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miR-126-3p Inhibits Thyroid Cancer Cell Growth and Metastasis, and Is Associated with Aggressive Thyroid Cancer.

Xiong Y, Kotian S, Zeiger MA, Zhang L, Kebebew E - PLoS ONE (2015)

Bottom Line: We found that miR-126-3p expression was significantly lower in larger tumors, in tumor samples with extrathyroidal invasion, and in higher risk group thyroid cancer in 496 papillary thyroid cancer samples from The Cancer Genome Atlas study cohort.In an independent sample set, lower miR-126-3p expression was observed in follicular thyroid cancers (which have capsular and angioinvasion) as compared to follicular adenomas.Of these 14 genes, SLC7A5 and ADAM9 were confirmed to be inhibited by miR-126-3p overexpression and to be direct targets of miR-136-3p.

View Article: PubMed Central - PubMed

Affiliation: Endocrine Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.

ABSTRACT

Background: Previous studies have shown that microRNAs are dysregulated in thyroid cancer and play important roles in the post-transcriptional regulation of target oncogenes and/or tumor suppressor genes.

Methodology/principal findings: We studied the function of miR-126-3p in thyroid cancer cells, and as a marker of disease aggressiveness. We found that miR-126-3p expression was significantly lower in larger tumors, in tumor samples with extrathyroidal invasion, and in higher risk group thyroid cancer in 496 papillary thyroid cancer samples from The Cancer Genome Atlas study cohort. In an independent sample set, lower miR-126-3p expression was observed in follicular thyroid cancers (which have capsular and angioinvasion) as compared to follicular adenomas. Mechanistically, ectopic overexpression of miR-126-3p significantly inhibited thyroid cancer cell proliferation, in vitro (p<0.01) and in vivo (p<0.01), colony formation (p<0.01), tumor spheroid formation (p<0.05), cellular migration (p<0.05), VEGF secretion and endothelial tube formation, and lung metastasis in vivo. We found 14 predicted target genes, which were significantly altered upon miR-126-3p transfection in thyroid cancer cells, and which are involved in cancer biology. Of these 14 genes, SLC7A5 and ADAM9 were confirmed to be inhibited by miR-126-3p overexpression and to be direct targets of miR-136-3p.

Conclusions/significance: To our knowledge, this is the first study to demonstrate that miR-126-3p has a tumor-suppressive function in thyroid cancer cells, and is associated with aggressive disease phenotype.

No MeSH data available.


Related in: MedlinePlus

miR-126-3p regulates and directly targets SLC7A5 and ADAM9 protein expression in thyroid cancer cells in vitro and in vivo.(A) Immunoblots of SLC7A5 and GAPDH in TPC-1 and XTC-1 cell lines, which were transfected with either miR-126-3p or miR-NC for 72 hours. The FTC-133 cell line had no detectable protein expression for SLC7A5. (B) Immunoblots for ADAM9 and GAPDH in TPC-1, FTC-133 and XTC-1 cell lines, which were transfected with either miR-126-3p or miR-NC for 72 hours in vitro. (C) Immunoblots for detecting ADAM9 and GAPDH in FTC-133-Luc2 tumor xenografts that had been inoculated subcutaneously into the flanks of athymic nude mice and allowed to develop for 10 days. (D) Luciferase activity of pEZX-SLC7A5-3′UTR and pEZX-SLC7A5-3′UTR in FTC-133 cells when co-transfected with miR-126-3p or miR-NC. All luciferase measurements were made in triplicate and readings were performed 24 hours post-transfection. Error bars represent SEM (*** indicates p<0.001). (E) The expression level of miR-126-3p is significantly inversely associated with the expression level of SLC7A5 in 481 papillary thyroid cancer samples from the TCGA dataset. *** indicates p<0.001.
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pone.0130496.g005: miR-126-3p regulates and directly targets SLC7A5 and ADAM9 protein expression in thyroid cancer cells in vitro and in vivo.(A) Immunoblots of SLC7A5 and GAPDH in TPC-1 and XTC-1 cell lines, which were transfected with either miR-126-3p or miR-NC for 72 hours. The FTC-133 cell line had no detectable protein expression for SLC7A5. (B) Immunoblots for ADAM9 and GAPDH in TPC-1, FTC-133 and XTC-1 cell lines, which were transfected with either miR-126-3p or miR-NC for 72 hours in vitro. (C) Immunoblots for detecting ADAM9 and GAPDH in FTC-133-Luc2 tumor xenografts that had been inoculated subcutaneously into the flanks of athymic nude mice and allowed to develop for 10 days. (D) Luciferase activity of pEZX-SLC7A5-3′UTR and pEZX-SLC7A5-3′UTR in FTC-133 cells when co-transfected with miR-126-3p or miR-NC. All luciferase measurements were made in triplicate and readings were performed 24 hours post-transfection. Error bars represent SEM (*** indicates p<0.001). (E) The expression level of miR-126-3p is significantly inversely associated with the expression level of SLC7A5 in 481 papillary thyroid cancer samples from the TCGA dataset. *** indicates p<0.001.

