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SMAR1 binds to T(C/G) repeat and inhibits tumor progression by regulating miR-371-373 cluster

View Article: PubMed Central - PubMed

ABSTRACT

Chromatin architecture and dynamics are regulated by various histone and non-histone proteins. The matrix attachment region binding proteins (MARBPs) play a central role in chromatin organization and function through numerous regulatory proteins. In the present study, we demonstrate that nuclear matrix protein SMAR1 orchestrates global gene regulation as determined by massively parallel ChIP-sequencing. The study revealed that SMAR1 binds to T(C/G) repeat and targets genes involved in diverse biological pathways. We observe that SMAR1 binds and targets distinctly different genes based on the availability of p53. Our data suggest that SMAR1 binds and regulates one of the imperative microRNA clusters in cancer and metastasis, miR-371-373. It negatively regulates miR-371-373 transcription as confirmed by SMAR1 overexpression and knockdown studies. Further, deletion studies indicate that a ~200 bp region in the miR-371-373 promoter is necessary for SMAR1 binding and transcriptional repression. Recruitment of HDAC1/mSin3A complex by SMAR1, concomitant with alteration of histone marks results in downregulation of the miRNA cluster. The regulation of miR-371-373 by SMAR1 inhibits breast cancer tumorigenesis and metastasis as determined by in vivo experiments. Overall, our study highlights the binding of SMAR1 to T(C/G) repeat and its role in cancer through miR-371-373.

No MeSH data available.


Related in: MedlinePlus

SMAR1 inhibits cancer cells in vitro and in vivo by regulating miR-371-373 cluster.(A) (i) In vitro wound healing assay in control and Adeno-SMAR1 transduced MDA-MB-231 cells showed reduced cell migration upon SMAR1 over expression. (ii) Graphical representation of the distance migrated (microns). (B) In vitro cell invasion assay performed in control MDA-MB-231 cells against different treatments of SMAR1 and miR-371-373 overexpression and/or knockdown alone and in combination. (C) In vivo imaging showing reduction in lung metastasis in mice injected with Adeno-SM transduced MDA-MB-231/Luc cells as compared to the mice injected with control MDA-MB-231/Luc cells. (D) Hematoxylin and Eosin staining of the lung sections of control mice showing alveolar collapse. Lung sections of Adeno-SM treated mice showing normal lung and alveolar architecture. (E) Quantitative real time PCR analysis showing increase in SMAR1 levels in the lung tissues of Adeno-SM treated mice. (F) Quantitative real time PCR analysis showing reduction in the miR-371-373 transcript levels in the lung tissues of Adeno-SM treated mice. (G) Kaplan-Meier relapse free survival analysis for SMAR1 in 1660 breast cancer patients. Lower expression levels of SMAR1 correlated with poor survival (p value = 0.00043). (H) Overall survival analysis for miR-371-5p in 99 breast cancer patients using MIRUMIR prediction tool. Higher expression levels corresponded to poor prognosis of patients (p value = 0.0143). (I) Disease free survival analysis for miR-373 in 99 breast carcinoma patients using MIRUMIR tool. Higher expression levels led to decreased survival of patients (p value = 0.0181). (J) Model depicting the inhibition of tumor progression and metastasis by SMAR1 mediated regulation of miR-371-373 cluster. Under normal scenario or in benign breast cancer, SMAR1 is present in physiologically normal levels in the cell thereby forming a repressor complex at the miR-371-373 promoter and repressing its expression. This allows the tumor suppressor mRNA targets of this cluster like LATS2 (Large Antigen Tumor Suppressor 2), CD44 to be translated preventing cellular transformation and maintaining homeostasis.
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f6: SMAR1 inhibits cancer cells in vitro and in vivo by regulating miR-371-373 cluster.(A) (i) In vitro wound healing assay in control and Adeno-SMAR1 transduced MDA-MB-231 cells showed reduced cell migration upon SMAR1 over expression. (ii) Graphical representation of the distance migrated (microns). (B) In vitro cell invasion assay performed in control MDA-MB-231 cells against different treatments of SMAR1 and miR-371-373 overexpression and/or knockdown alone and in combination. (C) In vivo imaging showing reduction in lung metastasis in mice injected with Adeno-SM transduced MDA-MB-231/Luc cells as compared to the mice injected with control MDA-MB-231/Luc cells. (D) Hematoxylin and Eosin staining of the lung sections of control mice showing alveolar collapse. Lung sections of Adeno-SM treated mice showing normal lung and alveolar architecture. (E) Quantitative real time PCR analysis showing increase in SMAR1 levels in the lung tissues of Adeno-SM treated mice. (F) Quantitative real time PCR analysis showing reduction in the miR-371-373 transcript levels in the lung tissues of Adeno-SM treated mice. (G) Kaplan-Meier relapse free survival analysis for SMAR1 in 1660 breast cancer patients. Lower expression levels of SMAR1 correlated with poor survival (p value = 0.00043). (H) Overall survival analysis for miR-371-5p in 99 breast cancer patients using MIRUMIR prediction tool. Higher expression levels corresponded to poor prognosis of patients (p value = 0.0143). (I) Disease free survival analysis for miR-373 in 99 breast carcinoma patients using MIRUMIR tool. Higher expression levels led to decreased survival of patients (p value = 0.0181). (J) Model depicting the inhibition of tumor progression and metastasis by SMAR1 mediated regulation of miR-371-373 cluster. Under normal scenario or in benign breast cancer, SMAR1 is present in physiologically normal levels in the cell thereby forming a repressor complex at the miR-371-373 promoter and repressing its expression. This allows the tumor suppressor mRNA targets of this cluster like LATS2 (Large Antigen Tumor Suppressor 2), CD44 to be translated preventing cellular transformation and maintaining homeostasis.

