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MicroRNA-19a mediates gastric carcinoma cell proliferation through the activation of nuclear factor-κB.

Yang F, Wang H, Jiang Z, Hu A, Chu L, Sun Y, Han J - Mol Med Rep (2015)

Bottom Line: The present study predominantly focussed on the effects of microRNA (miR)‑19a on NF‑κB activation.The results of the western blot analysis demonstrated that the protein levels of p65 increased when the MGC‑803 cells were transfected with miR‑19a mimics.In addition, the downstream target genes of miR‑19a, including intercellular adhesion molecule, vascular cell adhesion molecule and monocyte chemoattractant protein‑1, were upregulated.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Research and Therapy Center, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China.

ABSTRACT
In gastric carcinoma, the nuclear factor‑κB (NF‑κB) signaling pathway is highly active, and the constitutive activation of NF‑κB prompts malignant cell proliferation. MicroRNAs are considered to be important mediators in the regulation of the NF‑κB signaling pathway. The present study predominantly focussed on the effects of microRNA (miR)‑19a on NF‑κB activation. Reverse transcription‑quantitative polymerase chain reaction was used to quantify the relative levels of miR‑19a in gastric carcinoma cells. MTT assays were used to determine the effect of miR‑19a on cellular proliferation. To detect the activation of NF‑κB, western blotting was performed to measure the protein levels of NF‑κB and the products of its downstream target genes. To define the target genes, luciferase reporter assays were used. miR‑19a was found to be markedly upregulated in gastric carcinoma cells. The overexpression of miR‑19a resulted in proliferation and enhanced migratory capabilities of the MGC‑803 gastric carcinoma cell line. The results of the western blot analysis demonstrated that the protein levels of p65 increased when the MGC‑803 cells were transfected with miR‑19a mimics. In addition, the downstream target genes of miR‑19a, including intercellular adhesion molecule, vascular cell adhesion molecule and monocyte chemoattractant protein‑1, were upregulated. The results of the luciferase assay indicated that IκB‑α was the target gene of miR‑19a. Therefore, the results of the present study suggested that miR‑19a enhances malignant gastric cell proliferation by constitutively activating the NF‑κB signaling pathway.

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NF-κB signaling pathway is activated when miR-19a is overexpressed in the MGC-803 human gastric carcinoma cell line. (A) Reverse transcription-quantitative polymerase chain reaction was used to determine the relative levels of miR-19a when MGC-803 cells were treated with 10 ng/μl TNF-α for 48 h. (B) Western blot analysis of NF-κB activation and its downstream regulators when miR-19a was overexpressed. (C) Western blot analysis of siRNA targeting NF-κB. (D) MTT assay demonstrated a low cellular proliferation rate in cells cotransfected with si-NF-κB and miR-19a mimics. Data are presented as the mean ± standard error of three independent experiments. *P<0.05, vs. control. TNF-α, tumor necrosis factor-α; NF-κB, nuclear factor-κB; VCAM, vascular cell adhesion molecule; ICAM, intercellular adhesion molecule; MCP, monocyte chemoattractant protein; miR, microRNA; si, small interfering; p-, phosphorylated; NC, negative control; Con, control.
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f3-mmr-12-04-5780: NF-κB signaling pathway is activated when miR-19a is overexpressed in the MGC-803 human gastric carcinoma cell line. (A) Reverse transcription-quantitative polymerase chain reaction was used to determine the relative levels of miR-19a when MGC-803 cells were treated with 10 ng/μl TNF-α for 48 h. (B) Western blot analysis of NF-κB activation and its downstream regulators when miR-19a was overexpressed. (C) Western blot analysis of siRNA targeting NF-κB. (D) MTT assay demonstrated a low cellular proliferation rate in cells cotransfected with si-NF-κB and miR-19a mimics. Data are presented as the mean ± standard error of three independent experiments. *P<0.05, vs. control. TNF-α, tumor necrosis factor-α; NF-κB, nuclear factor-κB; VCAM, vascular cell adhesion molecule; ICAM, intercellular adhesion molecule; MCP, monocyte chemoattractant protein; miR, microRNA; si, small interfering; p-, phosphorylated; NC, negative control; Con, control.

