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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

Predictive, ontological, and qPCR screen for hsa-mir-30c reveals NOV as a target of hsa-mir-30c. (A) The log2 of the fold change in mean with SEM detection levels (hsa-mir-30c/non-targeting miRNA) is plotted for each set of primers for each transcript normalized to HPRT using delta-delta Ct method [51]. (B) Representative Western blots for lysates extracted from MDA-MB-231 cells following 48 h of transient transfection with either non-targeting miRNA (NT) or hsa-mir-30c (30c). Top blot: NOV, bottom blot: Lamin C. (C) qPCR for NOV levels for two technical replicates each for three biological replicates following 48 h transient transfections of non-targeting miRNA (NT) or hsa-mir-30c (30c) normalized to HPRT using delta-delta Ct method [51]. Mean with SEM. ** = p-value from Student’s t-test with Welch correction < 0.01. (D) Alignment of hsa-mir-30c (top sequences) with the 3’-UTR of NOV (bottom sequences) with the 5’-positions within the NOV 3’-UTR being relative to the 5’-start of the 3’-UTR for each of the three predicted targeting sites. Target scores are provided by mirSVR. Uppercase letters linked with a “/” character indicates a perfect match, while uppercase letters linked with a “:” character indicates a wobble pair.
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Figure 3: Predictive, ontological, and qPCR screen for hsa-mir-30c reveals NOV as a target of hsa-mir-30c. (A) The log2 of the fold change in mean with SEM detection levels (hsa-mir-30c/non-targeting miRNA) is plotted for each set of primers for each transcript normalized to HPRT using delta-delta Ct method [51]. (B) Representative Western blots for lysates extracted from MDA-MB-231 cells following 48 h of transient transfection with either non-targeting miRNA (NT) or hsa-mir-30c (30c). Top blot: NOV, bottom blot: Lamin C. (C) qPCR for NOV levels for two technical replicates each for three biological replicates following 48 h transient transfections of non-targeting miRNA (NT) or hsa-mir-30c (30c) normalized to HPRT using delta-delta Ct method [51]. Mean with SEM. ** = p-value from Student’s t-test with Welch correction < 0.01. (D) Alignment of hsa-mir-30c (top sequences) with the 3’-UTR of NOV (bottom sequences) with the 5’-positions within the NOV 3’-UTR being relative to the 5’-start of the 3’-UTR for each of the three predicted targeting sites. Target scores are provided by mirSVR. Uppercase letters linked with a “/” character indicates a perfect match, while uppercase letters linked with a “:” character indicates a wobble pair.

Mentions: To identify potential targets of hsa-mir-30c participating in the invasive phenotype associated with hsa-mir-30c expression in MDA-MB-231 cells, we performed a screen in three steps: 1) generated a list of potential targets based on seed sequence targeting potential using the top 300 human mRNA targets from www.microrna.org[21]; 2) filtered the list based on known functions and ontological terms for mRNAs that code for proteins associated with invasion, adhesion, or migration, as well as mRNAs that code for transcription factors [22],[23] (Table 1); 3) used qPCR to measure the relative mRNA levels of potential targets in MDA-MB-231 cells that had been transfected with either non-targeting miRNA or hsa-mir-30c. Using this screening approach, we observed that NOV mRNA was one of the two most down regulated in silico-identified target upon transfection of hsa-mir-30c (Figure 3A). We also examined several in silico-predicted targets related to cancer at the protein level and found these to be unaffected by the levels of hsa-mir-30c (Additional file 1: Figure S1). We focused on NOV, which is a member of the CCN family of matricellular signaling proteins, and has context-specific functions as an oncogene/tumor suppressor [24]–[28]. While CELSR3 had similar response trends to hsa-mir-30c overexpression and is involved in cellular adhesion [29], we did not focus on CELSR3 because the expression levels were very low in MDA-MB-231 cells (data not shown) and CELSR3 was previously shown to not affect invasion in metastatic melanoma cells [30]. In parallel we also looked at in silico-predicted targets of hsa-mir-30c that have known functions in cancer and found that none of these predicted targets were affected by overexpression of hsa-mir-30c (Additional file 1: Figure S1). Using Western blotting to detect the levels of NOV protein, we also observed NOV protein to be reduced following transfection of MDA-MB-231 cells with hsa-mir-30c (Figure 3B). The observation that NOV mRNA was reduced upon transfection of hsa-mir-30c was highly reproducible in multiple biological replicates (Figure 3C), consistent with the reduction in protein levels.


