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Tumor suppressive microRNA-137 negatively regulates Musashi-1 and colorectal cancer progression.

Smith AR, Marquez RT, Tsao WC, Pathak S, Roy A, Ping J, Wilkerson B, Lan L, Meng W, Neufeld KL, Sun XF, Xu L - Oncotarget (2015)

Bottom Line: MicroRNA miR-137 was identified as a MSI1-targeting microRNA by immunoblotting and luciferase reporter assays.In addition to reduced MSI1 protein, exogenous expression of miR-137 inhibited cell growth, colony formation, and tumorsphere growth of colon cancer cells.Finally, in vivo studies demonstrated that induction of miR-137 can decrease growth of human colon cancer xenografts.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA.

ABSTRACT
Stem cell marker, Musashi-1 (MSI1) is over-expressed in many cancer types; however the molecular mechanisms involved in MSI1 over-expression are not well understood. We investigated the microRNA (miRNA) regulation of MSI1 and the implications this regulation plays in colorectal cancer. MicroRNA miR-137 was identified as a MSI1-targeting microRNA by immunoblotting and luciferase reporter assays. MSI1 protein was found to be highly expressed in 79% of primary rectal tumors (n=146), while miR-137 expression was decreased in 84% of the rectal tumor tissues (n=68) compared to paired normal mucosal samples. In addition to reduced MSI1 protein, exogenous expression of miR-137 inhibited cell growth, colony formation, and tumorsphere growth of colon cancer cells. Finally, in vivo studies demonstrated that induction of miR-137 can decrease growth of human colon cancer xenografts. Our results demonstrate that miR-137 acts as a tumor-suppressive miRNA in colorectal cancers and negatively regulates oncogenic MSI1.

No MeSH data available.


Related in: MedlinePlus

miRNA regulation of MSI1(A) Expression of MSI1 mRNA and protein analyzed in a panel of colon cancer cell lines using quantitative real-time PCR and Western blotting. mRNA normalized to GAPDH and set relative to expression in normal colon epithelial cell line, CCD-841. (B) Venn diagram displaying highly conserved miRNAs predicted to bind to MSI1 3′UTR using miRanda, TargetScan and PicTar prediction software. (C) MSI1 protein expression analyzed in HCT-116 and DLD-1 colon cancer cell lines transfected with a panel of miRNA mimics as compared to cells transfected with a NC miRNA mimic. (D) MSI1 protein expression analyzed in HT29 and HCT-116 β/W colon cancer cells lines transfected with miR-137 mimic as compared to cells transfected with a NC miRNA mimic. (E) Precursor and mature miR-137 expression was analyzed in a panel of cell lines using qRT-PCR and Taqman PCR. Pre-miR-137 was normalized to GAPDH and mature miR-137 expression was normalized to RNU6b. Expression data was set relative to CCD-841.
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Figure 1: miRNA regulation of MSI1(A) Expression of MSI1 mRNA and protein analyzed in a panel of colon cancer cell lines using quantitative real-time PCR and Western blotting. mRNA normalized to GAPDH and set relative to expression in normal colon epithelial cell line, CCD-841. (B) Venn diagram displaying highly conserved miRNAs predicted to bind to MSI1 3′UTR using miRanda, TargetScan and PicTar prediction software. (C) MSI1 protein expression analyzed in HCT-116 and DLD-1 colon cancer cell lines transfected with a panel of miRNA mimics as compared to cells transfected with a NC miRNA mimic. (D) MSI1 protein expression analyzed in HT29 and HCT-116 β/W colon cancer cells lines transfected with miR-137 mimic as compared to cells transfected with a NC miRNA mimic. (E) Precursor and mature miR-137 expression was analyzed in a panel of cell lines using qRT-PCR and Taqman PCR. Pre-miR-137 was normalized to GAPDH and mature miR-137 expression was normalized to RNU6b. Expression data was set relative to CCD-841.

Mentions: MSI1 mRNA and protein is over-expressed in a panel of colon cancer cell lines compared to normal colon epithelial cell line, CCD-841 (Figure 1A). An apparent uncoupling of the mRNA and protein levels of MSI1 is revealed in this panel of cell lines, suggesting post-transcriptional regulation of MSI1. The molecular mechanism for MSI1 over-expression in colorectal cancer is not well understood. One possibility is a dysregulation of miRNA regulation. We utilized three miRNA targeting prediction programs (miRanda, TargetScan, PicTar) to identify highly conserved putative miRNA binding sites in MSI1 3′UTR. Using a variety of computational algorithms based on seed sequence position, pairing and conservation, these programs predict miRNA sites within target genes 3′UTR [17-19]. Among the three prediction programs, five overlapping miRNAs contained conserved, potential binding sites within MSI1 3′UTR; miR-125b, miR-137, miR-144, miR-185, and miR-342-3p (Figure 1B, Supplemental Table 1).


