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MicroRNA-133a suppresses multiple oncogenic membrane receptors and cell invasion in non-small cell lung carcinoma.

Wang LK, Hsiao TH, Hong TM, Chen HY, Kao SH, Wang WL, Yu SL, Lin CW, Yang PC - PLoS ONE (2014)

Bottom Line: The cell invasive ability is suppressed in IGF-1R- and TGFBR1-repressed cells and this phenomenon is mediated through AKT signaling in highly invasive cell lines.In addition, by using the in vivo animal model, we find that ectopically-expressing miR-133a dramatically suppresses the metastatic ability of lung cancer cells.Accordingly, patients with NSCLCs who have higher expression levels of miR-133a have longer survival rates compared with those who have lower miR-133a expression levels.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.

ABSTRACT
Non-small cell lung cancers (NSCLCs) cause high mortality worldwide, and the cancer progression can be activated by several genetic events causing receptor dysregulation, including mutation or amplification. MicroRNAs are a group of small non-coding RNA molecules that function in gene silencing and have emerged as the fine-tuning regulators during cancer progression. MiR-133a is known as a key regulator in skeletal and cardiac myogenesis, and it acts as a tumor suppressor in various cancers. This study demonstrates that miR-133a expression negatively correlates with cell invasiveness in both transformed normal bronchial epithelial cells and lung cancer cell lines. The oncogenic receptors in lung cancer cells, including insulin-like growth factor 1 receptor (IGF-1R), TGF-beta receptor type-1 (TGFBR1), and epidermal growth factor receptor (EGFR), are direct targets of miR-133a. MiR-133a can inhibit cell invasiveness and cell growth through suppressing the expressions of IGF-1R, TGFBR1 and EGFR, which then influences the downstream signaling in lung cancer cell lines. The cell invasive ability is suppressed in IGF-1R- and TGFBR1-repressed cells and this phenomenon is mediated through AKT signaling in highly invasive cell lines. In addition, by using the in vivo animal model, we find that ectopically-expressing miR-133a dramatically suppresses the metastatic ability of lung cancer cells. Accordingly, patients with NSCLCs who have higher expression levels of miR-133a have longer survival rates compared with those who have lower miR-133a expression levels. In summary, we identified the tumor suppressor role of miR-133a in lung cancer outcome prognosis, and we demonstrated that it targets several membrane receptors, which generally produce an activating signaling network during the progression of lung cancer.

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Oncogenic receptor expression and activation are regulated by miR-133a in lung cancer cell lines.(A) The target genes of miR-133a were determined from Targetscan prediction software and the two database containing mRNA expression levels of receptors in miR-133a-expressing cells. (B) Co-transfection of CL1-0 cells with the AS2-Neo vector (Ctl) or AS2-Neo-miR-133a-expressing plasmid with firefly luciferase fused with 3′UTR sequences of putative miR-133a target genes. Luciferase activity was measured, and the relative ratio of the activity in the miR-133a groups to that in the control vector group is presented. (C) 72 hours after transient infection with the miR-133a-expressing virus, the protein expression levels of receptors in CL1-5 and A549 cells were determined. (D) The phosphorylation level of IGF-1R was determined in CL1-5/AS2-Neo- or CL1-5/miR-133a-expressing cells by the Phospho-Receptor Tyrosine Kinase Array.
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pone-0096765-g002: Oncogenic receptor expression and activation are regulated by miR-133a in lung cancer cell lines.(A) The target genes of miR-133a were determined from Targetscan prediction software and the two database containing mRNA expression levels of receptors in miR-133a-expressing cells. (B) Co-transfection of CL1-0 cells with the AS2-Neo vector (Ctl) or AS2-Neo-miR-133a-expressing plasmid with firefly luciferase fused with 3′UTR sequences of putative miR-133a target genes. Luciferase activity was measured, and the relative ratio of the activity in the miR-133a groups to that in the control vector group is presented. (C) 72 hours after transient infection with the miR-133a-expressing virus, the protein expression levels of receptors in CL1-5 and A549 cells were determined. (D) The phosphorylation level of IGF-1R was determined in CL1-5/AS2-Neo- or CL1-5/miR-133a-expressing cells by the Phospho-Receptor Tyrosine Kinase Array.

