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MiR-139-5p inhibits migration and invasion of colorectal cancer by downregulating AMFR and NOTCH1.

Song M, Yin Y, Zhang J, Zhang B, Bian Z, Quan C, Zhou L, Hu Y, Wang Q, Ni S, Fei B, Wang W, Du X, Hua D, Huang Z - Protein Cell (2014)

Bottom Line: We found that miR-139-5p was significantly downregulated in 73.8% CRC samples compared with adjacent noncancerous tissues (NCTs), and decreased miR-139-5p was associated with poor prognosis.Furthermore, the protein levels of the two genes were upregulated in CRC samples compared with NCTs, and inversely correlated with the miR-139-5p expression.Increased NOTCH1 protein expression was correlated with poor prognosis of CRC patients.

View Article: PubMed Central - PubMed

Affiliation: Wuxi Oncology Institute, the Affiliated Hospital of Jiangnan University, Wuxi, 214062, China.

ABSTRACT
MicroRNAs (miRNAs) that exert function by posttranscriptional suppression have recently brought insight in our understanding of the role of non-protein-coding RNAs in carcinogenesis and metastasis. In this study, we described the function and molecular mechanism of miR-139-5p in colorectal cancer (CRC) and its potential clinical application in CRC. We found that miR-139-5p was significantly downregulated in 73.8% CRC samples compared with adjacent noncancerous tissues (NCTs), and decreased miR-139-5p was associated with poor prognosis. Functional analyses demonstrated that ectopic expression of miR-139-5p suppressed CRC cell migration and invasion in vitro and metastasis in vivo. Mechanistic investigations revealed that miR-139-5p suppress CRC cell invasion and metastasis by targeting AMFR and NOTCH1. Knockdown of the two genes phenocopied the inhibitory effect of miR-139-5p on CRC metastasis. Furthermore, the protein levels of the two genes were upregulated in CRC samples compared with NCTs, and inversely correlated with the miR-139-5p expression. Increased NOTCH1 protein expression was correlated with poor prognosis of CRC patients. Together, our data indicate that miR-139-5p is a potential tumor suppressor and prognostic factor for CRC, and targeting miR-139-5p may repress the metastasis of CRC and improve survival.

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AMFR and NOTCH1 are the direct target genes of miR-139-5p. (A) Initial screening of miR-139-5p target genes using a microarray assay, bioinformatics predictions and the luciferase reporter assay. Five downregulated genes (AMFR, NOTCH1, HNRNPF, TOP1, and LAPTM4B) were selected from the 31 genes in the initial screening based on the functional analysis of these genes, and their 3′UTRs were assessed using the luciferase reporter assay. (B and C) Analyses of the luciferase activity of the luciferase reporter plasmids containing either wild-type (WT) or mutant-type (MT) 3′UTRs of AMFR and NOTCH1 in HEK-293T and HCT-116 cells. A mutation was generated in the site complementary to the miR-139-5p seed region of the 3′UTR of AMFR or NOTCH1, as indicated. (D) The protein levels of AMFR and NOTCH1 were determined by Western blot assays using LoVo and HCT-116 cells transfected with miR-139-5p mimic, miR-139-5p inhibitor (anti-miR-139-5p) or their corresponding negative control (NC or anti-NC). Beta-actin protein was used as an internal control
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Fig3: AMFR and NOTCH1 are the direct target genes of miR-139-5p. (A) Initial screening of miR-139-5p target genes using a microarray assay, bioinformatics predictions and the luciferase reporter assay. Five downregulated genes (AMFR, NOTCH1, HNRNPF, TOP1, and LAPTM4B) were selected from the 31 genes in the initial screening based on the functional analysis of these genes, and their 3′UTRs were assessed using the luciferase reporter assay. (B and C) Analyses of the luciferase activity of the luciferase reporter plasmids containing either wild-type (WT) or mutant-type (MT) 3′UTRs of AMFR and NOTCH1 in HEK-293T and HCT-116 cells. A mutation was generated in the site complementary to the miR-139-5p seed region of the 3′UTR of AMFR or NOTCH1, as indicated. (D) The protein levels of AMFR and NOTCH1 were determined by Western blot assays using LoVo and HCT-116 cells transfected with miR-139-5p mimic, miR-139-5p inhibitor (anti-miR-139-5p) or their corresponding negative control (NC or anti-NC). Beta-actin protein was used as an internal control

