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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 inhibits colon cancer growth and clonogenic growth by inhibiting MSI1(A) Cell growth curve in HCT-116 cells transfected with miR-137 and NC mimic. Cells were collected and counted every day for 4 days. Data are means ± SE; n = 3; *** P < 0.001. (B) Cell growth curve in HCT-116 cells co-transfected with miR-137/NC mimic and EV-GFP/MSI1-GFP expression vectors. Cells were collected and counted every day for 4 days. Data are means ± SE; n = 2; **P < 0.01, *** P < 0.001. (C) Cell viability was measured using a MTT colorimetric assay in HCT-116 cells transfected with miR-137 and NC mimic. (D) Colony formation assay in HCT-116 and DLD-1 cells transfected with miR-137 and NC mimic. Image of representative colonies are shown in right panel. (E) Tumorsphere assay in HCT-116 cells transfected with miR-137 and NC mimic. Data are means ± SE; n = 3; *** P < 0.001. (F) Colony formation on Tet-on miR-137 cells transfected with MSI1-GFP or EV-GFP constructs. Cells were plated in media with and without 1 μg/ml DOX. Data are means ± SE; n = 2; *P < 0.05, **P<0.01 and ***P < 0.001.
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Figure 4: miR-137 inhibits colon cancer growth and clonogenic growth by inhibiting MSI1(A) Cell growth curve in HCT-116 cells transfected with miR-137 and NC mimic. Cells were collected and counted every day for 4 days. Data are means ± SE; n = 3; *** P < 0.001. (B) Cell growth curve in HCT-116 cells co-transfected with miR-137/NC mimic and EV-GFP/MSI1-GFP expression vectors. Cells were collected and counted every day for 4 days. Data are means ± SE; n = 2; **P < 0.01, *** P < 0.001. (C) Cell viability was measured using a MTT colorimetric assay in HCT-116 cells transfected with miR-137 and NC mimic. (D) Colony formation assay in HCT-116 and DLD-1 cells transfected with miR-137 and NC mimic. Image of representative colonies are shown in right panel. (E) Tumorsphere assay in HCT-116 cells transfected with miR-137 and NC mimic. Data are means ± SE; n = 3; *** P < 0.001. (F) Colony formation on Tet-on miR-137 cells transfected with MSI1-GFP or EV-GFP constructs. Cells were plated in media with and without 1 μg/ml DOX. Data are means ± SE; n = 2; *P < 0.05, **P<0.01 and ***P < 0.001.

Mentions: Based on our preliminary data, we hypothesized that miR-137 acts as a tumor suppressor miRNA by negatively regulating MSI1. To examine the effect of miR-137 restoration on colon cancer cells, in vitro cell growth and cell viability assays were utilized. miR-137 mimic transfected HCT-116 cells grew significantly less than cells treated with NC mimic (P < .0001) (Figure 4A). To examine whether the miR-137-induced cell growth inhibition was via MSI1 down-regulation, a phenotype rescue experiment was performed. miR-137 mediated inhibition of cell growth was partially restored when HCT-116 cells were co-transfected with a MSI1 cDNA expression vector that lacks the 3′UTR as compared to cells co-transfected with an empty vector (Figure 4B). This data suggests that miR-137 tumor suppressive function is mediated in part by negatively regulating MSI1. We also show that cell viability was significantly reduced upon miR-137 restoration as measured by a MTT cell viability assay (P < .0001) (Figure 4C).


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 inhibits colon cancer growth and clonogenic growth by inhibiting MSI1(A) Cell growth curve in HCT-116 cells transfected with miR-137 and NC mimic. Cells were collected and counted every day for 4 days. Data are means ± SE; n = 3; *** P < 0.001. (B) Cell growth curve in HCT-116 cells co-transfected with miR-137/NC mimic and EV-GFP/MSI1-GFP expression vectors. Cells were collected and counted every day for 4 days. Data are means ± SE; n = 2; **P < 0.01, *** P < 0.001. (C) Cell viability was measured using a MTT colorimetric assay in HCT-116 cells transfected with miR-137 and NC mimic. (D) Colony formation assay in HCT-116 and DLD-1 cells transfected with miR-137 and NC mimic. Image of representative colonies are shown in right panel. (E) Tumorsphere assay in HCT-116 cells transfected with miR-137 and NC mimic. Data are means ± SE; n = 3; *** P < 0.001. (F) Colony formation on Tet-on miR-137 cells transfected with MSI1-GFP or EV-GFP constructs. Cells were plated in media with and without 1 μg/ml DOX. Data are means ± SE; n = 2; *P < 0.05, **P<0.01 and ***P < 0.001.
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Figure 4: miR-137 inhibits colon cancer growth and clonogenic growth by inhibiting MSI1(A) Cell growth curve in HCT-116 cells transfected with miR-137 and NC mimic. Cells were collected and counted every day for 4 days. Data are means ± SE; n = 3; *** P < 0.001. (B) Cell growth curve in HCT-116 cells co-transfected with miR-137/NC mimic and EV-GFP/MSI1-GFP expression vectors. Cells were collected and counted every day for 4 days. Data are means ± SE; n = 2; **P < 0.01, *** P < 0.001. (C) Cell viability was measured using a MTT colorimetric assay in HCT-116 cells transfected with miR-137 and NC mimic. (D) Colony formation assay in HCT-116 and DLD-1 cells transfected with miR-137 and NC mimic. Image of representative colonies are shown in right panel. (E) Tumorsphere assay in HCT-116 cells transfected with miR-137 and NC mimic. Data are means ± SE; n = 3; *** P < 0.001. (F) Colony formation on Tet-on miR-137 cells transfected with MSI1-GFP or EV-GFP constructs. Cells were plated in media with and without 1 μg/ml DOX. Data are means ± SE; n = 2; *P < 0.05, **P<0.01 and ***P < 0.001.
Mentions: Based on our preliminary data, we hypothesized that miR-137 acts as a tumor suppressor miRNA by negatively regulating MSI1. To examine the effect of miR-137 restoration on colon cancer cells, in vitro cell growth and cell viability assays were utilized. miR-137 mimic transfected HCT-116 cells grew significantly less than cells treated with NC mimic (P < .0001) (Figure 4A). To examine whether the miR-137-induced cell growth inhibition was via MSI1 down-regulation, a phenotype rescue experiment was performed. miR-137 mediated inhibition of cell growth was partially restored when HCT-116 cells were co-transfected with a MSI1 cDNA expression vector that lacks the 3′UTR as compared to cells co-transfected with an empty vector (Figure 4B). This data suggests that miR-137 tumor suppressive function is mediated in part by negatively regulating MSI1. We also show that cell viability was significantly reduced upon miR-137 restoration as measured by a MTT cell viability assay (P < .0001) (Figure 4C).

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