Limits...
hsa-mir-30c promotes the invasive phenotype of metastatic breast cancer cells by targeting NOV/CCN3.

Dobson JR, Taipaleenmäki H, Hu YJ, Hong D, van Wijnen AJ, Stein JL, Stein GS, Lian JB, Pratap J - Cancer Cell Int. (2014)

Bottom Line: Significant effects were determined using Student's T-tests with Welch's correction for unequal variance.Depleting NOV by siRNA caused a significant increase in the invasiveness of MDA-MB-231 cells is a regulatory protein associated with the extracellular matrix.Our findings allow for mechanistic insight into the clinical observation of poor survival of patients with elevated hsa-mir-30c levels, which can be considered for miRNA-based translational studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, 55 Lake Ave, North, Worcester 01655, MA, USA ; Current address: Center for Computational Molecular Biology, Department of Molecular Biology, Cell Biology, and Biochemistry, and Department of Computer Science, Brown University, 115 Waterman Street, Providence 02912, RI, USA.

ABSTRACT

Background: For treatment and prevention of metastatic disease, one of the premier challenges is the identification of pathways and proteins to target for clinical intervention. Micro RNAs (miRNAs) are short, non-coding RNAs, which regulate cellular activities by either mRNA degradation or translational inhibition. Our studies focused on the invasive properties of hsa-mir30c based on its high expression in MDA-MB-231 metastatic cells and our bioinformatic analysis of the Cancer Genome Atlas that identified aberrant hsa-mir-30c to be associated with poor survival.

Methods: Contributions of hsa-mir-30c to breast cancer cell invasion were examined by Matrigel invasion transwell assays following modulation of hsa-mir-30c or hsa-mir-30c* levels in MDA-MB-231 cells. hsa-mir-30c in silico predicted targets linked to cell invasion were screened for targeting by hsa-mir-30c in metastatic breast cancer cells by RT-qPCR. The contribution to invasion by a target of hsa-mir-30c, Nephroblastoma overexpressed (NOV), was characterized by siRNA and invasion assays. Significant effects were determined using Student's T-tests with Welch's correction for unequal variance.

Results: MCF-7 and MDA-MB-231 cells were used as models of poorly invasive and late-stage metastatic disease, respectively. By modulating the levels of hsa-mir-30c in these cells, we observed concomitant changes in breast cancer cell invasiveness. From predicted targets of hsa-mir-30c that were related to cellular migration and invasion, NOV/CCN3 was identified as a novel target of hsa-mir-30c. Depleting NOV by siRNA caused a significant increase in the invasiveness of MDA-MB-231 cells is a regulatory protein associated with the extracellular matrix.

Conclusions: NOV/CCN3 expression, which protects cells from invasion, is known in patient tumors to inversely correlate with advanced breast cancer and metastasis. This study has identified a novel target of hsa-mir-30c, NOV, which is an inhibitor of the invasiveness of metastatic breast cancer cells. Thus, hsa-mir-30c-mediated inhibition of NOV levels promotes the invasive phenotype of MDA-MB-231 cells and significantly, the miR-30/NOV pathways is independent of RUNX2, a known target of hsa-mir-30c that promotes osteolytic disease in metastatic breast cancer cells. Our findings allow for mechanistic insight into the clinical observation of poor survival of patients with elevated hsa-mir-30c levels, which can be considered for miRNA-based translational studies.

No MeSH data available.


Related in: MedlinePlus

NOV inhibits the invasiveness of MDA-MB-231 cells. (A) Representative Western blot for NOV (upper blot) and tubulin (lower blot) 48 h post-transfection with siRNA. Vertical dashed line indicates where image of gel was cut for figure. (B) Representative image of HEMA-3 stained cells, which migrated through either the control inserts (upper row) or Matrigel inserts (lower row) after 48 h of transfection with siRNA. (C) Quantification of 4 technical replicates of 2 biological replicates measuring the percent of cells that migrated through the control inserts (100 % being the number of cells loaded into the inserts). (D) Quantification of 4 technical replicates of 2 biological replicates measuring the percent of cells that invaded through the Matrigel normalized by the number of cells migrated through the control inserts. (A-D) NS = Non-silencing siRNA, siNOV = NOV siRNA. (C, D) Bars equal mean, error bars equal SEM. ** = Wilcoxon Rank Sum p-value < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4129468&req=5

