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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 overexpression inhibits cellular proliferation, and colony and spheroid formation.(A–C) Thyroid cancer cell line proliferation with miR-126-3p overexpression. The Y axis represents the cell number. Error bars represent the standard error of the mean (SEM). (* indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001). (D) miR-126-3p overexpression inhibits colony formation in thyroid cancer cells. Colony numbers in FTC-133 cell lines. The Y axis represents the number of colonies per field. Error bars represent SEM (*** indicates p<0.001). (E) miR-126-3p overexpression decreases the size and number of spheroids. Top panel: representative image of spheroids in culture with miR-126-3p overexpression (FTC-133 cells). Lower panel: Quantification of spheroid differences between XTC-1 and FTC-133 cells with miR-126-3p overexpression. The total area occupied by the spheroids within an image was measured by circumscribing the perimeter of each spheroid, marking the entire area, and calculating the pixel numbers with ImageJ software (National Institutes of Health, Bethesda, MD, USA). The Y axis represents the size and number of the spheroids. Error bars represent SEM (* indicates p<0.05).
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pone.0130496.g002: miR-126-3p overexpression inhibits cellular proliferation, and colony and spheroid formation.(A–C) Thyroid cancer cell line proliferation with miR-126-3p overexpression. The Y axis represents the cell number. Error bars represent the standard error of the mean (SEM). (* indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001). (D) miR-126-3p overexpression inhibits colony formation in thyroid cancer cells. Colony numbers in FTC-133 cell lines. The Y axis represents the number of colonies per field. Error bars represent SEM (*** indicates p<0.001). (E) miR-126-3p overexpression decreases the size and number of spheroids. Top panel: representative image of spheroids in culture with miR-126-3p overexpression (FTC-133 cells). Lower panel: Quantification of spheroid differences between XTC-1 and FTC-133 cells with miR-126-3p overexpression. The total area occupied by the spheroids within an image was measured by circumscribing the perimeter of each spheroid, marking the entire area, and calculating the pixel numbers with ImageJ software (National Institutes of Health, Bethesda, MD, USA). The Y axis represents the size and number of the spheroids. Error bars represent SEM (* indicates p<0.05).

Mentions: Given the association between miR-126-3p expression and aggressive thyroid cancer disease phenotype, we wanted to determine if the function of miR-126-3p in thyroid cancer could mechanistically explain this association. We overexpressed miR-126-3p in three well-characterized and authenticated thyroid cancer cell lines (TPC-1, FTC-133 and XTC-1) using miR-NC as a negative control to determine its effect on cellular proliferation. miR-126-3p overexpression inhibited cell proliferation significantly in TPC-1 and FTC-133 cells at 120 hours, by 52% (p<0.001) and 37% (p<0.001), respectively; however, it inhibited proliferation only by 16% in XTC-1 cells at 168 hours (p<0.001) (Fig 2A, 2B and 2C). A soft agar colony formation assay was performed to evaluate anchorage-independent growth in FTC-133, which is a colony-forming thyroid cancer cell line. We found a significantly lower number of colonies in FTC-133 cell lines overexpressing miR-126-3p (Fig 2D). We also studied the effect of miR-126-3p on thyroid cancer cell tumor spheroid formation. The FTC-133 and XTC-1 cell lines form spheroids when cultured in ultra-low adherent culture flasks, and after transfection with miR-126-3p, the number and size of spheroids were significantly decreased (Fig 2E).


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 overexpression inhibits cellular proliferation, and colony and spheroid formation.(A–C) Thyroid cancer cell line proliferation with miR-126-3p overexpression. The Y axis represents the cell number. Error bars represent the standard error of the mean (SEM). (* indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001). (D) miR-126-3p overexpression inhibits colony formation in thyroid cancer cells. Colony numbers in FTC-133 cell lines. The Y axis represents the number of colonies per field. Error bars represent SEM (*** indicates p<0.001). (E) miR-126-3p overexpression decreases the size and number of spheroids. Top panel: representative image of spheroids in culture with miR-126-3p overexpression (FTC-133 cells). Lower panel: Quantification of spheroid differences between XTC-1 and FTC-133 cells with miR-126-3p overexpression. The total area occupied by the spheroids within an image was measured by circumscribing the perimeter of each spheroid, marking the entire area, and calculating the pixel numbers with ImageJ software (National Institutes of Health, Bethesda, MD, USA). The Y axis represents the size and number of the spheroids. Error bars represent SEM (* indicates p<0.05).
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pone.0130496.g002: miR-126-3p overexpression inhibits cellular proliferation, and colony and spheroid formation.(A–C) Thyroid cancer cell line proliferation with miR-126-3p overexpression. The Y axis represents the cell number. Error bars represent the standard error of the mean (SEM). (* indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001). (D) miR-126-3p overexpression inhibits colony formation in thyroid cancer cells. Colony numbers in FTC-133 cell lines. The Y axis represents the number of colonies per field. Error bars represent SEM (*** indicates p<0.001). (E) miR-126-3p overexpression decreases the size and number of spheroids. Top panel: representative image of spheroids in culture with miR-126-3p overexpression (FTC-133 cells). Lower panel: Quantification of spheroid differences between XTC-1 and FTC-133 cells with miR-126-3p overexpression. The total area occupied by the spheroids within an image was measured by circumscribing the perimeter of each spheroid, marking the entire area, and calculating the pixel numbers with ImageJ software (National Institutes of Health, Bethesda, MD, USA). The Y axis represents the size and number of the spheroids. Error bars represent SEM (* indicates p<0.05).
Mentions: Given the association between miR-126-3p expression and aggressive thyroid cancer disease phenotype, we wanted to determine if the function of miR-126-3p in thyroid cancer could mechanistically explain this association. We overexpressed miR-126-3p in three well-characterized and authenticated thyroid cancer cell lines (TPC-1, FTC-133 and XTC-1) using miR-NC as a negative control to determine its effect on cellular proliferation. miR-126-3p overexpression inhibited cell proliferation significantly in TPC-1 and FTC-133 cells at 120 hours, by 52% (p<0.001) and 37% (p<0.001), respectively; however, it inhibited proliferation only by 16% in XTC-1 cells at 168 hours (p<0.001) (Fig 2A, 2B and 2C). A soft agar colony formation assay was performed to evaluate anchorage-independent growth in FTC-133, which is a colony-forming thyroid cancer cell line. We found a significantly lower number of colonies in FTC-133 cell lines overexpressing miR-126-3p (Fig 2D). We also studied the effect of miR-126-3p on thyroid cancer cell tumor spheroid formation. The FTC-133 and XTC-1 cell lines form spheroids when cultured in ultra-low adherent culture flasks, and after transfection with miR-126-3p, the number and size of spheroids were significantly decreased (Fig 2E).

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