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miR156a Mimic Represses the Epithelial-Mesenchymal Transition of Human Nasopharyngeal Cancer Cells by Targeting Junctional Adhesion Molecule A.

Tian Y, Cai L, Tian Y, Tu Y, Qiu H, Xie G, Huang D, Zheng R, Zhang W - PLoS ONE (2016)

Bottom Line: We demonstrated that a total of 84 conserved miRNAs and 184 putative novel miRNAs were found in broccoli by sequencing technology.Knockdown of JAMA was consistent with the effects of miR156a mimic on the EMT of NPC, and the up-regulation of JAMA could partially restore EMT repressed by miR156a mimic.In conclusion, these results indicate that the miR156a mimic inhibits the EMT of NPC cells by targeting the 3' UTR of JAMA.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Cancer Center of Guangzhou Medical University, Guangzhou, Guangdong Province, People's Republic of China.

ABSTRACT
MicroRNAs (miRNAs) have been documented as having an important role in the development of cancer. Broccoli is very popular in large groups of the population and has anticancer properties. Junctional adhesion molecule A (JAMA) is preferentially concentrated at tight junctions and influences cell morphology and migration. Epithelial-mesenchymal transition (EMT) is a developmental program associated with cancer progression and metastasis. In this study we aimed to investigate the role of miRNAs from broccoli in human nasopharyngeal cancer (NPC). We demonstrated that a total of 84 conserved miRNAs and 184 putative novel miRNAs were found in broccoli by sequencing technology. Among these, miR156a was expressed the most. In addition, synthetic miR156a mimic inhibited the EMT of NPC cells in vitro. Furthermore, it was confirmed that JAMA was the target of miR156a mimic as validated by 3' UTR luciferase reporter assays and western blotting. Knockdown of JAMA was consistent with the effects of miR156a mimic on the EMT of NPC, and the up-regulation of JAMA could partially restore EMT repressed by miR156a mimic. In conclusion, these results indicate that the miR156a mimic inhibits the EMT of NPC cells by targeting the 3' UTR of JAMA. These miRNA profiles of broccoli provide a fundamental basis for further research. Moreover, the discovery of miR156a may have clinical implications for the treatment of patients with NPC.

No MeSH data available.


Related in: MedlinePlus

miR-156a mimic directly regulates JAMA by binding to the 3’ UTR of JAMA.(A) Schematic description of the hypothesized duplexes formed by interactions between the 3’ UTR and miR156a. Note that the potential binding site of MIR156a to JAMA mRNA is highly conserved across species. (B) Western blotting analysis of JAMA protein levels in miR168a-transfected NPC cells. (C) Real-time RT-PCR analysis of JAMA mRNA levels in miR168a-transfected NPC cells. (D) Diagram of the luciferase reporter plasmid carrying the firefly luciferase-coding sequence attached to the wild type or mutant miR156a complementary site. (E) Luciferase activities in 293T cells co-transfected with luciferase reporters and miR156a or NC (n = 6). *P < 0.05; **P < 0.01 compared with negative controls.
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pone.0157686.g003: miR-156a mimic directly regulates JAMA by binding to the 3’ UTR of JAMA.(A) Schematic description of the hypothesized duplexes formed by interactions between the 3’ UTR and miR156a. Note that the potential binding site of MIR156a to JAMA mRNA is highly conserved across species. (B) Western blotting analysis of JAMA protein levels in miR168a-transfected NPC cells. (C) Real-time RT-PCR analysis of JAMA mRNA levels in miR168a-transfected NPC cells. (D) Diagram of the luciferase reporter plasmid carrying the firefly luciferase-coding sequence attached to the wild type or mutant miR156a complementary site. (E) Luciferase activities in 293T cells co-transfected with luciferase reporters and miR156a or NC (n = 6). *P < 0.05; **P < 0.01 compared with negative controls.

Mentions: To understand the underlying molecular mechanism by which miR156a suppresses NPC cell invasion and metastasis, we searched for miR156a targets using different computational methods according to a previous study [6] and the basic local alignment search tool (BLAST) of NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). These methods identified 40 candidate genes that were commonly predicted to be possible targets of miR156a (S3 Table). The most highly conserved sequence of a putative binding site among various species is located in the 3’ UTR of JAMA (Fig 3A). The minimum free binding energy was calculated to be –27.3 kcal/mol. First, we evaluated whether miR156a mimic could negatively regulate JAMA expression. As shown in Fig 3B and 3C, the decrease in JAMA protein expression was confirmed by western blotting, whereas JAMA mRNA expression was not affected. Moreover, a luciferase reporter assay was performed to determine whether miR156a had an effect on the 3’ UTR of JAMA. The wild-type or mutant miR156a complementary sites (CS) were cloned into a luciferase reporter vector and transfected into 293T cells combined with miR156a mimic (Fig 3D). As expected, compared with the negative controls, co-transfection of miR156a mimic with the human JAMA 3’ UTR wild type reporter (psiCHECK2-JAMA 3’ UTR-WT) resulted in a highly significant decrease in luciferase activity. A consistent lack of decrease in luciferase activity was observed when miR156a mimic or negative control was co-transfected with the empty vector or the mutant reporter (psiCHECK-JAMA-3’ UTR-MUT), indicating that the predicted site is a direct target of miR156a (Fig 3E).


miR156a Mimic Represses the Epithelial-Mesenchymal Transition of Human Nasopharyngeal Cancer Cells by Targeting Junctional Adhesion Molecule A.

