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The microRNA-218~Survivin axis regulates migration, invasion, and lymph node metastasis in cervical cancer.

Kogo R, How C, Chaudary N, Bruce J, Shi W, Hill RP, Zahedi P, Yip KW, Liu FF - Oncotarget (2015)

Bottom Line: Lower miR-218 expression was associated significantly with worse overall survival (OS), disease-free survival (DFS), and pelvic/aortic lymph node recurrence.In vitro, miR-218 over-expression decreased clonogenicity, migration, and invasion.YM155, a small molecule survivin inhibitor, significantly suppressed tumor growth and lymph node metastasis in vivo.

View Article: PubMed Central - PubMed

Affiliation: Ontario Cancer Institute, University Health Network (UHN), Toronto, Ontario, Canada.

ABSTRACT
Cervical cancer is the third most common cancer in women worldwide. In the present study, global microRNA profiling for 79 cervical cancer patient samples led to the identification of miR-218 down-regulation in cervical cancer tissues compared to normal cervical tissues. Lower miR-218 expression was associated significantly with worse overall survival (OS), disease-free survival (DFS), and pelvic/aortic lymph node recurrence. In vitro, miR-218 over-expression decreased clonogenicity, migration, and invasion. Survivin (BIRC5) was subsequently identified as an important cervical cancer target of miR-218 using in silico prediction, mRNA profiling, and quantitative real-time PCR (qRT-PCR). Concordant with miR-218 over-expression, survivin knockdown by siRNA decreased clonogenicity, migration, and invasion. YM155, a small molecule survivin inhibitor, significantly suppressed tumor growth and lymph node metastasis in vivo. Our findings demonstrate that the miR-218~survivin axis inhibits cervical cancer progression by regulating clonogenicity, migration, and invasion, and suggest that the inhibition of survivin could be a potential therapeutic strategy to improve outcome in this disease.

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Related in: MedlinePlus

Survivin is a direct target of miR-218A) Identification of miR-218 targets in cervical cancer. Cancer (Up): mRNA expression greater than 2-fold compared to normal cervix, from GeneChip Human Genome U133 Plus 2.0 Array data for 79 cervical cancer tissues and 11 normal cervix tissues; in silico: predicted targets of miR-218 by miRDB (http://mirdb.org/miRDB/). B) mRNA (left) and protein (right) survivin expression after 48 hrs miR-218 or miR-NC (10 nM each) transfection in SiHa and ME-180 cells. Survivin mRNA expression levels were normalized to GAPDH. *P<0.05, bars represent mean ± SEM from triplicates. C) Schema of pMIR-REPORT vectors for the luciferase assay. Wild type (upper) and/or Mutant (lower) survivin 3′UTR were cloned downstream of firefly luciferase gene. miR-218 directly binds to wild type survivin 3′UTR and inhibits luciferase gene expression; on the other hand, miR-218 cannot bind to Mutant 3′UTR and express luciferase gene. D) Relative luciferase activity in SiHa and ME-180 cells after co-transfection with pMIR-REPORT (empty), pMIR-survivin-3′UTR-wild-type (survivin-WT), or pMIR-survivin-3′UTR-mutant (survivin-MT) and miR-NC or miR-218. The values represent the luciferase activity of miR-218 and pMIR-REPORT/miR-NC and pMIR-REPORT. Data were normalized to the luciferase activity of empty pMIR-REPORT transfected cells. *P<0.05, bars represent mean ± SEM from six replicates.
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Figure 3: Survivin is a direct target of miR-218A) Identification of miR-218 targets in cervical cancer. Cancer (Up): mRNA expression greater than 2-fold compared to normal cervix, from GeneChip Human Genome U133 Plus 2.0 Array data for 79 cervical cancer tissues and 11 normal cervix tissues; in silico: predicted targets of miR-218 by miRDB (http://mirdb.org/miRDB/). B) mRNA (left) and protein (right) survivin expression after 48 hrs miR-218 or miR-NC (10 nM each) transfection in SiHa and ME-180 cells. Survivin mRNA expression levels were normalized to GAPDH. *P<0.05, bars represent mean ± SEM from triplicates. C) Schema of pMIR-REPORT vectors for the luciferase assay. Wild type (upper) and/or Mutant (lower) survivin 3′UTR were cloned downstream of firefly luciferase gene. miR-218 directly binds to wild type survivin 3′UTR and inhibits luciferase gene expression; on the other hand, miR-218 cannot bind to Mutant 3′UTR and express luciferase gene. D) Relative luciferase activity in SiHa and ME-180 cells after co-transfection with pMIR-REPORT (empty), pMIR-survivin-3′UTR-wild-type (survivin-WT), or pMIR-survivin-3′UTR-mutant (survivin-MT) and miR-NC or miR-218. The values represent the luciferase activity of miR-218 and pMIR-REPORT/miR-NC and pMIR-REPORT. Data were normalized to the luciferase activity of empty pMIR-REPORT transfected cells. *P<0.05, bars represent mean ± SEM from six replicates.

Mentions: In order to identify biologically relevant miR-218 targets, we first examined in silico predicted targets using miRDB (http://mirdb.org/miRDB/) [19, 20]. These data were combined with mRNA array (GeneChip Human Genome U133 Plus 2.0) data generated from the same 79 cervical cancer tissues and 11 normal cervix tissues used for TLDA [17]. At the intersection between the in silico predicted targets and mRNAs that were up-regulated by greater than 2 fold were 35 candidate targets (Figure 3A; Table S1). For these candidate targets, their in silico prediction scores and expression levels were used to rank the genes independently, then these ranks were summed for a cumulative final rank (Table S1).


