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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

miR-137 negatively regulates MSI1(A) MSI1 protein expression in HCT-116 cells transfected with increasing concentrations of miR-137 mimic as compared to cells transfected with NC mimic. (B) Top, MSI1 mRNA and protein expression analyzed in HCT-116 cells transfected with NC mimic, miR-137 mimic and positive control, MSI1-siRNA. mRNA expression analyzed using qRT-PCR, normalized to GAPDH and set relative to NC transfected cells. Bottom, MSI1 protein analyzed using Western blotting with β-actin as loading control. (C) MSI1 protein expression in HEK-293FT cells transfected with miR-137 antagomiR (137-Inh.), negative control antagomiR (NC-Inh.), miR-137 mimic (miR-137), negative control mimic (NC), MSI1 siRNA (si-MSI1) and a negative control siRNA (si-NC). Intensity of MSI1 protein was normalized to β-actin and set relative to control. Change in MSI1 protein shown in red. (D) MSI1 protein expression in HCT-116 transfected with NC-Inh, miR-137-Inh, NC-mimic and miR-137 mimic. (E) HCT-116 cells were transfected with wild-type (WT) or mutant (mut) pSGG-MSI1 3′UTR luciferase construct with miR-137 or NC mimic. Data are means ± SE; n = 3; *** P < 0.001.
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Figure 2: miR-137 negatively regulates MSI1(A) MSI1 protein expression in HCT-116 cells transfected with increasing concentrations of miR-137 mimic as compared to cells transfected with NC mimic. (B) Top, MSI1 mRNA and protein expression analyzed in HCT-116 cells transfected with NC mimic, miR-137 mimic and positive control, MSI1-siRNA. mRNA expression analyzed using qRT-PCR, normalized to GAPDH and set relative to NC transfected cells. Bottom, MSI1 protein analyzed using Western blotting with β-actin as loading control. (C) MSI1 protein expression in HEK-293FT cells transfected with miR-137 antagomiR (137-Inh.), negative control antagomiR (NC-Inh.), miR-137 mimic (miR-137), negative control mimic (NC), MSI1 siRNA (si-MSI1) and a negative control siRNA (si-NC). Intensity of MSI1 protein was normalized to β-actin and set relative to control. Change in MSI1 protein shown in red. (D) MSI1 protein expression in HCT-116 transfected with NC-Inh, miR-137-Inh, NC-mimic and miR-137 mimic. (E) HCT-116 cells were transfected with wild-type (WT) or mutant (mut) pSGG-MSI1 3′UTR luciferase construct with miR-137 or NC mimic. Data are means ± SE; n = 3; *** P < 0.001.

Mentions: Since miR-137 significantly decreased MSI1 protein expression in both HCT-116 and DLD-1 compared to the other mimics; we focused this study on understanding the miR-137-mediated regulation of MSI1. miR-137 reduced MSI1 protein expression in a dose-dependent manner (Figure 2A). Furthermore, miR-137 decreased MSI1 mRNA levels more than cells transfected with NC mimic (P < .0001) and similarly as cells transfected with MSI1 siRNA (Figure 2B). Alternatively, inhibiting endogenous miR-137 in HEK-293FT and HCT-116 using antagomiRs increased MSI1 protein expression (Figure 2C and 2D).


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)

miR-137 negatively regulates MSI1(A) MSI1 protein expression in HCT-116 cells transfected with increasing concentrations of miR-137 mimic as compared to cells transfected with NC mimic. (B) Top, MSI1 mRNA and protein expression analyzed in HCT-116 cells transfected with NC mimic, miR-137 mimic and positive control, MSI1-siRNA. mRNA expression analyzed using qRT-PCR, normalized to GAPDH and set relative to NC transfected cells. Bottom, MSI1 protein analyzed using Western blotting with β-actin as loading control. (C) MSI1 protein expression in HEK-293FT cells transfected with miR-137 antagomiR (137-Inh.), negative control antagomiR (NC-Inh.), miR-137 mimic (miR-137), negative control mimic (NC), MSI1 siRNA (si-MSI1) and a negative control siRNA (si-NC). Intensity of MSI1 protein was normalized to β-actin and set relative to control. Change in MSI1 protein shown in red. (D) MSI1 protein expression in HCT-116 transfected with NC-Inh, miR-137-Inh, NC-mimic and miR-137 mimic. (E) HCT-116 cells were transfected with wild-type (WT) or mutant (mut) pSGG-MSI1 3′UTR luciferase construct with miR-137 or NC mimic. Data are means ± SE; n = 3; *** P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: miR-137 negatively regulates MSI1(A) MSI1 protein expression in HCT-116 cells transfected with increasing concentrations of miR-137 mimic as compared to cells transfected with NC mimic. (B) Top, MSI1 mRNA and protein expression analyzed in HCT-116 cells transfected with NC mimic, miR-137 mimic and positive control, MSI1-siRNA. mRNA expression analyzed using qRT-PCR, normalized to GAPDH and set relative to NC transfected cells. Bottom, MSI1 protein analyzed using Western blotting with β-actin as loading control. (C) MSI1 protein expression in HEK-293FT cells transfected with miR-137 antagomiR (137-Inh.), negative control antagomiR (NC-Inh.), miR-137 mimic (miR-137), negative control mimic (NC), MSI1 siRNA (si-MSI1) and a negative control siRNA (si-NC). Intensity of MSI1 protein was normalized to β-actin and set relative to control. Change in MSI1 protein shown in red. (D) MSI1 protein expression in HCT-116 transfected with NC-Inh, miR-137-Inh, NC-mimic and miR-137 mimic. (E) HCT-116 cells were transfected with wild-type (WT) or mutant (mut) pSGG-MSI1 3′UTR luciferase construct with miR-137 or NC mimic. Data are means ± SE; n = 3; *** P < 0.001.
Mentions: Since miR-137 significantly decreased MSI1 protein expression in both HCT-116 and DLD-1 compared to the other mimics; we focused this study on understanding the miR-137-mediated regulation of MSI1. miR-137 reduced MSI1 protein expression in a dose-dependent manner (Figure 2A). Furthermore, miR-137 decreased MSI1 mRNA levels more than cells transfected with NC mimic (P < .0001) and similarly as cells transfected with MSI1 siRNA (Figure 2B). Alternatively, inhibiting endogenous miR-137 in HEK-293FT and HCT-116 using antagomiRs increased MSI1 protein expression (Figure 2C and 2D).

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