Figure 5: NOV inhibits the invasiveness of MDA-MB-231 cells. (A) Representative Western blot for NOV (upper blot) and tubulin (lower blot) 48 h post-transfection with siRNA. Vertical dashed line indicates where image of gel was cut for figure. (B) Representative image of HEMA-3 stained cells, which migrated through either the control inserts (upper row) or Matrigel inserts (lower row) after 48 h of transfection with siRNA. (C) Quantification of 4 technical replicates of 2 biological replicates measuring the percent of cells that migrated through the control inserts (100 % being the number of cells loaded into the inserts). (D) Quantification of 4 technical replicates of 2 biological replicates measuring the percent of cells that invaded through the Matrigel normalized by the number of cells migrated through the control inserts. (A-D) NS = Non-silencing siRNA, siNOV = NOV siRNA. (C, D) Bars equal mean, error bars equal SEM. ** = Wilcoxon Rank Sum p-value < 0.01.

Mentions: To determine the extent to which hsa-mir-30c targeting and reduction of NOV was involved in the increased invasion of MDA-MB-231 cells following transfection of hsa-mir-30c, we transfected MDA-MB-231 cells with NOV-specific siRNA to reduce NOV protein levels without directly affecting other targets of hsa-mir-30c (Figure 5A). We postulate if hsa-mir-30c-mediated down-regulation of NOV levels is a key contributor to the invasiveness of MDA-MB-231 cells, we should therefore observe that siRNA-mediated knockdown of NOV results in an invasive phenotype similar to overexpression of hsa-mir-30c. The levels of NOV were significantly reduced by the siRNA (Figure 5A), and using Matrigel invasion assay, we observed an increase in the number of MDA-MB-231 that invaded through the Matrigel (Figure 5B-D). MDA-MB-231 cells lacking NOV were significantly more invasive (Figure 5D), yet were less migratory (Figure 5C). While it is often observed that trends in invasion and migration are positively correlated, we observed less migration coupled with increased invasion in the absence of NOV. These results suggest that in MDA-MB-231 cells, NOV has distinct regulatory roles for the pathways governing invasion and migration. Taken together, our findings support the conclusion that the targeting of NOV by hsa-mir-30c is an important factor in the invasive phenotype evidenced by the MDA-MB-231 cells as illustrated in Figure 6.


hsa-mir-30c promotes the invasive phenotype of metastatic breast cancer cells by targeting NOV/CCN3.

Dobson JR, Taipaleenmäki H, Hu YJ, Hong D, van Wijnen AJ, Stein JL, Stein GS, Lian JB, Pratap J - Cancer Cell Int. (2014)

NOV inhibits the invasiveness of MDA-MB-231 cells. (A) Representative Western blot for NOV (upper blot) and tubulin (lower blot) 48 h post-transfection with siRNA. Vertical dashed line indicates where image of gel was cut for figure. (B) Representative image of HEMA-3 stained cells, which migrated through either the control inserts (upper row) or Matrigel inserts (lower row) after 48 h of transfection with siRNA. (C) Quantification of 4 technical replicates of 2 biological replicates measuring the percent of cells that migrated through the control inserts (100 % being the number of cells loaded into the inserts). (D) Quantification of 4 technical replicates of 2 biological replicates measuring the percent of cells that invaded through the Matrigel normalized by the number of cells migrated through the control inserts. (A-D) NS = Non-silencing siRNA, siNOV = NOV siRNA. (C, D) Bars equal mean, error bars equal SEM. ** = Wilcoxon Rank Sum p-value < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4129468&req=5

