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MicroRNA-218 inhibits the proliferation and metastasis of esophageal squamous cell carcinoma cells by targeting BMI1.

Wang T, Chen T, Niu H, Li C, Xu C, Li Y, Huang R, Zhao J, Wu S - Int. J. Mol. Med. (2015)

Bottom Line: MicroRNAs (miRNAs or miRs) play a pivotal role in esophageal carcinogenesis either as oncogenes or as tumor suppressor genes.Furthermore, the decreased expression of miR-218 was found to be associated with an enhanced ESCC cell proliferation and metastasis.In the present study, our findings confirm miR-218 as a tumor suppressor and identify BMI1 as a novel target of miR-218 in ESCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Medical College of Soochow University, Suzhou, Jiangsu 215123, P.R. China.

ABSTRACT
MicroRNAs (miRNAs or miRs) play a pivotal role in esophageal carcinogenesis either as oncogenes or as tumor suppressor genes. In the present study, we found that the expression level of miR-218 was significantly reduced in esophageal squamous cell carcinoma (ESCC) tissues and ESCC cell lines. Moreover, its expression was found to correlate with the clinicopathological stage of ESCC; miR-218 expression was lower in the stage III tissue samples than in the stage I and II tissue samples. Furthermore, the decreased expression of miR-218 was found to be associated with an enhanced ESCC cell proliferation and metastasis. Western blot analysis and luciferase reporter assay revealed that miR-218 decreased BMI1 expression by binding to the putative binding sites in its 3'-untranslated region (3'-UTR). The BMI1 mRNA expression levels were markedly increased and negatively correlated with the miR-218 expression level in the ESCC tissues. Functional analyses revealed that the restoration of miR-218 expression inhibited ESCC cell proliferation, migration and invasion and promoted apoptosis. The knockdown of BMI1 by siRNA showed the same phenocopy as the effect of miR-218 on ESCC cells, indicating that BMI1 was a major target of miR-218. In the present study, our findings confirm miR-218 as a tumor suppressor and identify BMI1 as a novel target of miR-218 in ESCC. Therefore, miR-218 may prove to be a useful biomarker for monitoring the initiation and development of ESCC, and may thus be an effective therapeutic target in ESCC.

No MeSH data available.


Related in: MedlinePlus

Identification of BMI1 as target of miR-218 in EC109 cells and ESCC tissues. (A) Detection of BMI1 expression by western blot analysis following transfection of EC109 cells with miR-218 mimics, BMI1-siRNA, or co-transfection of EC109 cells with miR-218 mimics and BMI1-siRNA. (B) Prediction of the potential binding sites of miR-218 in the 3′-UTR of BMI1 using bioinformatics software. (C) Schematic diagram showing cloning strategy of the predicted miR-218 binding sites of BMI1-3′-UTR into psiCHECK-2 luciferase vector. Predicted duplex formation between miR-218 and the wild-type/mutant of miR-218 binding sites was indicated. (D) Luciferase activity of the wild-type or mutant BMI1-3′-UTR reporter gene in EC109 cells transfected with control oligo or miR-218 mimics. AB-WT represents wild-type of both site A and site B; A-MT and B-MT represent single mutant type of site A or site B, respectively. AB-MT represents double mutant type of both site A and site B. Differences in luciferase activity were assessed by a two-tailed t-test. Data are presented as the means ± SD from 3 replicate samples. A scrambled sequence was used as a control. Relative Renilla luciferase activity was obtained after normalizing to Firefly luciferase activity. (E) Average mRNA expression level of BMI1 in ESCC tissue samples and adjacent normal tissue samples. *P<0.05 and **P<0.01. (F) Inverse correlation between miR-218 and BMI1 mRNA levels in the 33 ESCC tissue samples shown by Spearman’s correlation analysis.
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f3-ijmm-36-01-0093: Identification of BMI1 as target of miR-218 in EC109 cells and ESCC tissues. (A) Detection of BMI1 expression by western blot analysis following transfection of EC109 cells with miR-218 mimics, BMI1-siRNA, or co-transfection of EC109 cells with miR-218 mimics and BMI1-siRNA. (B) Prediction of the potential binding sites of miR-218 in the 3′-UTR of BMI1 using bioinformatics software. (C) Schematic diagram showing cloning strategy of the predicted miR-218 binding sites of BMI1-3′-UTR into psiCHECK-2 luciferase vector. Predicted duplex formation between miR-218 and the wild-type/mutant of miR-218 binding sites was indicated. (D) Luciferase activity of the wild-type or mutant BMI1-3′-UTR reporter gene in EC109 cells transfected with control oligo or miR-218 mimics. AB-WT represents wild-type of both site A and site B; A-MT and B-MT represent single mutant type of site A or site B, respectively. AB-MT represents double mutant type of both site A and site B. Differences in luciferase activity were assessed by a two-tailed t-test. Data are presented as the means ± SD from 3 replicate samples. A scrambled sequence was used as a control. Relative Renilla luciferase activity was obtained after normalizing to Firefly luciferase activity. (E) Average mRNA expression level of BMI1 in ESCC tissue samples and adjacent normal tissue samples. *P<0.05 and **P<0.01. (F) Inverse correlation between miR-218 and BMI1 mRNA levels in the 33 ESCC tissue samples shown by Spearman’s correlation analysis.

