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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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miR-19a targets IκBα in MGC-803 human gastric carcinoma cells. (A) Immunofluorescence analysis of the expression of NF-κB when MGC-803 cells were transfected with miR-19a or negative control (magnification, ×100). Western blot analysis of IκBα was performed on the MGC-803 cells transfected with (B) miR-19a mimics or (C) miR-19a inhibitors and negative controls. (D) Luciferase reporter assay to detect the effect of miR-19a on IκBα-3′-UTR. Data are presented as the mean ± standard error of three independent experiments. *P<0.05, vs. control. NF-κB, nuclear factor-κB; miR, microRNA; NFKR, NF-κB-repressing factor; UTR, untranslated region; NC, negative control; Con, control.
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f4-mmr-12-04-5780: miR-19a targets IκBα in MGC-803 human gastric carcinoma cells. (A) Immunofluorescence analysis of the expression of NF-κB when MGC-803 cells were transfected with miR-19a or negative control (magnification, ×100). Western blot analysis of IκBα was performed on the MGC-803 cells transfected with (B) miR-19a mimics or (C) miR-19a inhibitors and negative controls. (D) Luciferase reporter assay to detect the effect of miR-19a on IκBα-3′-UTR. Data are presented as the mean ± standard error of three independent experiments. *P<0.05, vs. control. NF-κB, nuclear factor-κB; miR, microRNA; NFKR, NF-κB-repressing factor; UTR, untranslated region; NC, negative control; Con, control.

Mentions: To further confirm that NF-κB was activated upon miR-19a overexpression, immunofluorescence was measured. As shown in Fig. 4A, enhanced NF-κB (p65) protein levels were detected when the MGC-803 cells were transfected with miR-19a mimics. According to TargetScan, miR-19a was predicted to target IκBα. IκB is an NF-κB inhibitory protein (19). In the resting state, IκB-α combines with p65, P50, resulting in the inactivation of NF-κB in the cytoplasm. Through the activation of IKK, IκBα is degraded, which drives the two subunits of NF-κB to translocate between the cytoplasm and the nucleus, particularly the p65 subunit, inducing a downstream signaling pathway. Therefore, the effects of miR-19a on the expression of IκBα were assessed. When the MGC-803 human gastric carcinoma cell line was transfected with miR-19a mimics for 48 h, the density of IκBα was significantly reduced, compared with the negative control (Fig. 4B). Furthermore, 48 h following transfection of the MGC-803 cells with miR-19a, the expression level of IκBα was reduced by 56%. Conversely, when miR-19a was inhibited in the MGC-803 cells, the expression level of IκBα was increased by almost 1-fold (Fig. 4C). A luciferase assay was also performed to detect the effect of miR-19a on the 3′-UTR region of IκBα. As shown in Fig. 4D, miR-19a significantly reduced IκBα-3′-UTR-luciferase reporter activity. Based on the above analysis, it was suggested that miR-19a induced NF-κB activation, predominantly by targeting IκBα, in the human gastric carcinoma cells.


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)

miR-19a targets IκBα in MGC-803 human gastric carcinoma cells. (A) Immunofluorescence analysis of the expression of NF-κB when MGC-803 cells were transfected with miR-19a or negative control (magnification, ×100). Western blot analysis of IκBα was performed on the MGC-803 cells transfected with (B) miR-19a mimics or (C) miR-19a inhibitors and negative controls. (D) Luciferase reporter assay to detect the effect of miR-19a on IκBα-3′-UTR. Data are presented as the mean ± standard error of three independent experiments. *P<0.05, vs. control. NF-κB, nuclear factor-κB; miR, microRNA; NFKR, NF-κB-repressing factor; UTR, untranslated region; NC, negative control; Con, control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4-mmr-12-04-5780: miR-19a targets IκBα in MGC-803 human gastric carcinoma cells. (A) Immunofluorescence analysis of the expression of NF-κB when MGC-803 cells were transfected with miR-19a or negative control (magnification, ×100). Western blot analysis of IκBα was performed on the MGC-803 cells transfected with (B) miR-19a mimics or (C) miR-19a inhibitors and negative controls. (D) Luciferase reporter assay to detect the effect of miR-19a on IκBα-3′-UTR. Data are presented as the mean ± standard error of three independent experiments. *P<0.05, vs. control. NF-κB, nuclear factor-κB; miR, microRNA; NFKR, NF-κB-repressing factor; UTR, untranslated region; NC, negative control; Con, control.
Mentions: To further confirm that NF-κB was activated upon miR-19a overexpression, immunofluorescence was measured. As shown in Fig. 4A, enhanced NF-κB (p65) protein levels were detected when the MGC-803 cells were transfected with miR-19a mimics. According to TargetScan, miR-19a was predicted to target IκBα. IκB is an NF-κB inhibitory protein (19). In the resting state, IκB-α combines with p65, P50, resulting in the inactivation of NF-κB in the cytoplasm. Through the activation of IKK, IκBα is degraded, which drives the two subunits of NF-κB to translocate between the cytoplasm and the nucleus, particularly the p65 subunit, inducing a downstream signaling pathway. Therefore, the effects of miR-19a on the expression of IκBα were assessed. When the MGC-803 human gastric carcinoma cell line was transfected with miR-19a mimics for 48 h, the density of IκBα was significantly reduced, compared with the negative control (Fig. 4B). Furthermore, 48 h following transfection of the MGC-803 cells with miR-19a, the expression level of IκBα was reduced by 56%. Conversely, when miR-19a was inhibited in the MGC-803 cells, the expression level of IκBα was increased by almost 1-fold (Fig. 4C). A luciferase assay was also performed to detect the effect of miR-19a on the 3′-UTR region of IκBα. As shown in Fig. 4D, miR-19a significantly reduced IκBα-3′-UTR-luciferase reporter activity. Based on the above analysis, it was suggested that miR-19a induced NF-κB activation, predominantly by targeting IκBα, in the human gastric carcinoma cells.

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