non-malignant cells. Moreover, we demonstrate that p32 regulates the expression levels of genes tightly related to malignant phenotypes such as HAS-2 and PDCD4. Remarkably, we demonstrate that knockdown of p32 negatively affects Akt/mTOR signaling activation, inhibits the migration ability of colon malignant cells, and sensitizes them to cell death induced by oxidative stress and chemotherapeutic agents, but not to cell death induced by nutritional stress. In addition, knockdown of p32 significantly decreased clonogenic capacity and in vivo tumorigenesis in a xenograft mice model. Altogether, our results demonstrate that p32 is an important promoter of malignant phenotype in colorectal cancer cells, suggesting that it could be used as a therapeutic target in colorectal cancer treatment.
| S-EPMC8201776 | biostudies-literature
Project description:Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. However, science has not yet been able to substantially improve the prognosis of lung cancer patients. Accumulating evidence suggests that microRNAs (miRNAs) are key players in the regulation of tumor development and metastasis. Expression of six miRNAs previously shown to play roles in tumor development (miR-146b-5p, miR-128b, miR-21, miR-221, miR-34a, and Let-7a) in other tumor types was examined using real-time RT-PCR in 78 specimens of NSCLC. The results revealed that patients with low expression of miR-146b-5p had significant shorter median and mean survival time than those with high miR-146b-5p expression (33.00 and 30.44 months versus 42.0 and 36.90 months, respectively; log-rank test P=0.048), thus low miR-146b-5p expression level was associated with poor prognosis in NSCLC patients. Univariate Cox hazard regression analysis demonstrated that miR-146b-5p expression levels tended to be a significant prognostic indicator of NSCLC (adjusted hazard ratio=0.482, 95% CI: 1.409- 29.593, P=0.016). Multivariate Cox proportional hazard regression analysis showed that miR-146b-5p expression levels were an independent prognostic factor for NSCLC patients (hazard ratio=0.259, 95% CI: 0.083-0.809, P=0.020). Furthermore, the effects of miR-146b-5p and miR-146b-3p on NSCLC cell growth and invasion in vitro were investigated. Our findings demonstrate that ectopic expression of miR-146b-5p suppressed cell proliferation, clonogenicity, migration/ invasion and also induced G1 arrest in vitro, but did not induce cell apoptosis; whereas enforced expression of miR-146b-3p did not have a significant effect on cell growth and metastasis. Further experiments indicated that miR-146b-5p could reduce mRNA levels of MMP16 and TRAF6 in vitro and was negatively related to the expression of TRAF6 in human NSCLC tissues. In a mouse model, Ago-miR-146b-5p could significantly inhibit the growth of lung cancer xenografts in nude mice. In conclusion, our findings demonstrate that miR-146b-5p functions as a suppressor miRNA and prognosis predictor in NSCLC.
| S-EPMC5535726 | biostudies-literature
Project description:Increasing evidence highlights the multiple roles of microRNAs (miRs) in the tumorigenesis of colorectal cancer (CRC); however, the molecular mechanism, particularly the target of miR-146b-5p in CRC has not been fully elucidated. The present study aimed to elucidate the influence of miR-146b-5p via regulating tumor necrosis factor receptor-associated factor 6 (TRAF6) in CRC. The expression levels of miR-146b-5p and TRAF6 in CRC tissue and cells were determined by reverse transcription quantitative PCR and western blotting. Binding between miR-146b-5p and TRAF6 was examined using a dual luciferase reporter gene assay. The impact of miR-146b-5p and TRAF6 on proliferation and migration of CRC cells was determined using Cell Counting Kit-8 and Transwell assays, respectively. An animal model of CRC was established to determine the carcinogenic effect of the miR-146b-5p-TRAF6 axis. The results demonstrated that miR-146b-5p was highly expressed in CRC tissue samples compared with in normal adjacent tissue samples and in CRC cells compared with in the normal NCM460 cell line, whereas TRAF6 was expressed at low levels. Overexpression of miR-146b-5p decreased TRAF6 expression in CRC HT29 and SW620 cells. miR-146b-5p targeted and inhibited TRAF6 expression in CRC cells. Furthermore, transfection with a miR-146b-5p mimic promoted the proliferation, migration and invasion of CRC cells and tumor growth; however, these effects were abolished by TRAF6 overexpression. Transfection with a miR-146b-5p inhibitor suppressed the proliferation of CRC cells. Taken together, the results from the present study demonstrated that miR-146b-5p could enhance the initiation and tumorigenesis of CRC by targeting TRAF6. These results will help elucidate the mechanisms underlying CRC development and will facilitate the development of targeted therapy for CRC.
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Project description:Some microRNAs (miRNAs) are known to suppress breast cancer. However, whether the expressions of these tumor suppressive miRNAs translate to patient survival were not investigated in large cohort. Nine miRNAs (miR-30a, miR-30c, miR-31, miR-126, miR-140, miR-146b, miR-200c, miR-206, and miR-335) known to be tumor suppressive miRNAs in breast cancer were investigated in Genomic Data Common data portal miRNA-Seq dataset and The Cancer Genome Atlas (TCGA) (n = 1052). Of the 9 miRNAs, miR-30a, miR-30c, miR-126, miR-140, miR-206, and miR-335 were found to have significantly lower expression in breast cancer tissues compared to paired normal breast tissue. High expression of miR-30a or miR-200c was associated with significantly better overall survival (OS). Gene Set Enrichment Analysis (GSEA) demonstrated that low expression levels of miR-30a had the tendency to associate with gene enrichment of EMT, while miR-200c did not, in TCGA cohort, and our findings support the need of validation using large cohort to use miRNA as prognostic biomarker for patients with breast cancer.
| S-EPMC5698306 | biostudies-literature
Project description:MicroRNA (miR)-421 has been reported to serve various important roles in numerous types of cancer, including neuroblastoma and gastric cancer. However, to the best of our knowledge, few reports have determined the role of miR-421 in lung cancer. The aim of the current study was to analyze the expression levels of miR-421 in A549 lung cancer cells, to determine the target gene of miR-421, and to investigate the function and mechanism of miR-421 in cellular cytotoxicity. miR-421 expression levels were analyzed in A549 lung cancer cells using reverse transcription-quantitative PCR, a MTT assay was performed to determine the effect of miR-421 on A549 cell cytotoxicity and the protein expression levels of forkhead box O1 (FOXO1) were determined via western blotting. The target gene of miR-421 was predicted and verified using TargetScan and a dual-luciferase reporter assay, respectively. The results revealed that miR-421 expression levels were significantly upregulated in A549 lung cancer cell lines compared with the normal cells (P<0.01). Additionally, it was discovered that miR-421 promoted A549 cell viability (P<0.01) compared with A549 transfected with negative control. miR-421 was also identified to bind to the 3'-untranslated region of FOXO1. In A549 cells transfected with miR-421-mimics, the expression levels of phosphorylated (p)-AKT, p-glycogen synthase kinase-3?, p-retinoblastoma and cyclin D1 were significantly upregulated (P<0.01), whereas the expression levels of FOXO1 and p21 were significantly downregulated (P<0.01) compared with the control group. In conclusion, the results of the present study suggested that miR-421 may promote the viability of A549 lung cancer cells by targeting FOXO1 and modulating cell cycle, indicating that targeting miR-421 and FOXO1 may represent future therapeutic strategies for the treatment of patients with lung cancer.
| S-EPMC7573922 | biostudies-literature