Mentions: To investigate the mechanism of miR-133a-mediated suppression of cancer cell invasion, we used TargetScan (http://www.targetscan.org/) to predict miR-133a downstream targets. Among them, a target network containing several receptor tyrosine kinases (RTKs), such as FGFR1, EGFR, IGF-1R, insulin receptor (INSR), and a serine/threonine kinase receptor, TGFBR1, are presented as possible candidates, all of which possess several putative miR-133a binding sites at their 3′- untranslated regions (3′-UTR) (Figure S2A). We further investigated the mRNA levels of these receptors in miR-133a-overexpressing cancer cell lines by combining the microarray database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE20028 and http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE26032) (Figure S2B). The mRNA levels were consistent with the in silico prediction results, suggesting that FGFR1, EGFR, IGF-1R and TGFBR1 may be downregulated by miR-133a (Figure 2A). To evaluate whether miR-133a directly regulates the expression of these receptors, the 3-′UTRs of these receptors containing the predicted miR-133a target sites were cloned into the luciferase reporter plasmid for a reporter assay. The luciferase activity of the constructs containing the 3′UTRs of EGFR, FGFR1, IGF-1R, TGFBR1 were significantly reduced in the presence of miR-133a, although the fold change in FGFR1 was not as dramatic as the others, and the luciferase activity of INSR was not affected by miR-133a (Figure 2B). We further evaluated whether TGFBR1 may be directly regulated by miR-133a since this correlation has never been reported. The luciferase activity was decreased in the presence of miR-133a, but this repression was alleviated by the addition of an anti-miR-133a inhibitor (Figure S3A). These results indicate that miR-133a may regulate the expression of FGFR1, EGFR, IGF-1R, and TGFBR1.


MicroRNA-133a suppresses multiple oncogenic membrane receptors and cell invasion in non-small cell lung carcinoma.

Wang LK, Hsiao TH, Hong TM, Chen HY, Kao SH, Wang WL, Yu SL, Lin CW, Yang PC - PLoS ONE (2014)

Oncogenic receptor expression and activation are regulated by miR-133a in lung cancer cell lines.(A) The target genes of miR-133a were determined from Targetscan prediction software and the two database containing mRNA expression levels of receptors in miR-133a-expressing cells. (B) Co-transfection of CL1-0 cells with the AS2-Neo vector (Ctl) or AS2-Neo-miR-133a-expressing plasmid with firefly luciferase fused with 3′UTR sequences of putative miR-133a target genes. Luciferase activity was measured, and the relative ratio of the activity in the miR-133a groups to that in the control vector group is presented. (C) 72 hours after transient infection with the miR-133a-expressing virus, the protein expression levels of receptors in CL1-5 and A549 cells were determined. (D) The phosphorylation level of IGF-1R was determined in CL1-5/AS2-Neo- or CL1-5/miR-133a-expressing cells by the Phospho-Receptor Tyrosine Kinase Array.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016005&req=5