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)

Predictive, ontological, and qPCR screen for hsa-mir-30c reveals NOV as a target of hsa-mir-30c. (A) The log2 of the fold change in mean with SEM detection levels (hsa-mir-30c/non-targeting miRNA) is plotted for each set of primers for each transcript normalized to HPRT using delta-delta Ct method [51]. (B) Representative Western blots for lysates extracted from MDA-MB-231 cells following 48 h of transient transfection with either non-targeting miRNA (NT) or hsa-mir-30c (30c). Top blot: NOV, bottom blot: Lamin C. (C) qPCR for NOV levels for two technical replicates each for three biological replicates following 48 h transient transfections of non-targeting miRNA (NT) or hsa-mir-30c (30c) normalized to HPRT using delta-delta Ct method [51]. Mean with SEM. ** = p-value from Student’s t-test with Welch correction < 0.01. (D) Alignment of hsa-mir-30c (top sequences) with the 3’-UTR of NOV (bottom sequences) with the 5’-positions within the NOV 3’-UTR being relative to the 5’-start of the 3’-UTR for each of the three predicted targeting sites. Target scores are provided by mirSVR. Uppercase letters linked with a “/” character indicates a perfect match, while uppercase letters linked with a “:” character indicates a wobble pair.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Predictive, ontological, and qPCR screen for hsa-mir-30c reveals NOV as a target of hsa-mir-30c. (A) The log2 of the fold change in mean with SEM detection levels (hsa-mir-30c/non-targeting miRNA) is plotted for each set of primers for each transcript normalized to HPRT using delta-delta Ct method [51]. (B) Representative Western blots for lysates extracted from MDA-MB-231 cells following 48 h of transient transfection with either non-targeting miRNA (NT) or hsa-mir-30c (30c). Top blot: NOV, bottom blot: Lamin C. (C) qPCR for NOV levels for two technical replicates each for three biological replicates following 48 h transient transfections of non-targeting miRNA (NT) or hsa-mir-30c (30c) normalized to HPRT using delta-delta Ct method [51]. Mean with SEM. ** = p-value from Student’s t-test with Welch correction < 0.01. (D) Alignment of hsa-mir-30c (top sequences) with the 3’-UTR of NOV (bottom sequences) with the 5’-positions within the NOV 3’-UTR being relative to the 5’-start of the 3’-UTR for each of the three predicted targeting sites. Target scores are provided by mirSVR. Uppercase letters linked with a “/” character indicates a perfect match, while uppercase letters linked with a “:” character indicates a wobble pair.
Mentions: To identify potential targets of hsa-mir-30c participating in the invasive phenotype associated with hsa-mir-30c expression in MDA-MB-231 cells, we performed a screen in three steps: 1) generated a list of potential targets based on seed sequence targeting potential using the top 300 human mRNA targets from www.microrna.org[21]; 2) filtered the list based on known functions and ontological terms for mRNAs that code for proteins associated with invasion, adhesion, or migration, as well as mRNAs that code for transcription factors [22],[23] (Table 1); 3) used qPCR to measure the relative mRNA levels of potential targets in MDA-MB-231 cells that had been transfected with either non-targeting miRNA or hsa-mir-30c. Using this screening approach, we observed that NOV mRNA was one of the two most down regulated in silico-identified target upon transfection of hsa-mir-30c (Figure 3A). We also examined several in silico-predicted targets related to cancer at the protein level and found these to be unaffected by the levels of hsa-mir-30c (Additional file 1: Figure S1). We focused on NOV, which is a member of the CCN family of matricellular signaling proteins, and has context-specific functions as an oncogene/tumor suppressor [24]–[28]. While CELSR3 had similar response trends to hsa-mir-30c overexpression and is involved in cellular adhesion [29], we did not focus on CELSR3 because the expression levels were very low in MDA-MB-231 cells (data not shown) and CELSR3 was previously shown to not affect invasion in metastatic melanoma cells [30]. In parallel we also looked at in silico-predicted targets of hsa-mir-30c that have known functions in cancer and found that none of these predicted targets were affected by overexpression of hsa-mir-30c (Additional file 1: Figure S1). Using Western blotting to detect the levels of NOV protein, we also observed NOV protein to be reduced following transfection of MDA-MB-231 cells with hsa-mir-30c (Figure 3B). The observation that NOV mRNA was reduced upon transfection of hsa-mir-30c was highly reproducible in multiple biological replicates (Figure 3C), consistent with the reduction in protein levels.

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