Tian Y, Cai L, Tian Y, Tu Y, Qiu H, Xie G, Huang D, Zheng R, Zhang W - PLoS ONE (2016)

miR-156a mimic directly regulates JAMA by binding to the 3’ UTR of JAMA.(A) Schematic description of the hypothesized duplexes formed by interactions between the 3’ UTR and miR156a. Note that the potential binding site of MIR156a to JAMA mRNA is highly conserved across species. (B) Western blotting analysis of JAMA protein levels in miR168a-transfected NPC cells. (C) Real-time RT-PCR analysis of JAMA mRNA levels in miR168a-transfected NPC cells. (D) Diagram of the luciferase reporter plasmid carrying the firefly luciferase-coding sequence attached to the wild type or mutant miR156a complementary site. (E) Luciferase activities in 293T cells co-transfected with luciferase reporters and miR156a or NC (n = 6). *P < 0.05; **P < 0.01 compared with negative controls.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4920421&req=5

pone.0157686.g003: miR-156a mimic directly regulates JAMA by binding to the 3’ UTR of JAMA.(A) Schematic description of the hypothesized duplexes formed by interactions between the 3’ UTR and miR156a. Note that the potential binding site of MIR156a to JAMA mRNA is highly conserved across species. (B) Western blotting analysis of JAMA protein levels in miR168a-transfected NPC cells. (C) Real-time RT-PCR analysis of JAMA mRNA levels in miR168a-transfected NPC cells. (D) Diagram of the luciferase reporter plasmid carrying the firefly luciferase-coding sequence attached to the wild type or mutant miR156a complementary site. (E) Luciferase activities in 293T cells co-transfected with luciferase reporters and miR156a or NC (n = 6). *P < 0.05; **P < 0.01 compared with negative controls.
Mentions: To understand the underlying molecular mechanism by which miR156a suppresses NPC cell invasion and metastasis, we searched for miR156a targets using different computational methods according to a previous study [6] and the basic local alignment search tool (BLAST) of NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). These methods identified 40 candidate genes that were commonly predicted to be possible targets of miR156a (S3 Table). The most highly conserved sequence of a putative binding site among various species is located in the 3’ UTR of JAMA (Fig 3A). The minimum free binding energy was calculated to be –27.3 kcal/mol. First, we evaluated whether miR156a mimic could negatively regulate JAMA expression. As shown in Fig 3B and 3C, the decrease in JAMA protein expression was confirmed by western blotting, whereas JAMA mRNA expression was not affected. Moreover, a luciferase reporter assay was performed to determine whether miR156a had an effect on the 3’ UTR of JAMA. The wild-type or mutant miR156a complementary sites (CS) were cloned into a luciferase reporter vector and transfected into 293T cells combined with miR156a mimic (Fig 3D). As expected, compared with the negative controls, co-transfection of miR156a mimic with the human JAMA 3’ UTR wild type reporter (psiCHECK2-JAMA 3’ UTR-WT) resulted in a highly significant decrease in luciferase activity. A consistent lack of decrease in luciferase activity was observed when miR156a mimic or negative control was co-transfected with the empty vector or the mutant reporter (psiCHECK-JAMA-3’ UTR-MUT), indicating that the predicted site is a direct target of miR156a (Fig 3E).

Bottom Line: We demonstrated that a total of 84 conserved miRNAs and 184 putative novel miRNAs were found in broccoli by sequencing technology.Knockdown of JAMA was consistent with the effects of miR156a mimic on the EMT of NPC, and the up-regulation of JAMA could partially restore EMT repressed by miR156a mimic.In conclusion, these results indicate that the miR156a mimic inhibits the EMT of NPC cells by targeting the 3' UTR of JAMA.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Cancer Center of Guangzhou Medical University, Guangzhou, Guangdong Province, People's Republic of China.

ABSTRACT
MicroRNAs (miRNAs) have been documented as having an important role in the development of cancer. Broccoli is very popular in large groups of the population and has anticancer properties. Junctional adhesion molecule A (JAMA) is preferentially concentrated at tight junctions and influences cell morphology and migration. Epithelial-mesenchymal transition (EMT) is a developmental program associated with cancer progression and metastasis. In this study we aimed to investigate the role of miRNAs from broccoli in human nasopharyngeal cancer (NPC). We demonstrated that a total of 84 conserved miRNAs and 184 putative novel miRNAs were found in broccoli by sequencing technology. Among these, miR156a was expressed the most. In addition, synthetic miR156a mimic inhibited the EMT of NPC cells in vitro. Furthermore, it was confirmed that JAMA was the target of miR156a mimic as validated by 3' UTR luciferase reporter assays and western blotting. Knockdown of JAMA was consistent with the effects of miR156a mimic on the EMT of NPC, and the up-regulation of JAMA could partially restore EMT repressed by miR156a mimic. In conclusion, these results indicate that the miR156a mimic inhibits the EMT of NPC cells by targeting the 3' UTR of JAMA. These miRNA profiles of broccoli provide a fundamental basis for further research. Moreover, the discovery of miR156a may have clinical implications for the treatment of patients with NPC.

No MeSH data available.


Related in: MedlinePlus