Mentions: Integrated analyses of the genome-wide gene expression data and target scan prediction revealed 14 genes as targets of miR-126-3p (S1 Table). We then performed a pathway analysis with these 14 genes. The top diseases and biological functions identified by IPA are summarized in S2 Table. Cancer was the top disease category, with four molecules involved in this pathway that were significantly reduced by miR-126-3p overexpression. Furthermore, among the 14 genes, we found that SLC7A5 had the highest fold changes in both cell lines upon miR-126-3p overexpression, and ADAM9 had the second highest fold change (2.8-fold) in FTC-133 cells, which were used for both in vitro and in vivo assays in the present study. Both ADAM9 and SLC7A5 play important roles in several cancers and have been shown to be targeted by miR-126-3p [13–16]. Thus, we were interested in determining whether SLC7A5 and ADAM9 protein levels were altered upon miR-126-3p overexpression. We found that miR-126-3p overexpression reduced SLC7A5 protein expression in TPC-1 and XTC-1 cells (Fig 5A) and reduced ADAM9 protein expression in all three cell lines (TPC-1, FTC-133, and XTC-1) (Fig 5B). We also found that miR-126-3p overexpression downregulated ADAM9 protein expression in FTC-133-luc2 tumor xenografts that had been inoculated subcutaneously into the flanks of athymic nude mice and allowed to develop for 10 days (Fig 5C). Given these results, we next determined whether ADAM9 and/or SLC7A5 were direct targets of miR-126-3p. We performed luciferase assays on FTC-133 cells co-transfected with pEZX-ADAM9-3′UTR (vector with 3′-UTR of ADAM9) and miR-126-3p or miR-NC. We found that miR-126-3p overexpression significantly decreased luciferase activity as compared to the negative control (Fig 5D), suggesting that miR-126-3p directly targets the 3′-UTR region of ADAM9 and SLC7A5 in thyroid cancer cells. We next analyzed whether there was an association between miR-126-3p expression and ADAM9 and SLC7A5 mRNA expression in the TCGA papillary thyroid cancer dataset, and found a significant inverse association with SLC7A5 mRNA expression (r = −0.257, p<0.01) but not with ADAM9 mRNA expression (Fig 5E).


miR-126-3p Inhibits Thyroid Cancer Cell Growth and Metastasis, and Is Associated with Aggressive Thyroid Cancer.

Xiong Y, Kotian S, Zeiger MA, Zhang L, Kebebew E - PLoS ONE (2015)

miR-126-3p regulates and directly targets SLC7A5 and ADAM9 protein expression in thyroid cancer cells in vitro and in vivo.(A) Immunoblots of SLC7A5 and GAPDH in TPC-1 and XTC-1 cell lines, which were transfected with either miR-126-3p or miR-NC for 72 hours. The FTC-133 cell line had no detectable protein expression for SLC7A5. (B) Immunoblots for ADAM9 and GAPDH in TPC-1, FTC-133 and XTC-1 cell lines, which were transfected with either miR-126-3p or miR-NC for 72 hours in vitro. (C) Immunoblots for detecting ADAM9 and GAPDH in FTC-133-Luc2 tumor xenografts that had been inoculated subcutaneously into the flanks of athymic nude mice and allowed to develop for 10 days. (D) Luciferase activity of pEZX-SLC7A5-3′UTR and pEZX-SLC7A5-3′UTR in FTC-133 cells when co-transfected with miR-126-3p or miR-NC. All luciferase measurements were made in triplicate and readings were performed 24 hours post-transfection. Error bars represent SEM (*** indicates p<0.001). (E) The expression level of miR-126-3p is significantly inversely associated with the expression level of SLC7A5 in 481 papillary thyroid cancer samples from the TCGA dataset. *** indicates p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526518&req=5

