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MircoRNA-33a inhibits epithelial-to-mesenchymal transition and metastasis and could be a prognostic marker in non-small cell lung cancer.

Yang L, Yang J, Li J, Shen X, Le Y, Zhou C, Wang S, Zhang S, Xu D, Gong Z - Sci Rep (2015)

Bottom Line: Here we found that miR-33a, an intronic miRNA located within the sterol regulatory element-binding protein 2 (SREBP-2) gene, is expressed at low levels in metastatic non-small cell lung cancer (NSCLC) cells and is inversely correlated with Twist1 expression.Additionally, Twist1 knockdown blocks EMT-related metastasis and forced expression of miR-33a inhibits lung cancer metastasis in a xenograft animal model.Clinically, miR-33a is found to be at low levels in NSCLC patients and down-regulation of miR-33a predicts a poor prognosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Molecular Biology.

ABSTRACT
Understanding the molecular mechanism by which epithelial mesenchymal transition (EMT)-mediated cancer metastasis and how microRNA (miRNA) regulates lung cancer progression via Twist1-activated EMT may provide potential therapeutic targets for cancer therapy. Here we found that miR-33a, an intronic miRNA located within the sterol regulatory element-binding protein 2 (SREBP-2) gene, is expressed at low levels in metastatic non-small cell lung cancer (NSCLC) cells and is inversely correlated with Twist1 expression. Conversely, miR-33a knockdown induces EMT and miR-33a overexpression blocks EMT by regulating of Twist1 expression in NSCLC cells. Bioinformatical prediction and luciferase reporter assay confirm that Twist1 is a direct target of miR-33a. Additionally, Twist1 knockdown blocks EMT-related metastasis and forced expression of miR-33a inhibits lung cancer metastasis in a xenograft animal model. Clinically, miR-33a is found to be at low levels in NSCLC patients and down-regulation of miR-33a predicts a poor prognosis. These findings suggest that miR-33a targets Twist1 and inhibits invasion and metastasis in NSCLC. Thus, miR-33a might be a potential prognostic marker and of therapeutic relevance for NSCLC metastasis intervention.

No MeSH data available.


Related in: MedlinePlus

miR-33a negatively regulates Twist1 expression.Quantitative RT-PCRs of Twist1 mRNA expressions in SPC-A-1 and NCI-H1299 cells were performed after transfections of anti-miR-33a (A) and miR-33a (B). Western blotting (C,D) and semi-quantitative analyses (E,F) of Twist1 protein in SPC-A-1 and NCI-H1299 cells treated as described above. *P < 0.05, **P < 0.01.
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f3: miR-33a negatively regulates Twist1 expression.Quantitative RT-PCRs of Twist1 mRNA expressions in SPC-A-1 and NCI-H1299 cells were performed after transfections of anti-miR-33a (A) and miR-33a (B). Western blotting (C,D) and semi-quantitative analyses (E,F) of Twist1 protein in SPC-A-1 and NCI-H1299 cells treated as described above. *P < 0.05, **P < 0.01.

Mentions: In an effort to elucidate the mechanism of the suppression of EMT and metastasis by miR-33a, we predicted 417 potential targets for miR-33a using the TargetScan tool (http://www.targetscan.org). To further narrow down the list of potential targets, another prediction tool (http://www.microrna.org) was used. Among these candidates, Twist1 (NM_000474) was the only EMT-inducing gene. Thus, we investigated the effect of miR-33a on Twist1 gene expression. In the low-metastasis SPC-A-1 cell line, the quantitative RT-PCR and Western blot analyses indicated that miR-33a knockdown resulted in an increase in Twist1 expression at both the mRNA and protein levels (Fig. 3A,C,E). In contrast, miR-33a overexpression in NCI-H1299 cells resulted in a decrease of Twist1 gene expression (Fig. 3B,D,F). Similarly, the endogenous transcript level of Twist1 in response to miR-33a was rescued when miR-33a was cotransfected with the miR-33a inhibitor (Supplementary Fig. 2). As Pim-1, another target of miR-33a, is up-regulated in NSCLC cells and down-regulation of Pim-1 by miR-33a may contribute to the invasion and metastasis in vitro1214, we compared the Pim-1 levels in NCI-H1299 cells with and without miR-33a replacement, and in SPC-A-1 cells with and without anti-miR-33a. Unfortunately, transfection of miR-33a mimics provided no evidence for a significant down-regulation of Pim-1 in NCI-H1299 cells (Supplementary Fig. 3A). Meanwhile, inhibition of miR-33a did not result in an obvious change of Pim-1 in SPC-A-1 cells (Supplementary Fig. 3B). These results indicated miR-33a negatively regulates Twist1 expression, but not Pim-1 in these two tested NSCLC cell lines. In other words, miR-33a expression is specifically and inversely correlated with Twist1 expression in NSCLC cells.


