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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 negatively regulates the transcription of the miR-371-373 cluster.(A) (i) Endogenous transcript and (ii) protein levels of SMAR1 detected in the four cell lines HEK293, MCF-7, MDA-MB-231 and T47D by quantitative real time PCR and western blot, respectively. (B) Quantitative PCR analysis to detect endogenous miR-371-373 transcript levels in these four cell lines. (C) Reduced levels of miR-371-373 transcripts upon ectopic expression of SMAR1 using Adeno-SM as determined by quantitative real time PCR in all four cell lines. Empty adenoviral vector (Ad-V) was used as control. (D) Upregulation of miR-371-373 transcript levels upon SMAR1 knockdown using sh1077 construct observed in four cell lines by quantitative real time PCR. shRNA construct was used as control. (E) Ectopic SMAR1 expression and knockdown was confirmed at transcript levels in HEK293, MCF-7, MDA-MB-231 and T47D by quantitative real time PCR. (F) SMAR1 overexpression and knockdown was confirmed at protein levels in HEK293, MCF-7, MDA-MB-231 and T47D by western blot assay. All the western blots are cropped representation of the original blots shown in Supplementary Fig. S8. All the gels are run under same experimental conditions.
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f5: SMAR1 negatively regulates the transcription of the miR-371-373 cluster.(A) (i) Endogenous transcript and (ii) protein levels of SMAR1 detected in the four cell lines HEK293, MCF-7, MDA-MB-231 and T47D by quantitative real time PCR and western blot, respectively. (B) Quantitative PCR analysis to detect endogenous miR-371-373 transcript levels in these four cell lines. (C) Reduced levels of miR-371-373 transcripts upon ectopic expression of SMAR1 using Adeno-SM as determined by quantitative real time PCR in all four cell lines. Empty adenoviral vector (Ad-V) was used as control. (D) Upregulation of miR-371-373 transcript levels upon SMAR1 knockdown using sh1077 construct observed in four cell lines by quantitative real time PCR. shRNA construct was used as control. (E) Ectopic SMAR1 expression and knockdown was confirmed at transcript levels in HEK293, MCF-7, MDA-MB-231 and T47D by quantitative real time PCR. (F) SMAR1 overexpression and knockdown was confirmed at protein levels in HEK293, MCF-7, MDA-MB-231 and T47D by western blot assay. All the western blots are cropped representation of the original blots shown in Supplementary Fig. S8. All the gels are run under same experimental conditions.

Mentions: As the cell lines HEK293, MCF-7, MDA-MB-231 and T47D have an inherent difference in their metastasis potential, we wanted to estimate the endogenous levels of SMAR1 and miR-371-373 transcripts in these lines. We observed that endogenous SMAR1 transcript and protein levels significantly decreased in the metastatic lines as compared to the non-metastatic ones, suggesting a negative correlation (Fig. 5A (i) and (ii)). The quantitative PCR analysis of the miR-371-373 transcripts indicated that the levels of miR-371-373 significantly increase with the metastatic ability of the breast cancer cell line (Fig. 5B). Thus, HEK293 which represented the normal cells showed the least expression of these miR transcripts endogenously and the maximum SMAR1 expression. On the other hand, the metastatic lines MDA-MB-231 and T47D showed the maximum expression of the miR-371-373 transcripts, with T47D showing the highest expression. We next wanted to determine the effect of SMAR1 on the transcription of the microRNAs in this cluster. It was observed that the transcript levels of miR-371-373 cluster were drastically reduced upon ectopic expression of SMAR1 in all the four cell lines (Fig. 5C). The knock down of SMAR1 was found to reverse this effect resulting in increased levels of these transcripts (Fig. 5D). Overexpression and knock down of SMAR1 was confirmed by quantitative real time PCR as well as immunoblot assays (Fig. 5E,F). Thus these results together indicate that SMAR1 regulates the expression of miR-371-373, transcriptionally.