pone.0130496.g005: miR-126-3p regulates and directly targets SLC7A5 and ADAM9 protein expression in thyroid cancer cells in vitro and in vivo.(A) Immunoblots of SLC7A5 and GAPDH in TPC-1 and XTC-1 cell lines, which were transfected with either miR-126-3p or miR-NC for 72 hours. The FTC-133 cell line had no detectable protein expression for SLC7A5. (B) Immunoblots for ADAM9 and GAPDH in TPC-1, FTC-133 and XTC-1 cell lines, which were transfected with either miR-126-3p or miR-NC for 72 hours in vitro. (C) Immunoblots for detecting ADAM9 and GAPDH in FTC-133-Luc2 tumor xenografts that had been inoculated subcutaneously into the flanks of athymic nude mice and allowed to develop for 10 days. (D) Luciferase activity of pEZX-SLC7A5-3′UTR and pEZX-SLC7A5-3′UTR in FTC-133 cells when co-transfected with miR-126-3p or miR-NC. All luciferase measurements were made in triplicate and readings were performed 24 hours post-transfection. Error bars represent SEM (*** indicates p<0.001). (E) The expression level of miR-126-3p is significantly inversely associated with the expression level of SLC7A5 in 481 papillary thyroid cancer samples from the TCGA dataset. *** indicates p<0.001.
Mentions: Integrated analyses of the genome-wide gene expression data and target scan prediction revealed 14 genes as targets of miR-126-3p (S1 Table). We then performed a pathway analysis with these 14 genes. The top diseases and biological functions identified by IPA are summarized in S2 Table. Cancer was the top disease category, with four molecules involved in this pathway that were significantly reduced by miR-126-3p overexpression. Furthermore, among the 14 genes, we found that SLC7A5 had the highest fold changes in both cell lines upon miR-126-3p overexpression, and ADAM9 had the second highest fold change (2.8-fold) in FTC-133 cells, which were used for both in vitro and in vivo assays in the present study. Both ADAM9 and SLC7A5 play important roles in several cancers and have been shown to be targeted by miR-126-3p [13–16]. Thus, we were interested in determining whether SLC7A5 and ADAM9 protein levels were altered upon miR-126-3p overexpression. We found that miR-126-3p overexpression reduced SLC7A5 protein expression in TPC-1 and XTC-1 cells (Fig 5A) and reduced ADAM9 protein expression in all three cell lines (TPC-1, FTC-133, and XTC-1) (Fig 5B). We also found that miR-126-3p overexpression downregulated ADAM9 protein expression in FTC-133-luc2 tumor xenografts that had been inoculated subcutaneously into the flanks of athymic nude mice and allowed to develop for 10 days (Fig 5C). Given these results, we next determined whether ADAM9 and/or SLC7A5 were direct targets of miR-126-3p. We performed luciferase assays on FTC-133 cells co-transfected with pEZX-ADAM9-3′UTR (vector with 3′-UTR of ADAM9) and miR-126-3p or miR-NC. We found that miR-126-3p overexpression significantly decreased luciferase activity as compared to the negative control (Fig 5D), suggesting that miR-126-3p directly targets the 3′-UTR region of ADAM9 and SLC7A5 in thyroid cancer cells. We next analyzed whether there was an association between miR-126-3p expression and ADAM9 and SLC7A5 mRNA expression in the TCGA papillary thyroid cancer dataset, and found a significant inverse association with SLC7A5 mRNA expression (r = −0.257, p<0.01) but not with ADAM9 mRNA expression (Fig 5E).

Bottom Line: We found that miR-126-3p expression was significantly lower in larger tumors, in tumor samples with extrathyroidal invasion, and in higher risk group thyroid cancer in 496 papillary thyroid cancer samples from The Cancer Genome Atlas study cohort.In an independent sample set, lower miR-126-3p expression was observed in follicular thyroid cancers (which have capsular and angioinvasion) as compared to follicular adenomas.Of these 14 genes, SLC7A5 and ADAM9 were confirmed to be inhibited by miR-126-3p overexpression and to be direct targets of miR-136-3p.

View Article: PubMed Central - PubMed

Affiliation: Endocrine Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.

ABSTRACT

Background: Previous studies have shown that microRNAs are dysregulated in thyroid cancer and play important roles in the post-transcriptional regulation of target oncogenes and/or tumor suppressor genes.

Methodology/principal findings: We studied the function of miR-126-3p in thyroid cancer cells, and as a marker of disease aggressiveness. We found that miR-126-3p expression was significantly lower in larger tumors, in tumor samples with extrathyroidal invasion, and in higher risk group thyroid cancer in 496 papillary thyroid cancer samples from The Cancer Genome Atlas study cohort. In an independent sample set, lower miR-126-3p expression was observed in follicular thyroid cancers (which have capsular and angioinvasion) as compared to follicular adenomas. Mechanistically, ectopic overexpression of miR-126-3p significantly inhibited thyroid cancer cell proliferation, in vitro (p<0.01) and in vivo (p<0.01), colony formation (p<0.01), tumor spheroid formation (p<0.05), cellular migration (p<0.05), VEGF secretion and endothelial tube formation, and lung metastasis in vivo. We found 14 predicted target genes, which were significantly altered upon miR-126-3p transfection in thyroid cancer cells, and which are involved in cancer biology. Of these 14 genes, SLC7A5 and ADAM9 were confirmed to be inhibited by miR-126-3p overexpression and to be direct targets of miR-136-3p.

Conclusions/significance: To our knowledge, this is the first study to demonstrate that miR-126-3p has a tumor-suppressive function in thyroid cancer cells, and is associated with aggressive disease phenotype.

No MeSH data available.


Related in: MedlinePlus