Mentions: To investigate the mechanism by which miR-139-5p exerts anti-metastasis function in CRC, we searched for the potential targets of miR-139-5p using bioinformatics algorithms (TargetScan and miRanda) and a microarray assay. Genomic-wide expression profiling was performed in NC- and miR-139-5p-transfected LoVo cells using a microarray. A total of 1443 downregulated transcripts (>2-fold change) were identified in miR-139-5p-transfected cells compared with the control (Table S3). Among the top 500 targets of miR-139-5p predicted by the two algorithms, 31 genes were downregulated in the miR-139-5p-transfected cells. Then, 5 potential target genes with tumor-promoting function (AMFR, NOTCH1, HNRNPF, TOP1, and LAPTM4B) were selected from the 31 genes and their 3′UTRs containing the complementary binding sites of miR-139-5p were cloned into a luciferase reporter vector to evaluate the influence of miR-139-5p on the expression of a reporter gene using a luciferase assay (Fig. 3A). The expression of the reporter gene in the recombinant plasmids containing AMFR and NOTCH1 3′UTRs were significantly repressed by miR-139-5p. To further confirm that AMFR and NOTCH1 are direct targets of miR-139-5p in CRC, we mutated the predicted binding sites of miR-139-5p in the 3′UTRs of AMFR and NOTCH1 that are conserved among mammals, and found that the mutant 3′UTRs were completely refractory to miR-139-5p-mediated luciferase reporter repression in HEK-293T and HCT-116 cells (Figs. 3B, 3C and S3). In line with these results, the endogenous AMFR and NOTCH1 protein levels were also decreased in miR-139-5p-overexpressing CRC cells and could be restored in miR-139-5p-depleted cells (Fig. 3D).Figure 3


MiR-139-5p inhibits migration and invasion of colorectal cancer by downregulating AMFR and NOTCH1.

Song M, Yin Y, Zhang J, Zhang B, Bian Z, Quan C, Zhou L, Hu Y, Wang Q, Ni S, Fei B, Wang W, Du X, Hua D, Huang Z - Protein Cell (2014)

AMFR and NOTCH1 are the direct target genes of miR-139-5p. (A) Initial screening of miR-139-5p target genes using a microarray assay, bioinformatics predictions and the luciferase reporter assay. Five downregulated genes (AMFR, NOTCH1, HNRNPF, TOP1, and LAPTM4B) were selected from the 31 genes in the initial screening based on the functional analysis of these genes, and their 3′UTRs were assessed using the luciferase reporter assay. (B and C) Analyses of the luciferase activity of the luciferase reporter plasmids containing either wild-type (WT) or mutant-type (MT) 3′UTRs of AMFR and NOTCH1 in HEK-293T and HCT-116 cells. A mutation was generated in the site complementary to the miR-139-5p seed region of the 3′UTR of AMFR or NOTCH1, as indicated. (D) The protein levels of AMFR and NOTCH1 were determined by Western blot assays using LoVo and HCT-116 cells transfected with miR-139-5p mimic, miR-139-5p inhibitor (anti-miR-139-5p) or their corresponding negative control (NC or anti-NC). Beta-actin protein was used as an internal control
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Related In: Results  -  Collection