Mentions: In order to establish the biological relevance of SMAR1 binding and repressing an important cluster of microRNAs reported to have a role in tumorigenesis and metastasis, we studied the role of SMAR1 in cell migration by wound healing experiment. MDA-MB-231 control and Adeno-SMAR1 transduced confluent cell monolayers were subjected to a wound and time lapse assay was performed to check for cell migration and wound healing. In comparison to the control cells, the SMAR1 overexpressed cells showed significantly retarded cell migration and wound healing (Fig. 6A (i) and (ii)). Next, we compared the cell invasion ability of control MDA-MB-231 cells against different treatments of SMAR1 and miR-371-373 overexpression and/or knockdown alone and in combination (Fig. 6B). It was observed that over expression of SMAR1 severely compromised the invasive capacity of these cells, while SMAR1 knockdown led to an increased invasiveness. The ectopic expression of miR-371, miR-372 and miR-373 enhanced cell invasion, and their respective antagomiRs inhibited it significantly. Over expression of miR-371, 372 and 373 along with Adeno-SMAR1 surpassed the inhibitory effects of SMAR1, making cells as invasive as the control. Similarly, the use of antagomiRs along with SMAR1 knockdown, compensated for the enhanced cell invasion potential, now making it comparable or less invasive than control MDA-MB-231 cells. Similarly, the in vivo tumorigenicity and metastasis experiments in SCID mice using control and Adeno-SMAR1 transduced MDA-MB-231/Luc cells indicated a perturbed metastases in the mice injected with SMAR1 overexpressed cells, while the control mice showed metastatic foci (Fig. 6C). Infiltration and metastasis of cells, accompanied by severe alveolar collapse is evident from the Hematoxylin and Eosin staining of the lung sections of control mice, while the Adeno-SMAR1 treated mice showed comparably normal lung and alveolar architecture (Fig. 6D). Quantitative real time PCR confirmed the increased levels of SMAR1 in the lung tissues of Adeno-SMAR1 treated mice (Fig. 6E). Parallely, we detected severely diminished levels of miR-371-373 transcripts in these lung tissues (Fig. 6F).