Mentions: In gastric carcinoma, the NF-κB signaling pathway is constitutively activated. In previous studies, several miRNAs have been reported to activate NF-κB. For example, miR-301a has been demonstrated to target NF-κB-repressing factor and to correlate with abnormal NF-κB activation (17). In the present study, the MGC-803 gastric carcinoma cells were treated with 10 ng/μl TNF-α for 48 h, following which the relative levels of miR-19a were analyzed. As shown in Fig. 3A, the relative levels of miR-19a increased ~4-fold following TNF-α treatment. In addition, western blotting was performed to ascertain whether TNF-α treatment significantly activated the NF-κB signaling pathway. To address whether miR-19a contributes to NF-κB activation, the MGC-803 gastric carcinoma cells were transfected with miR-19a mimics. Based on the results of the western blot analysis, it was concluded that when miR-19a was overexpressed, NF-κB was significantly activated. As shown in Fig. 3A, compared with the negative control, the phosphorylated-NF-κB/NF-κB ratio was increased by >1-fold. Vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM) and monocyte chemoattractant protein-1 (MCP-1) are among the important adhesion molecules that are aberrantly expressed in various types of cancer (18). To validate the abnormal NF-κB activation, the protein levels of VCAM, ICAM and MCP-1 were measured. As shown in Fig. 3B, their relative levels were all increased ~1-fold. Based on the above results, it was concluded that miR-19a contributed to TNF-α-stimulated NF-κB activation. In order to investigate the effect of NF-κB on cellular proliferation, a small interfering (si)RNA-targeting NF-κB was selected. As shown in Fig. 3C, following knockdown of NF-κB, cell viability was significantly reduced, even in the cells transfected with the miR-19a mimics. The MTT assay demonstrated that cell viability was significantly reduced in the cells transfected with si-NF-κB. These data suggested that miR-19a enhanced cellular proliferation through activation of the NF-κB signaling pathway.


MicroRNA-19a mediates gastric carcinoma cell proliferation through the activation of nuclear factor-κB.

Yang F, Wang H, Jiang Z, Hu A, Chu L, Sun Y, Han J - Mol Med Rep (2015)

NF-κB signaling pathway is activated when miR-19a is overexpressed in the MGC-803 human gastric carcinoma cell line. (A) Reverse transcription-quantitative polymerase chain reaction was used to determine the relative levels of miR-19a when MGC-803 cells were treated with 10 ng/μl TNF-α for 48 h. (B) Western blot analysis of NF-κB activation and its downstream regulators when miR-19a was overexpressed. (C) Western blot analysis of siRNA targeting NF-κB. (D) MTT assay demonstrated a low cellular proliferation rate in cells cotransfected with si-NF-κB and miR-19a mimics. Data are presented as the mean ± standard error of three independent experiments. *P<0.05, vs. control. TNF-α, tumor necrosis factor-α; NF-κB, nuclear factor-κB; VCAM, vascular cell adhesion molecule; ICAM, intercellular adhesion molecule; MCP, monocyte chemoattractant protein; miR, microRNA; si, small interfering; p-, phosphorylated; NC, negative control; Con, control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581753&req=5