Tumor suppressive microRNA-137 negatively regulates Musashi-1 and colorectal cancer progression.

Smith AR, Marquez RT, Tsao WC, Pathak S, Roy A, Ping J, Wilkerson B, Lan L, Meng W, Neufeld KL, Sun XF, Xu L - Oncotarget (2015)

miRNA regulation of MSI1(A) Expression of MSI1 mRNA and protein analyzed in a panel of colon cancer cell lines using quantitative real-time PCR and Western blotting. mRNA normalized to GAPDH and set relative to expression in normal colon epithelial cell line, CCD-841. (B) Venn diagram displaying highly conserved miRNAs predicted to bind to MSI1 3′UTR using miRanda, TargetScan and PicTar prediction software. (C) MSI1 protein expression analyzed in HCT-116 and DLD-1 colon cancer cell lines transfected with a panel of miRNA mimics as compared to cells transfected with a NC miRNA mimic. (D) MSI1 protein expression analyzed in HT29 and HCT-116 β/W colon cancer cells lines transfected with miR-137 mimic as compared to cells transfected with a NC miRNA mimic. (E) Precursor and mature miR-137 expression was analyzed in a panel of cell lines using qRT-PCR and Taqman PCR. Pre-miR-137 was normalized to GAPDH and mature miR-137 expression was normalized to RNU6b. Expression data was set relative to CCD-841.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494958&req=5

Figure 1: miRNA regulation of MSI1(A) Expression of MSI1 mRNA and protein analyzed in a panel of colon cancer cell lines using quantitative real-time PCR and Western blotting. mRNA normalized to GAPDH and set relative to expression in normal colon epithelial cell line, CCD-841. (B) Venn diagram displaying highly conserved miRNAs predicted to bind to MSI1 3′UTR using miRanda, TargetScan and PicTar prediction software. (C) MSI1 protein expression analyzed in HCT-116 and DLD-1 colon cancer cell lines transfected with a panel of miRNA mimics as compared to cells transfected with a NC miRNA mimic. (D) MSI1 protein expression analyzed in HT29 and HCT-116 β/W colon cancer cells lines transfected with miR-137 mimic as compared to cells transfected with a NC miRNA mimic. (E) Precursor and mature miR-137 expression was analyzed in a panel of cell lines using qRT-PCR and Taqman PCR. Pre-miR-137 was normalized to GAPDH and mature miR-137 expression was normalized to RNU6b. Expression data was set relative to CCD-841.
Mentions: MSI1 mRNA and protein is over-expressed in a panel of colon cancer cell lines compared to normal colon epithelial cell line, CCD-841 (Figure 1A). An apparent uncoupling of the mRNA and protein levels of MSI1 is revealed in this panel of cell lines, suggesting post-transcriptional regulation of MSI1. The molecular mechanism for MSI1 over-expression in colorectal cancer is not well understood. One possibility is a dysregulation of miRNA regulation. We utilized three miRNA targeting prediction programs (miRanda, TargetScan, PicTar) to identify highly conserved putative miRNA binding sites in MSI1 3′UTR. Using a variety of computational algorithms based on seed sequence position, pairing and conservation, these programs predict miRNA sites within target genes 3′UTR [17-19]. Among the three prediction programs, five overlapping miRNAs contained conserved, potential binding sites within MSI1 3′UTR; miR-125b, miR-137, miR-144, miR-185, and miR-342-3p (Figure 1B, Supplemental Table 1).

Bottom Line: MicroRNA miR-137 was identified as a MSI1-targeting microRNA by immunoblotting and luciferase reporter assays.In addition to reduced MSI1 protein, exogenous expression of miR-137 inhibited cell growth, colony formation, and tumorsphere growth of colon cancer cells.Finally, in vivo studies demonstrated that induction of miR-137 can decrease growth of human colon cancer xenografts.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA.

ABSTRACT
Stem cell marker, Musashi-1 (MSI1) is over-expressed in many cancer types; however the molecular mechanisms involved in MSI1 over-expression are not well understood. We investigated the microRNA (miRNA) regulation of MSI1 and the implications this regulation plays in colorectal cancer. MicroRNA miR-137 was identified as a MSI1-targeting microRNA by immunoblotting and luciferase reporter assays. MSI1 protein was found to be highly expressed in 79% of primary rectal tumors (n=146), while miR-137 expression was decreased in 84% of the rectal tumor tissues (n=68) compared to paired normal mucosal samples. In addition to reduced MSI1 protein, exogenous expression of miR-137 inhibited cell growth, colony formation, and tumorsphere growth of colon cancer cells. Finally, in vivo studies demonstrated that induction of miR-137 can decrease growth of human colon cancer xenografts. Our results demonstrate that miR-137 acts as a tumor-suppressive miRNA in colorectal cancers and negatively regulates oncogenic MSI1.

No MeSH data available.


Related in: MedlinePlus