MircoRNA-33a inhibits epithelial-to-mesenchymal transition and metastasis and could be a prognostic marker in non-small cell lung cancer.

Yang L, Yang J, Li J, Shen X, Le Y, Zhou C, Wang S, Zhang S, Xu D, Gong Z - Sci Rep (2015)

miR-33a negatively regulates Twist1 expression.Quantitative RT-PCRs of Twist1 mRNA expressions in SPC-A-1 and NCI-H1299 cells were performed after transfections of anti-miR-33a (A) and miR-33a (B). Western blotting (C,D) and semi-quantitative analyses (E,F) of Twist1 protein in SPC-A-1 and NCI-H1299 cells treated as described above. *P < 0.05, **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556976&req=5

f3: miR-33a negatively regulates Twist1 expression.Quantitative RT-PCRs of Twist1 mRNA expressions in SPC-A-1 and NCI-H1299 cells were performed after transfections of anti-miR-33a (A) and miR-33a (B). Western blotting (C,D) and semi-quantitative analyses (E,F) of Twist1 protein in SPC-A-1 and NCI-H1299 cells treated as described above. *P < 0.05, **P < 0.01.
Mentions: In an effort to elucidate the mechanism of the suppression of EMT and metastasis by miR-33a, we predicted 417 potential targets for miR-33a using the TargetScan tool (http://www.targetscan.org). To further narrow down the list of potential targets, another prediction tool (http://www.microrna.org) was used. Among these candidates, Twist1 (NM_000474) was the only EMT-inducing gene. Thus, we investigated the effect of miR-33a on Twist1 gene expression. In the low-metastasis SPC-A-1 cell line, the quantitative RT-PCR and Western blot analyses indicated that miR-33a knockdown resulted in an increase in Twist1 expression at both the mRNA and protein levels (Fig. 3A,C,E). In contrast, miR-33a overexpression in NCI-H1299 cells resulted in a decrease of Twist1 gene expression (Fig. 3B,D,F). Similarly, the endogenous transcript level of Twist1 in response to miR-33a was rescued when miR-33a was cotransfected with the miR-33a inhibitor (Supplementary Fig. 2). As Pim-1, another target of miR-33a, is up-regulated in NSCLC cells and down-regulation of Pim-1 by miR-33a may contribute to the invasion and metastasis in vitro1214, we compared the Pim-1 levels in NCI-H1299 cells with and without miR-33a replacement, and in SPC-A-1 cells with and without anti-miR-33a. Unfortunately, transfection of miR-33a mimics provided no evidence for a significant down-regulation of Pim-1 in NCI-H1299 cells (Supplementary Fig. 3A). Meanwhile, inhibition of miR-33a did not result in an obvious change of Pim-1 in SPC-A-1 cells (Supplementary Fig. 3B). These results indicated miR-33a negatively regulates Twist1 expression, but not Pim-1 in these two tested NSCLC cell lines. In other words, miR-33a expression is specifically and inversely correlated with Twist1 expression in NSCLC cells.

Bottom Line: Here we found that miR-33a, an intronic miRNA located within the sterol regulatory element-binding protein 2 (SREBP-2) gene, is expressed at low levels in metastatic non-small cell lung cancer (NSCLC) cells and is inversely correlated with Twist1 expression.Additionally, Twist1 knockdown blocks EMT-related metastasis and forced expression of miR-33a inhibits lung cancer metastasis in a xenograft animal model.Clinically, miR-33a is found to be at low levels in NSCLC patients and down-regulation of miR-33a predicts a poor prognosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Molecular Biology.

ABSTRACT
Understanding the molecular mechanism by which epithelial mesenchymal transition (EMT)-mediated cancer metastasis and how microRNA (miRNA) regulates lung cancer progression via Twist1-activated EMT may provide potential therapeutic targets for cancer therapy. Here we found that miR-33a, an intronic miRNA located within the sterol regulatory element-binding protein 2 (SREBP-2) gene, is expressed at low levels in metastatic non-small cell lung cancer (NSCLC) cells and is inversely correlated with Twist1 expression. Conversely, miR-33a knockdown induces EMT and miR-33a overexpression blocks EMT by regulating of Twist1 expression in NSCLC cells. Bioinformatical prediction and luciferase reporter assay confirm that Twist1 is a direct target of miR-33a. Additionally, Twist1 knockdown blocks EMT-related metastasis and forced expression of miR-33a inhibits lung cancer metastasis in a xenograft animal model. Clinically, miR-33a is found to be at low levels in NSCLC patients and down-regulation of miR-33a predicts a poor prognosis. These findings suggest that miR-33a targets Twist1 and inhibits invasion and metastasis in NSCLC. Thus, miR-33a might be a potential prognostic marker and of therapeutic relevance for NSCLC metastasis intervention.

No MeSH data available.


Related in: MedlinePlus