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MicroRNA‑197 reverses the drug resistance of fluorouracil‑induced SGC7901 cells by targeting mitogen‑activated protein kinase 1.

Xiong HL, Zhou SW, Sun AH, He Y, Li J, Yuan X - Mol Med Rep (2015)

Bottom Line: A luciferase reporter assay confirmed that miR‑197 led to silencing of the MAPK1 gene by recognizing and then specifically binding to the predicted site of the MAPK1 mRNA 3'‑untranslated region.When miR‑197 was overexpressed in SGC7901 cells, the protein levels of MAPK1 were downregulated.Furthermore, MAPK1 knockdown significantly increased the growth inhibition rate of the SGC7901/5‑FU cells compared with those in the control group.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Huizhou Municipal Central Hospital of Guangdong Province, Huizhou, Guangdong 516000, P.R. China.

ABSTRACT
MicroRNAs (miRNAs) are a group of small non‑coding RNA molecules, which serve an important function in the development of multidrug resistance in cancer through the post‑transcriptional regulation of gene expression and RNA silencing. In the present study, the functional effects of miR‑197 were analyzed in chemo‑resistant gastric cancer cells. Low expression levels of miR‑197 were observed in the fluorouracil (5‑FU)‑resistant gastric cell line SGC7901/5‑FU when compared with those in the parental gastric cell line SGC7901. Overexpression of miR‑197 in SGC7901/5‑FU cells was identified to partially restore 5‑FU sensitivity. miRNA target prediction algorithms suggested that mitogen‑activated protein kinase 1 (MAPK1) is a candidate target gene for miR‑197. A luciferase reporter assay confirmed that miR‑197 led to silencing of the MAPK1 gene by recognizing and then specifically binding to the predicted site of the MAPK1 mRNA 3'‑untranslated region. When miR‑197 was overexpressed in SGC7901 cells, the protein levels of MAPK1 were downregulated. Furthermore, MAPK1 knockdown significantly increased the growth inhibition rate of the SGC7901/5‑FU cells compared with those in the control group. These results indicated that miR‑197 may influence the sensitivity of 5‑FU treatment in a gastric cancer cell line by targeting MAPK1.

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MAPK1 is important in 5-FU resistance in SGC7901/5-FU cells. (A) MAPK1 protein levels in the SGC7901/5-FU cells following treatment with MAPK1 siRNA or a scrambled siRNA. (B) SGC7901/5-FU cells were treated with various doses of 5-FU (0.5, 1.0 and 2.0 µmol/l) following 48 h of transfection. The cell growth inhibition rate was determined using an MTT assay. (C) Apoptosis detected by flow cytometry. SGC-7901/5-FU cells were untreated, transfected with miR-197 mimics or transfected with siRNA MAPK1 for 48 h, and apoptotic cells were detected by flow cytometry. Values are expressed as the mean ± standard deviation (n=3). *P<0.05 vs. control. MAPK1, mitogen-activated protein kinase 1; 5-FU, fluorouracil; SGC7901/5-FU, SGC7901 cells resistant to 5-FU; siR/siRNA, small interfering RNA; PI, propidium iodide.
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f4-mmr-12-04-5019: MAPK1 is important in 5-FU resistance in SGC7901/5-FU cells. (A) MAPK1 protein levels in the SGC7901/5-FU cells following treatment with MAPK1 siRNA or a scrambled siRNA. (B) SGC7901/5-FU cells were treated with various doses of 5-FU (0.5, 1.0 and 2.0 µmol/l) following 48 h of transfection. The cell growth inhibition rate was determined using an MTT assay. (C) Apoptosis detected by flow cytometry. SGC-7901/5-FU cells were untreated, transfected with miR-197 mimics or transfected with siRNA MAPK1 for 48 h, and apoptotic cells were detected by flow cytometry. Values are expressed as the mean ± standard deviation (n=3). *P<0.05 vs. control. MAPK1, mitogen-activated protein kinase 1; 5-FU, fluorouracil; SGC7901/5-FU, SGC7901 cells resistant to 5-FU; siR/siRNA, small interfering RNA; PI, propidium iodide.

Mentions: To validate whether MAPK1 serves a role in miR-197-induced 5-FU resistance, the MAPK1 gene was silenced to a certain degree in the SGC7901/5-FU cells. Western blot analysis demonstrated that MAPK1 siRNA effectively reduced the MAPK1 protein levels (Fig. 4A). Following MAPK1 siRNA transfection, the cell growth inhibition rate by was then detected following exposure to various concentrations of 5-FU (0.5–8 µM). Knockdown of MAPK1 significantly increased the growth inhibition rate of the SGC7901/5-FU cells compared with that in the control group (Fig. 4B). The SGC7901/5-FU cells transfected with miR-197 mimics in addition to those transfected with siR-MAPK1 demonstrated a significantly increased apoptotic rate compared with that in the negative control group (P<0.05). These results suggested that miR-197 may improve the 5-FU sensitivity of the SGC7901/5-FU cells by down-regulating MAPK1.