SMAR1 binds to T(C/G) repeat and inhibits tumor progression by regulating miR-371-373 cluster
SMAR1 inhibits cancer cells in vitro and in vivo by regulating miR-371-373 cluster.(A) (i) In vitro wound healing assay in control and Adeno-SMAR1 transduced MDA-MB-231 cells showed reduced cell migration upon SMAR1 over expression. (ii) Graphical representation of the distance migrated (microns). (B) In vitro cell invasion assay performed in control MDA-MB-231 cells against different treatments of SMAR1 and miR-371-373 overexpression and/or knockdown alone and in combination. (C) In vivo imaging showing reduction in lung metastasis in mice injected with Adeno-SM transduced MDA-MB-231/Luc cells as compared to the mice injected with control MDA-MB-231/Luc cells. (D) Hematoxylin and Eosin staining of the lung sections of control mice showing alveolar collapse. Lung sections of Adeno-SM treated mice showing normal lung and alveolar architecture. (E) Quantitative real time PCR analysis showing increase in SMAR1 levels in the lung tissues of Adeno-SM treated mice. (F) Quantitative real time PCR analysis showing reduction in the miR-371-373 transcript levels in the lung tissues of Adeno-SM treated mice. (G) Kaplan-Meier relapse free survival analysis for SMAR1 in 1660 breast cancer patients. Lower expression levels of SMAR1 correlated with poor survival (p value = 0.00043). (H) Overall survival analysis for miR-371-5p in 99 breast cancer patients using MIRUMIR prediction tool. Higher expression levels corresponded to poor prognosis of patients (p value = 0.0143). (I) Disease free survival analysis for miR-373 in 99 breast carcinoma patients using MIRUMIR tool. Higher expression levels led to decreased survival of patients (p value = 0.0181). (J) Model depicting the inhibition of tumor progression and metastasis by SMAR1 mediated regulation of miR-371-373 cluster. Under normal scenario or in benign breast cancer, SMAR1 is present in physiologically normal levels in the cell thereby forming a repressor complex at the miR-371-373 promoter and repressing its expression. This allows the tumor suppressor mRNA targets of this cluster like LATS2 (Large Antigen Tumor Suppressor 2), CD44 to be translated preventing cellular transformation and maintaining homeostasis.
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Related In: Results  -  Collection