SMAR1 binds to T(C/G) repeat and inhibits tumor progression by regulating miR-371-373 cluster
SMAR1 negatively regulates the transcription of the miR-371-373 cluster.(A) (i) Endogenous transcript and (ii) protein levels of SMAR1 detected in the four cell lines HEK293, MCF-7, MDA-MB-231 and T47D by quantitative real time PCR and western blot, respectively. (B) Quantitative PCR analysis to detect endogenous miR-371-373 transcript levels in these four cell lines. (C) Reduced levels of miR-371-373 transcripts upon ectopic expression of SMAR1 using Adeno-SM as determined by quantitative real time PCR in all four cell lines. Empty adenoviral vector (Ad-V) was used as control. (D) Upregulation of miR-371-373 transcript levels upon SMAR1 knockdown using sh1077 construct observed in four cell lines by quantitative real time PCR. shRNA construct was used as control. (E) Ectopic SMAR1 expression and knockdown was confirmed at transcript levels in HEK293, MCF-7, MDA-MB-231 and T47D by quantitative real time PCR. (F) SMAR1 overexpression and knockdown was confirmed at protein levels in HEK293, MCF-7, MDA-MB-231 and T47D by western blot assay. All the western blots are cropped representation of the original blots shown in Supplementary Fig. S8. All the gels are run under same experimental conditions.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037395&req=5

f5: SMAR1 negatively regulates the transcription of the miR-371-373 cluster.(A) (i) Endogenous transcript and (ii) protein levels of SMAR1 detected in the four cell lines HEK293, MCF-7, MDA-MB-231 and T47D by quantitative real time PCR and western blot, respectively. (B) Quantitative PCR analysis to detect endogenous miR-371-373 transcript levels in these four cell lines. (C) Reduced levels of miR-371-373 transcripts upon ectopic expression of SMAR1 using Adeno-SM as determined by quantitative real time PCR in all four cell lines. Empty adenoviral vector (Ad-V) was used as control. (D) Upregulation of miR-371-373 transcript levels upon SMAR1 knockdown using sh1077 construct observed in four cell lines by quantitative real time PCR. shRNA construct was used as control. (E) Ectopic SMAR1 expression and knockdown was confirmed at transcript levels in HEK293, MCF-7, MDA-MB-231 and T47D by quantitative real time PCR. (F) SMAR1 overexpression and knockdown was confirmed at protein levels in HEK293, MCF-7, MDA-MB-231 and T47D by western blot assay. All the western blots are cropped representation of the original blots shown in Supplementary Fig. S8. All the gels are run under same experimental conditions.
Mentions: As the cell lines HEK293, MCF-7, MDA-MB-231 and T47D have an inherent difference in their metastasis potential, we wanted to estimate the endogenous levels of SMAR1 and miR-371-373 transcripts in these lines. We observed that endogenous SMAR1 transcript and protein levels significantly decreased in the metastatic lines as compared to the non-metastatic ones, suggesting a negative correlation (Fig. 5A (i) and (ii)). The quantitative PCR analysis of the miR-371-373 transcripts indicated that the levels of miR-371-373 significantly increase with the metastatic ability of the breast cancer cell line (Fig. 5B). Thus, HEK293 which represented the normal cells showed the least expression of these miR transcripts endogenously and the maximum SMAR1 expression. On the other hand, the metastatic lines MDA-MB-231 and T47D showed the maximum expression of the miR-371-373 transcripts, with T47D showing the highest expression. We next wanted to determine the effect of SMAR1 on the transcription of the microRNAs in this cluster. It was observed that the transcript levels of miR-371-373 cluster were drastically reduced upon ectopic expression of SMAR1 in all the four cell lines (Fig. 5C). The knock down of SMAR1 was found to reverse this effect resulting in increased levels of these transcripts (Fig. 5D). Overexpression and knock down of SMAR1 was confirmed by quantitative real time PCR as well as immunoblot assays (Fig. 5E,F). Thus these results together indicate that SMAR1 regulates the expression of miR-371-373, transcriptionally.

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