MicroRNA‑197 reverses the drug resistance of fluorouracil‑induced SGC7901 cells by targeting mitogen‑activated protein kinase 1.

Xiong HL, Zhou SW, Sun AH, He Y, Li J, Yuan X - Mol Med Rep (2015)

MAPK1 is important in 5-FU resistance in SGC7901/5-FU cells. (A) MAPK1 protein levels in the SGC7901/5-FU cells following treatment with MAPK1 siRNA or a scrambled siRNA. (B) SGC7901/5-FU cells were treated with various doses of 5-FU (0.5, 1.0 and 2.0 µmol/l) following 48 h of transfection. The cell growth inhibition rate was determined using an MTT assay. (C) Apoptosis detected by flow cytometry. SGC-7901/5-FU cells were untreated, transfected with miR-197 mimics or transfected with siRNA MAPK1 for 48 h, and apoptotic cells were detected by flow cytometry. Values are expressed as the mean ± standard deviation (n=3). *P<0.05 vs. control. MAPK1, mitogen-activated protein kinase 1; 5-FU, fluorouracil; SGC7901/5-FU, SGC7901 cells resistant to 5-FU; siR/siRNA, small interfering RNA; PI, propidium iodide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581796&req=5

f4-mmr-12-04-5019: MAPK1 is important in 5-FU resistance in SGC7901/5-FU cells. (A) MAPK1 protein levels in the SGC7901/5-FU cells following treatment with MAPK1 siRNA or a scrambled siRNA. (B) SGC7901/5-FU cells were treated with various doses of 5-FU (0.5, 1.0 and 2.0 µmol/l) following 48 h of transfection. The cell growth inhibition rate was determined using an MTT assay. (C) Apoptosis detected by flow cytometry. SGC-7901/5-FU cells were untreated, transfected with miR-197 mimics or transfected with siRNA MAPK1 for 48 h, and apoptotic cells were detected by flow cytometry. Values are expressed as the mean ± standard deviation (n=3). *P<0.05 vs. control. MAPK1, mitogen-activated protein kinase 1; 5-FU, fluorouracil; SGC7901/5-FU, SGC7901 cells resistant to 5-FU; siR/siRNA, small interfering RNA; PI, propidium iodide.
Mentions: To validate whether MAPK1 serves a role in miR-197-induced 5-FU resistance, the MAPK1 gene was silenced to a certain degree in the SGC7901/5-FU cells. Western blot analysis demonstrated that MAPK1 siRNA effectively reduced the MAPK1 protein levels (Fig. 4A). Following MAPK1 siRNA transfection, the cell growth inhibition rate by was then detected following exposure to various concentrations of 5-FU (0.5–8 µM). Knockdown of MAPK1 significantly increased the growth inhibition rate of the SGC7901/5-FU cells compared with that in the control group (Fig. 4B). The SGC7901/5-FU cells transfected with miR-197 mimics in addition to those transfected with siR-MAPK1 demonstrated a significantly increased apoptotic rate compared with that in the negative control group (P<0.05). These results suggested that miR-197 may improve the 5-FU sensitivity of the SGC7901/5-FU cells by down-regulating MAPK1.

Bottom Line: A luciferase reporter assay confirmed that miR‑197 led to silencing of the MAPK1 gene by recognizing and then specifically binding to the predicted site of the MAPK1 mRNA 3'‑untranslated region.When miR‑197 was overexpressed in SGC7901 cells, the protein levels of MAPK1 were downregulated.Furthermore, MAPK1 knockdown significantly increased the growth inhibition rate of the SGC7901/5‑FU cells compared with those in the control group.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Huizhou Municipal Central Hospital of Guangdong Province, Huizhou, Guangdong 516000, P.R. China.

ABSTRACT
MicroRNAs (miRNAs) are a group of small non‑coding RNA molecules, which serve an important function in the development of multidrug resistance in cancer through the post‑transcriptional regulation of gene expression and RNA silencing. In the present study, the functional effects of miR‑197 were analyzed in chemo‑resistant gastric cancer cells. Low expression levels of miR‑197 were observed in the fluorouracil (5‑FU)‑resistant gastric cell line SGC7901/5‑FU when compared with those in the parental gastric cell line SGC7901. Overexpression of miR‑197 in SGC7901/5‑FU cells was identified to partially restore 5‑FU sensitivity. miRNA target prediction algorithms suggested that mitogen‑activated protein kinase 1 (MAPK1) is a candidate target gene for miR‑197. A luciferase reporter assay confirmed that miR‑197 led to silencing of the MAPK1 gene by recognizing and then specifically binding to the predicted site of the MAPK1 mRNA 3'‑untranslated region. When miR‑197 was overexpressed in SGC7901 cells, the protein levels of MAPK1 were downregulated. Furthermore, MAPK1 knockdown significantly increased the growth inhibition rate of the SGC7901/5‑FU cells compared with those in the control group. These results indicated that miR‑197 may influence the sensitivity of 5‑FU treatment in a gastric cancer cell line by targeting MAPK1.

Show MeSH
Related in: MedlinePlus