Figure 5: NOV inhibits the invasiveness of MDA-MB-231 cells. (A) Representative Western blot for NOV (upper blot) and tubulin (lower blot) 48 h post-transfection with siRNA. Vertical dashed line indicates where image of gel was cut for figure. (B) Representative image of HEMA-3 stained cells, which migrated through either the control inserts (upper row) or Matrigel inserts (lower row) after 48 h of transfection with siRNA. (C) Quantification of 4 technical replicates of 2 biological replicates measuring the percent of cells that migrated through the control inserts (100 % being the number of cells loaded into the inserts). (D) Quantification of 4 technical replicates of 2 biological replicates measuring the percent of cells that invaded through the Matrigel normalized by the number of cells migrated through the control inserts. (A-D) NS = Non-silencing siRNA, siNOV = NOV siRNA. (C, D) Bars equal mean, error bars equal SEM. ** = Wilcoxon Rank Sum p-value < 0.01.
Mentions: To determine the extent to which hsa-mir-30c targeting and reduction of NOV was involved in the increased invasion of MDA-MB-231 cells following transfection of hsa-mir-30c, we transfected MDA-MB-231 cells with NOV-specific siRNA to reduce NOV protein levels without directly affecting other targets of hsa-mir-30c (Figure 5A). We postulate if hsa-mir-30c-mediated down-regulation of NOV levels is a key contributor to the invasiveness of MDA-MB-231 cells, we should therefore observe that siRNA-mediated knockdown of NOV results in an invasive phenotype similar to overexpression of hsa-mir-30c. The levels of NOV were significantly reduced by the siRNA (Figure 5A), and using Matrigel invasion assay, we observed an increase in the number of MDA-MB-231 that invaded through the Matrigel (Figure 5B-D). MDA-MB-231 cells lacking NOV were significantly more invasive (Figure 5D), yet were less migratory (Figure 5C). While it is often observed that trends in invasion and migration are positively correlated, we observed less migration coupled with increased invasion in the absence of NOV. These results suggest that in MDA-MB-231 cells, NOV has distinct regulatory roles for the pathways governing invasion and migration. Taken together, our findings support the conclusion that the targeting of NOV by hsa-mir-30c is an important factor in the invasive phenotype evidenced by the MDA-MB-231 cells as illustrated in Figure 6.

Bottom Line: Significant effects were determined using Student's T-tests with Welch's correction for unequal variance.Depleting NOV by siRNA caused a significant increase in the invasiveness of MDA-MB-231 cells is a regulatory protein associated with the extracellular matrix.Our findings allow for mechanistic insight into the clinical observation of poor survival of patients with elevated hsa-mir-30c levels, which can be considered for miRNA-based translational studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, 55 Lake Ave, North, Worcester 01655, MA, USA ; Current address: Center for Computational Molecular Biology, Department of Molecular Biology, Cell Biology, and Biochemistry, and Department of Computer Science, Brown University, 115 Waterman Street, Providence 02912, RI, USA.

ABSTRACT

Background: For treatment and prevention of metastatic disease, one of the premier challenges is the identification of pathways and proteins to target for clinical intervention. Micro RNAs (miRNAs) are short, non-coding RNAs, which regulate cellular activities by either mRNA degradation or translational inhibition. Our studies focused on the invasive properties of hsa-mir30c based on its high expression in MDA-MB-231 metastatic cells and our bioinformatic analysis of the Cancer Genome Atlas that identified aberrant hsa-mir-30c to be associated with poor survival.

Methods: Contributions of hsa-mir-30c to breast cancer cell invasion were examined by Matrigel invasion transwell assays following modulation of hsa-mir-30c or hsa-mir-30c* levels in MDA-MB-231 cells. hsa-mir-30c in silico predicted targets linked to cell invasion were screened for targeting by hsa-mir-30c in metastatic breast cancer cells by RT-qPCR. The contribution to invasion by a target of hsa-mir-30c, Nephroblastoma overexpressed (NOV), was characterized by siRNA and invasion assays. Significant effects were determined using Student's T-tests with Welch's correction for unequal variance.

Results: MCF-7 and MDA-MB-231 cells were used as models of poorly invasive and late-stage metastatic disease, respectively. By modulating the levels of hsa-mir-30c in these cells, we observed concomitant changes in breast cancer cell invasiveness. From predicted targets of hsa-mir-30c that were related to cellular migration and invasion, NOV/CCN3 was identified as a novel target of hsa-mir-30c. Depleting NOV by siRNA caused a significant increase in the invasiveness of MDA-MB-231 cells is a regulatory protein associated with the extracellular matrix.

Conclusions: NOV/CCN3 expression, which protects cells from invasion, is known in patient tumors to inversely correlate with advanced breast cancer and metastasis. This study has identified a novel target of hsa-mir-30c, NOV, which is an inhibitor of the invasiveness of metastatic breast cancer cells. Thus, hsa-mir-30c-mediated inhibition of NOV levels promotes the invasive phenotype of MDA-MB-231 cells and significantly, the miR-30/NOV pathways is independent of RUNX2, a known target of hsa-mir-30c that promotes osteolytic disease in metastatic breast cancer cells. Our findings allow for mechanistic insight into the clinical observation of poor survival of patients with elevated hsa-mir-30c levels, which can be considered for miRNA-based translational studies.

No MeSH data available.


Related in: MedlinePlus