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f6: SMAR1 inhibits cancer cells in vitro and in vivo by regulating miR-371-373 cluster.(A) (i) In vitro wound healing assay in control and Adeno-SMAR1 transduced MDA-MB-231 cells showed reduced cell migration upon SMAR1 over expression. (ii) Graphical representation of the distance migrated (microns). (B) In vitro cell invasion assay performed in control MDA-MB-231 cells against different treatments of SMAR1 and miR-371-373 overexpression and/or knockdown alone and in combination. (C) In vivo imaging showing reduction in lung metastasis in mice injected with Adeno-SM transduced MDA-MB-231/Luc cells as compared to the mice injected with control MDA-MB-231/Luc cells. (D) Hematoxylin and Eosin staining of the lung sections of control mice showing alveolar collapse. Lung sections of Adeno-SM treated mice showing normal lung and alveolar architecture. (E) Quantitative real time PCR analysis showing increase in SMAR1 levels in the lung tissues of Adeno-SM treated mice. (F) Quantitative real time PCR analysis showing reduction in the miR-371-373 transcript levels in the lung tissues of Adeno-SM treated mice. (G) Kaplan-Meier relapse free survival analysis for SMAR1 in 1660 breast cancer patients. Lower expression levels of SMAR1 correlated with poor survival (p value = 0.00043). (H) Overall survival analysis for miR-371-5p in 99 breast cancer patients using MIRUMIR prediction tool. Higher expression levels corresponded to poor prognosis of patients (p value = 0.0143). (I) Disease free survival analysis for miR-373 in 99 breast carcinoma patients using MIRUMIR tool. Higher expression levels led to decreased survival of patients (p value = 0.0181). (J) Model depicting the inhibition of tumor progression and metastasis by SMAR1 mediated regulation of miR-371-373 cluster. Under normal scenario or in benign breast cancer, SMAR1 is present in physiologically normal levels in the cell thereby forming a repressor complex at the miR-371-373 promoter and repressing its expression. This allows the tumor suppressor mRNA targets of this cluster like LATS2 (Large Antigen Tumor Suppressor 2), CD44 to be translated preventing cellular transformation and maintaining homeostasis.
Mentions: In order to establish the biological relevance of SMAR1 binding and repressing an important cluster of microRNAs reported to have a role in tumorigenesis and metastasis, we studied the role of SMAR1 in cell migration by wound healing experiment. MDA-MB-231 control and Adeno-SMAR1 transduced confluent cell monolayers were subjected to a wound and time lapse assay was performed to check for cell migration and wound healing. In comparison to the control cells, the SMAR1 overexpressed cells showed significantly retarded cell migration and wound healing (Fig. 6A (i) and (ii)). Next, we compared the cell invasion ability of control MDA-MB-231 cells against different treatments of SMAR1 and miR-371-373 overexpression and/or knockdown alone and in combination (Fig. 6B). It was observed that over expression of SMAR1 severely compromised the invasive capacity of these cells, while SMAR1 knockdown led to an increased invasiveness. The ectopic expression of miR-371, miR-372 and miR-373 enhanced cell invasion, and their respective antagomiRs inhibited it significantly. Over expression of miR-371, 372 and 373 along with Adeno-SMAR1 surpassed the inhibitory effects of SMAR1, making cells as invasive as the control. Similarly, the use of antagomiRs along with SMAR1 knockdown, compensated for the enhanced cell invasion potential, now making it comparable or less invasive than control MDA-MB-231 cells. Similarly, the in vivo tumorigenicity and metastasis experiments in SCID mice using control and Adeno-SMAR1 transduced MDA-MB-231/Luc cells indicated a perturbed metastases in the mice injected with SMAR1 overexpressed cells, while the control mice showed metastatic foci (Fig. 6C). Infiltration and metastasis of cells, accompanied by severe alveolar collapse is evident from the Hematoxylin and Eosin staining of the lung sections of control mice, while the Adeno-SMAR1 treated mice showed comparably normal lung and alveolar architecture (Fig. 6D). Quantitative real time PCR confirmed the increased levels of SMAR1 in the lung tissues of Adeno-SMAR1 treated mice (Fig. 6E). Parallely, we detected severely diminished levels of miR-371-373 transcripts in these lung tissues (Fig. 6F).

View Article: PubMed Central - PubMed

ABSTRACT

Chromatin architecture and dynamics are regulated by various histone and non-histone proteins. The matrix attachment region binding proteins (MARBPs) play a central role in chromatin organization and function through numerous regulatory proteins. In the present study, we demonstrate that nuclear matrix protein SMAR1 orchestrates global gene regulation as determined by massively parallel ChIP-sequencing. The study revealed that SMAR1 binds to T(C/G) repeat and targets genes involved in diverse biological pathways. We observe that SMAR1 binds and targets distinctly different genes based on the availability of p53. Our data suggest that SMAR1 binds and regulates one of the imperative microRNA clusters in cancer and metastasis, miR-371-373. It negatively regulates miR-371-373 transcription as confirmed by SMAR1 overexpression and knockdown studies. Further, deletion studies indicate that a ~200 bp region in the miR-371-373 promoter is necessary for SMAR1 binding and transcriptional repression. Recruitment of HDAC1/mSin3A complex by SMAR1, concomitant with alteration of histone marks results in downregulation of the miRNA cluster. The regulation of miR-371-373 by SMAR1 inhibits breast cancer tumorigenesis and metastasis as determined by in vivo experiments. Overall, our study highlights the binding of SMAR1 to T(C/G) repeat and its role in cancer through miR-371-373.

No MeSH data available.


Related in: MedlinePlus