The microRNA-218~Survivin axis regulates migration, invasion, and lymph node metastasis in cervical cancer.

Kogo R, How C, Chaudary N, Bruce J, Shi W, Hill RP, Zahedi P, Yip KW, Liu FF - Oncotarget (2015)

Survivin is a direct target of miR-218A) Identification of miR-218 targets in cervical cancer. Cancer (Up): mRNA expression greater than 2-fold compared to normal cervix, from GeneChip Human Genome U133 Plus 2.0 Array data for 79 cervical cancer tissues and 11 normal cervix tissues; in silico: predicted targets of miR-218 by miRDB (http://mirdb.org/miRDB/). B) mRNA (left) and protein (right) survivin expression after 48 hrs miR-218 or miR-NC (10 nM each) transfection in SiHa and ME-180 cells. Survivin mRNA expression levels were normalized to GAPDH. *P<0.05, bars represent mean ± SEM from triplicates. C) Schema of pMIR-REPORT vectors for the luciferase assay. Wild type (upper) and/or Mutant (lower) survivin 3′UTR were cloned downstream of firefly luciferase gene. miR-218 directly binds to wild type survivin 3′UTR and inhibits luciferase gene expression; on the other hand, miR-218 cannot bind to Mutant 3′UTR and express luciferase gene. D) Relative luciferase activity in SiHa and ME-180 cells after co-transfection with pMIR-REPORT (empty), pMIR-survivin-3′UTR-wild-type (survivin-WT), or pMIR-survivin-3′UTR-mutant (survivin-MT) and miR-NC or miR-218. The values represent the luciferase activity of miR-218 and pMIR-REPORT/miR-NC and pMIR-REPORT. Data were normalized to the luciferase activity of empty pMIR-REPORT transfected cells. *P<0.05, bars represent mean ± SEM from six replicates.
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Related In: Results  -  Collection

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Figure 3: Survivin is a direct target of miR-218A) Identification of miR-218 targets in cervical cancer. Cancer (Up): mRNA expression greater than 2-fold compared to normal cervix, from GeneChip Human Genome U133 Plus 2.0 Array data for 79 cervical cancer tissues and 11 normal cervix tissues; in silico: predicted targets of miR-218 by miRDB (http://mirdb.org/miRDB/). B) mRNA (left) and protein (right) survivin expression after 48 hrs miR-218 or miR-NC (10 nM each) transfection in SiHa and ME-180 cells. Survivin mRNA expression levels were normalized to GAPDH. *P<0.05, bars represent mean ± SEM from triplicates. C) Schema of pMIR-REPORT vectors for the luciferase assay. Wild type (upper) and/or Mutant (lower) survivin 3′UTR were cloned downstream of firefly luciferase gene. miR-218 directly binds to wild type survivin 3′UTR and inhibits luciferase gene expression; on the other hand, miR-218 cannot bind to Mutant 3′UTR and express luciferase gene. D) Relative luciferase activity in SiHa and ME-180 cells after co-transfection with pMIR-REPORT (empty), pMIR-survivin-3′UTR-wild-type (survivin-WT), or pMIR-survivin-3′UTR-mutant (survivin-MT) and miR-NC or miR-218. The values represent the luciferase activity of miR-218 and pMIR-REPORT/miR-NC and pMIR-REPORT. Data were normalized to the luciferase activity of empty pMIR-REPORT transfected cells. *P<0.05, bars represent mean ± SEM from six replicates.
Mentions: In order to identify biologically relevant miR-218 targets, we first examined in silico predicted targets using miRDB (http://mirdb.org/miRDB/) [19, 20]. These data were combined with mRNA array (GeneChip Human Genome U133 Plus 2.0) data generated from the same 79 cervical cancer tissues and 11 normal cervix tissues used for TLDA [17]. At the intersection between the in silico predicted targets and mRNAs that were up-regulated by greater than 2 fold were 35 candidate targets (Figure 3A; Table S1). For these candidate targets, their in silico prediction scores and expression levels were used to rank the genes independently, then these ranks were summed for a cumulative final rank (Table S1).

Bottom Line: Lower miR-218 expression was associated significantly with worse overall survival (OS), disease-free survival (DFS), and pelvic/aortic lymph node recurrence.In vitro, miR-218 over-expression decreased clonogenicity, migration, and invasion.YM155, a small molecule survivin inhibitor, significantly suppressed tumor growth and lymph node metastasis in vivo.

View Article: PubMed Central - PubMed

Affiliation: Ontario Cancer Institute, University Health Network (UHN), Toronto, Ontario, Canada.

ABSTRACT
Cervical cancer is the third most common cancer in women worldwide. In the present study, global microRNA profiling for 79 cervical cancer patient samples led to the identification of miR-218 down-regulation in cervical cancer tissues compared to normal cervical tissues. Lower miR-218 expression was associated significantly with worse overall survival (OS), disease-free survival (DFS), and pelvic/aortic lymph node recurrence. In vitro, miR-218 over-expression decreased clonogenicity, migration, and invasion. Survivin (BIRC5) was subsequently identified as an important cervical cancer target of miR-218 using in silico prediction, mRNA profiling, and quantitative real-time PCR (qRT-PCR). Concordant with miR-218 over-expression, survivin knockdown by siRNA decreased clonogenicity, migration, and invasion. YM155, a small molecule survivin inhibitor, significantly suppressed tumor growth and lymph node metastasis in vivo. Our findings demonstrate that the miR-218~survivin axis inhibits cervical cancer progression by regulating clonogenicity, migration, and invasion, and suggest that the inhibition of survivin could be a potential therapeutic strategy to improve outcome in this disease.

Show MeSH
Related in: MedlinePlus