Mentions: To construct a luciferase reporter plasmid containing the BMI1 3′-UTR fused to the 3′ end of a luciferase reporter gene, the psiCHECK-2 dual luciferase vector (Promega, Madison, WI, USA) was used. Briefly, a 388-bp fragment containing 2 predicted miR-218 target sites (position 1470–1477 and position 1751–1758) was amplified by PCR using the following primers: forward, 5′-CCGCTCGAGTGTTCATCACCCATCAGTTATT-3′ (underlined letters indicate the XhoI site) and reverse, 5′-ATAAGAATGCGGCCGCAGCAATGTATTCTCTTTAACG-3′ (underlined letters indicate the NotI site). The XhoI and NotI-digested PCR product was then cloned into a psiCHECK-2 vector digested with XhoI and NotI to generate the psiCHECK-2-BMI1-3′-UTR-wild type. To prepare mutants, 4 bases in the predicted miR-218 target sites were changed (Fig. 3D). The mutated fragment was directly synthesized (Genewiz, Beijing, China), digested with XhoI and NotI, and cloned into the psiCHECK-2 vector to generate the psiCHECK-2-BMI1-3′-UTR-mutant. Subsequently, the EC109 cells were plated in a 24-well plate and co-transfected with 50 ng of psiCHECK-2-BMI1-3′-UTR-wild type or psiCHECK-2-BMI1-3′-UTR-mutant type and with 20 nM of either miR-218 mimics (5′-UUGUGCUUGAUCUAACCAUGU-3′) or the miR negative control (miR-NC). A scrambled sequence (5′-UUCUCCGAACGUGUCACGUTT-3′) was used as the miR-NC. At 48 h post-transfection, the EC109 were collected, and the luciferase activities were measured using the Dual-Luciferase Reporter assay kit (Promega) on a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA, USA).


MicroRNA-218 inhibits the proliferation and metastasis of esophageal squamous cell carcinoma cells by targeting BMI1.

Wang T, Chen T, Niu H, Li C, Xu C, Li Y, Huang R, Zhao J, Wu S - Int. J. Mol. Med. (2015)

Identification of BMI1 as target of miR-218 in EC109 cells and ESCC tissues. (A) Detection of BMI1 expression by western blot analysis following transfection of EC109 cells with miR-218 mimics, BMI1-siRNA, or co-transfection of EC109 cells with miR-218 mimics and BMI1-siRNA. (B) Prediction of the potential binding sites of miR-218 in the 3′-UTR of BMI1 using bioinformatics software. (C) Schematic diagram showing cloning strategy of the predicted miR-218 binding sites of BMI1-3′-UTR into psiCHECK-2 luciferase vector. Predicted duplex formation between miR-218 and the wild-type/mutant of miR-218 binding sites was indicated. (D) Luciferase activity of the wild-type or mutant BMI1-3′-UTR reporter gene in EC109 cells transfected with control oligo or miR-218 mimics. AB-WT represents wild-type of both site A and site B; A-MT and B-MT represent single mutant type of site A or site B, respectively. AB-MT represents double mutant type of both site A and site B. Differences in luciferase activity were assessed by a two-tailed t-test. Data are presented as the means ± SD from 3 replicate samples. A scrambled sequence was used as a control. Relative Renilla luciferase activity was obtained after normalizing to Firefly luciferase activity. (E) Average mRNA expression level of BMI1 in ESCC tissue samples and adjacent normal tissue samples. *P<0.05 and **P<0.01. (F) Inverse correlation between miR-218 and BMI1 mRNA levels in the 33 ESCC tissue samples shown by Spearman’s correlation analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4494586&req=5