pone-0096765-g002: Oncogenic receptor expression and activation are regulated by miR-133a in lung cancer cell lines.(A) The target genes of miR-133a were determined from Targetscan prediction software and the two database containing mRNA expression levels of receptors in miR-133a-expressing cells. (B) Co-transfection of CL1-0 cells with the AS2-Neo vector (Ctl) or AS2-Neo-miR-133a-expressing plasmid with firefly luciferase fused with 3′UTR sequences of putative miR-133a target genes. Luciferase activity was measured, and the relative ratio of the activity in the miR-133a groups to that in the control vector group is presented. (C) 72 hours after transient infection with the miR-133a-expressing virus, the protein expression levels of receptors in CL1-5 and A549 cells were determined. (D) The phosphorylation level of IGF-1R was determined in CL1-5/AS2-Neo- or CL1-5/miR-133a-expressing cells by the Phospho-Receptor Tyrosine Kinase Array.
Mentions: To investigate the mechanism of miR-133a-mediated suppression of cancer cell invasion, we used TargetScan (http://www.targetscan.org/) to predict miR-133a downstream targets. Among them, a target network containing several receptor tyrosine kinases (RTKs), such as FGFR1, EGFR, IGF-1R, insulin receptor (INSR), and a serine/threonine kinase receptor, TGFBR1, are presented as possible candidates, all of which possess several putative miR-133a binding sites at their 3′- untranslated regions (3′-UTR) (Figure S2A). We further investigated the mRNA levels of these receptors in miR-133a-overexpressing cancer cell lines by combining the microarray database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE20028 and http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE26032) (Figure S2B). The mRNA levels were consistent with the in silico prediction results, suggesting that FGFR1, EGFR, IGF-1R and TGFBR1 may be downregulated by miR-133a (Figure 2A). To evaluate whether miR-133a directly regulates the expression of these receptors, the 3-′UTRs of these receptors containing the predicted miR-133a target sites were cloned into the luciferase reporter plasmid for a reporter assay. The luciferase activity of the constructs containing the 3′UTRs of EGFR, FGFR1, IGF-1R, TGFBR1 were significantly reduced in the presence of miR-133a, although the fold change in FGFR1 was not as dramatic as the others, and the luciferase activity of INSR was not affected by miR-133a (Figure 2B). We further evaluated whether TGFBR1 may be directly regulated by miR-133a since this correlation has never been reported. The luciferase activity was decreased in the presence of miR-133a, but this repression was alleviated by the addition of an anti-miR-133a inhibitor (Figure S3A). These results indicate that miR-133a may regulate the expression of FGFR1, EGFR, IGF-1R, and TGFBR1.

Bottom Line: The cell invasive ability is suppressed in IGF-1R- and TGFBR1-repressed cells and this phenomenon is mediated through AKT signaling in highly invasive cell lines.In addition, by using the in vivo animal model, we find that ectopically-expressing miR-133a dramatically suppresses the metastatic ability of lung cancer cells.Accordingly, patients with NSCLCs who have higher expression levels of miR-133a have longer survival rates compared with those who have lower miR-133a expression levels.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.

ABSTRACT
Non-small cell lung cancers (NSCLCs) cause high mortality worldwide, and the cancer progression can be activated by several genetic events causing receptor dysregulation, including mutation or amplification. MicroRNAs are a group of small non-coding RNA molecules that function in gene silencing and have emerged as the fine-tuning regulators during cancer progression. MiR-133a is known as a key regulator in skeletal and cardiac myogenesis, and it acts as a tumor suppressor in various cancers. This study demonstrates that miR-133a expression negatively correlates with cell invasiveness in both transformed normal bronchial epithelial cells and lung cancer cell lines. The oncogenic receptors in lung cancer cells, including insulin-like growth factor 1 receptor (IGF-1R), TGF-beta receptor type-1 (TGFBR1), and epidermal growth factor receptor (EGFR), are direct targets of miR-133a. MiR-133a can inhibit cell invasiveness and cell growth through suppressing the expressions of IGF-1R, TGFBR1 and EGFR, which then influences the downstream signaling in lung cancer cell lines. The cell invasive ability is suppressed in IGF-1R- and TGFBR1-repressed cells and this phenomenon is mediated through AKT signaling in highly invasive cell lines. In addition, by using the in vivo animal model, we find that ectopically-expressing miR-133a dramatically suppresses the metastatic ability of lung cancer cells. Accordingly, patients with NSCLCs who have higher expression levels of miR-133a have longer survival rates compared with those who have lower miR-133a expression levels. In summary, we identified the tumor suppressor role of miR-133a in lung cancer outcome prognosis, and we demonstrated that it targets several membrane receptors, which generally produce an activating signaling network during the progression of lung cancer.

Show MeSH
Related in: MedlinePlus