f3-mmr-12-04-5780: NF-κB signaling pathway is activated when miR-19a is overexpressed in the MGC-803 human gastric carcinoma cell line. (A) Reverse transcription-quantitative polymerase chain reaction was used to determine the relative levels of miR-19a when MGC-803 cells were treated with 10 ng/μl TNF-α for 48 h. (B) Western blot analysis of NF-κB activation and its downstream regulators when miR-19a was overexpressed. (C) Western blot analysis of siRNA targeting NF-κB. (D) MTT assay demonstrated a low cellular proliferation rate in cells cotransfected with si-NF-κB and miR-19a mimics. Data are presented as the mean ± standard error of three independent experiments. *P<0.05, vs. control. TNF-α, tumor necrosis factor-α; NF-κB, nuclear factor-κB; VCAM, vascular cell adhesion molecule; ICAM, intercellular adhesion molecule; MCP, monocyte chemoattractant protein; miR, microRNA; si, small interfering; p-, phosphorylated; NC, negative control; Con, control.
Mentions: In gastric carcinoma, the NF-κB signaling pathway is constitutively activated. In previous studies, several miRNAs have been reported to activate NF-κB. For example, miR-301a has been demonstrated to target NF-κB-repressing factor and to correlate with abnormal NF-κB activation (17). In the present study, the MGC-803 gastric carcinoma cells were treated with 10 ng/μl TNF-α for 48 h, following which the relative levels of miR-19a were analyzed. As shown in Fig. 3A, the relative levels of miR-19a increased ~4-fold following TNF-α treatment. In addition, western blotting was performed to ascertain whether TNF-α treatment significantly activated the NF-κB signaling pathway. To address whether miR-19a contributes to NF-κB activation, the MGC-803 gastric carcinoma cells were transfected with miR-19a mimics. Based on the results of the western blot analysis, it was concluded that when miR-19a was overexpressed, NF-κB was significantly activated. As shown in Fig. 3A, compared with the negative control, the phosphorylated-NF-κB/NF-κB ratio was increased by >1-fold. Vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM) and monocyte chemoattractant protein-1 (MCP-1) are among the important adhesion molecules that are aberrantly expressed in various types of cancer (18). To validate the abnormal NF-κB activation, the protein levels of VCAM, ICAM and MCP-1 were measured. As shown in Fig. 3B, their relative levels were all increased ~1-fold. Based on the above results, it was concluded that miR-19a contributed to TNF-α-stimulated NF-κB activation. In order to investigate the effect of NF-κB on cellular proliferation, a small interfering (si)RNA-targeting NF-κB was selected. As shown in Fig. 3C, following knockdown of NF-κB, cell viability was significantly reduced, even in the cells transfected with the miR-19a mimics. The MTT assay demonstrated that cell viability was significantly reduced in the cells transfected with si-NF-κB. These data suggested that miR-19a enhanced cellular proliferation through activation of the NF-κB signaling pathway.

Bottom Line: The present study predominantly focussed on the effects of microRNA (miR)‑19a on NF‑κB activation.The results of the western blot analysis demonstrated that the protein levels of p65 increased when the MGC‑803 cells were transfected with miR‑19a mimics.In addition, the downstream target genes of miR‑19a, including intercellular adhesion molecule, vascular cell adhesion molecule and monocyte chemoattractant protein‑1, were upregulated.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Research and Therapy Center, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China.

ABSTRACT
In gastric carcinoma, the nuclear factor‑κB (NF‑κB) signaling pathway is highly active, and the constitutive activation of NF‑κB prompts malignant cell proliferation. MicroRNAs are considered to be important mediators in the regulation of the NF‑κB signaling pathway. The present study predominantly focussed on the effects of microRNA (miR)‑19a on NF‑κB activation. Reverse transcription‑quantitative polymerase chain reaction was used to quantify the relative levels of miR‑19a in gastric carcinoma cells. MTT assays were used to determine the effect of miR‑19a on cellular proliferation. To detect the activation of NF‑κB, western blotting was performed to measure the protein levels of NF‑κB and the products of its downstream target genes. To define the target genes, luciferase reporter assays were used. miR‑19a was found to be markedly upregulated in gastric carcinoma cells. The overexpression of miR‑19a resulted in proliferation and enhanced migratory capabilities of the MGC‑803 gastric carcinoma cell line. The results of the western blot analysis demonstrated that the protein levels of p65 increased when the MGC‑803 cells were transfected with miR‑19a mimics. In addition, the downstream target genes of miR‑19a, including intercellular adhesion molecule, vascular cell adhesion molecule and monocyte chemoattractant protein‑1, were upregulated. The results of the luciferase assay indicated that IκB‑α was the target gene of miR‑19a. Therefore, the results of the present study suggested that miR‑19a enhances malignant gastric cell proliferation by constitutively activating the NF‑κB signaling pathway.

Show MeSH
Related in: MedlinePlus