f3-ijmm-36-01-0093: Identification of BMI1 as target of miR-218 in EC109 cells and ESCC tissues. (A) Detection of BMI1 expression by western blot analysis following transfection of EC109 cells with miR-218 mimics, BMI1-siRNA, or co-transfection of EC109 cells with miR-218 mimics and BMI1-siRNA. (B) Prediction of the potential binding sites of miR-218 in the 3′-UTR of BMI1 using bioinformatics software. (C) Schematic diagram showing cloning strategy of the predicted miR-218 binding sites of BMI1-3′-UTR into psiCHECK-2 luciferase vector. Predicted duplex formation between miR-218 and the wild-type/mutant of miR-218 binding sites was indicated. (D) Luciferase activity of the wild-type or mutant BMI1-3′-UTR reporter gene in EC109 cells transfected with control oligo or miR-218 mimics. AB-WT represents wild-type of both site A and site B; A-MT and B-MT represent single mutant type of site A or site B, respectively. AB-MT represents double mutant type of both site A and site B. Differences in luciferase activity were assessed by a two-tailed t-test. Data are presented as the means ± SD from 3 replicate samples. A scrambled sequence was used as a control. Relative Renilla luciferase activity was obtained after normalizing to Firefly luciferase activity. (E) Average mRNA expression level of BMI1 in ESCC tissue samples and adjacent normal tissue samples. *P<0.05 and **P<0.01. (F) Inverse correlation between miR-218 and BMI1 mRNA levels in the 33 ESCC tissue samples shown by Spearman’s correlation analysis.
Mentions: To construct a luciferase reporter plasmid containing the BMI1 3′-UTR fused to the 3′ end of a luciferase reporter gene, the psiCHECK-2 dual luciferase vector (Promega, Madison, WI, USA) was used. Briefly, a 388-bp fragment containing 2 predicted miR-218 target sites (position 1470–1477 and position 1751–1758) was amplified by PCR using the following primers: forward, 5′-CCGCTCGAGTGTTCATCACCCATCAGTTATT-3′ (underlined letters indicate the XhoI site) and reverse, 5′-ATAAGAATGCGGCCGCAGCAATGTATTCTCTTTAACG-3′ (underlined letters indicate the NotI site). The XhoI and NotI-digested PCR product was then cloned into a psiCHECK-2 vector digested with XhoI and NotI to generate the psiCHECK-2-BMI1-3′-UTR-wild type. To prepare mutants, 4 bases in the predicted miR-218 target sites were changed (Fig. 3D). The mutated fragment was directly synthesized (Genewiz, Beijing, China), digested with XhoI and NotI, and cloned into the psiCHECK-2 vector to generate the psiCHECK-2-BMI1-3′-UTR-mutant. Subsequently, the EC109 cells were plated in a 24-well plate and co-transfected with 50 ng of psiCHECK-2-BMI1-3′-UTR-wild type or psiCHECK-2-BMI1-3′-UTR-mutant type and with 20 nM of either miR-218 mimics (5′-UUGUGCUUGAUCUAACCAUGU-3′) or the miR negative control (miR-NC). A scrambled sequence (5′-UUCUCCGAACGUGUCACGUTT-3′) was used as the miR-NC. At 48 h post-transfection, the EC109 were collected, and the luciferase activities were measured using the Dual-Luciferase Reporter assay kit (Promega) on a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA, USA).

Bottom Line: MicroRNAs (miRNAs or miRs) play a pivotal role in esophageal carcinogenesis either as oncogenes or as tumor suppressor genes.Furthermore, the decreased expression of miR-218 was found to be associated with an enhanced ESCC cell proliferation and metastasis.In the present study, our findings confirm miR-218 as a tumor suppressor and identify BMI1 as a novel target of miR-218 in ESCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Medical College of Soochow University, Suzhou, Jiangsu 215123, P.R. China.

ABSTRACT
MicroRNAs (miRNAs or miRs) play a pivotal role in esophageal carcinogenesis either as oncogenes or as tumor suppressor genes. In the present study, we found that the expression level of miR-218 was significantly reduced in esophageal squamous cell carcinoma (ESCC) tissues and ESCC cell lines. Moreover, its expression was found to correlate with the clinicopathological stage of ESCC; miR-218 expression was lower in the stage III tissue samples than in the stage I and II tissue samples. Furthermore, the decreased expression of miR-218 was found to be associated with an enhanced ESCC cell proliferation and metastasis. Western blot analysis and luciferase reporter assay revealed that miR-218 decreased BMI1 expression by binding to the putative binding sites in its 3'-untranslated region (3'-UTR). The BMI1 mRNA expression levels were markedly increased and negatively correlated with the miR-218 expression level in the ESCC tissues. Functional analyses revealed that the restoration of miR-218 expression inhibited ESCC cell proliferation, migration and invasion and promoted apoptosis. The knockdown of BMI1 by siRNA showed the same phenocopy as the effect of miR-218 on ESCC cells, indicating that BMI1 was a major target of miR-218. In the present study, our findings confirm miR-218 as a tumor suppressor and identify BMI1 as a novel target of miR-218 in ESCC. Therefore, miR-218 may prove to be a useful biomarker for monitoring the initiation and development of ESCC, and may thus be an effective therapeutic target in ESCC.

No MeSH data available.


Related in: MedlinePlus