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Fig3: AMFR and NOTCH1 are the direct target genes of miR-139-5p. (A) Initial screening of miR-139-5p target genes using a microarray assay, bioinformatics predictions and the luciferase reporter assay. Five downregulated genes (AMFR, NOTCH1, HNRNPF, TOP1, and LAPTM4B) were selected from the 31 genes in the initial screening based on the functional analysis of these genes, and their 3′UTRs were assessed using the luciferase reporter assay. (B and C) Analyses of the luciferase activity of the luciferase reporter plasmids containing either wild-type (WT) or mutant-type (MT) 3′UTRs of AMFR and NOTCH1 in HEK-293T and HCT-116 cells. A mutation was generated in the site complementary to the miR-139-5p seed region of the 3′UTR of AMFR or NOTCH1, as indicated. (D) The protein levels of AMFR and NOTCH1 were determined by Western blot assays using LoVo and HCT-116 cells transfected with miR-139-5p mimic, miR-139-5p inhibitor (anti-miR-139-5p) or their corresponding negative control (NC or anti-NC). Beta-actin protein was used as an internal control
Mentions: To investigate the mechanism by which miR-139-5p exerts anti-metastasis function in CRC, we searched for the potential targets of miR-139-5p using bioinformatics algorithms (TargetScan and miRanda) and a microarray assay. Genomic-wide expression profiling was performed in NC- and miR-139-5p-transfected LoVo cells using a microarray. A total of 1443 downregulated transcripts (>2-fold change) were identified in miR-139-5p-transfected cells compared with the control (Table S3). Among the top 500 targets of miR-139-5p predicted by the two algorithms, 31 genes were downregulated in the miR-139-5p-transfected cells. Then, 5 potential target genes with tumor-promoting function (AMFR, NOTCH1, HNRNPF, TOP1, and LAPTM4B) were selected from the 31 genes and their 3′UTRs containing the complementary binding sites of miR-139-5p were cloned into a luciferase reporter vector to evaluate the influence of miR-139-5p on the expression of a reporter gene using a luciferase assay (Fig. 3A). The expression of the reporter gene in the recombinant plasmids containing AMFR and NOTCH1 3′UTRs were significantly repressed by miR-139-5p. To further confirm that AMFR and NOTCH1 are direct targets of miR-139-5p in CRC, we mutated the predicted binding sites of miR-139-5p in the 3′UTRs of AMFR and NOTCH1 that are conserved among mammals, and found that the mutant 3′UTRs were completely refractory to miR-139-5p-mediated luciferase reporter repression in HEK-293T and HCT-116 cells (Figs. 3B, 3C and S3). In line with these results, the endogenous AMFR and NOTCH1 protein levels were also decreased in miR-139-5p-overexpressing CRC cells and could be restored in miR-139-5p-depleted cells (Fig. 3D).Figure 3

Bottom Line: We found that miR-139-5p was significantly downregulated in 73.8% CRC samples compared with adjacent noncancerous tissues (NCTs), and decreased miR-139-5p was associated with poor prognosis.Furthermore, the protein levels of the two genes were upregulated in CRC samples compared with NCTs, and inversely correlated with the miR-139-5p expression.Increased NOTCH1 protein expression was correlated with poor prognosis of CRC patients.

View Article: PubMed Central - PubMed

Affiliation: Wuxi Oncology Institute, the Affiliated Hospital of Jiangnan University, Wuxi, 214062, China.

ABSTRACT
MicroRNAs (miRNAs) that exert function by posttranscriptional suppression have recently brought insight in our understanding of the role of non-protein-coding RNAs in carcinogenesis and metastasis. In this study, we described the function and molecular mechanism of miR-139-5p in colorectal cancer (CRC) and its potential clinical application in CRC. We found that miR-139-5p was significantly downregulated in 73.8% CRC samples compared with adjacent noncancerous tissues (NCTs), and decreased miR-139-5p was associated with poor prognosis. Functional analyses demonstrated that ectopic expression of miR-139-5p suppressed CRC cell migration and invasion in vitro and metastasis in vivo. Mechanistic investigations revealed that miR-139-5p suppress CRC cell invasion and metastasis by targeting AMFR and NOTCH1. Knockdown of the two genes phenocopied the inhibitory effect of miR-139-5p on CRC metastasis. Furthermore, the protein levels of the two genes were upregulated in CRC samples compared with NCTs, and inversely correlated with the miR-139-5p expression. Increased NOTCH1 protein expression was correlated with poor prognosis of CRC patients. Together, our data indicate that miR-139-5p is a potential tumor suppressor and prognostic factor for CRC, and targeting miR-139-5p may repress the metastasis of CRC and improve survival.

Show MeSH
Related in: MedlinePlus