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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 a direct target of miR-197. (A) Predicted binding sites of miR-197 on MAPK1 mRNA. The mutant UTR contains 5 bases in the complementary seed sequences. (B) miR-197 mimics transfected together with luciferase reporter vector pGL3-MAPK1 3′UTR or the mutant vector pGL3-MAPK1 3′UTR mut into SGC7901 cells. The relative luciferase intensity was significantly different between the miR-197 and miR-control group. Values are expressed as the mean ± standard deviation (n=3). *P<0.05. (C) Reverse transcription-quantitative polymerase chain reaction was conducted to detect the expression levels of MAPK1 mRNA in SGC7901 and SGC7901/5-FU cells which were transfected with the miR-197 mimics or control. Values are expressed as the mean ± standard deviation (n=3). (D) MAPK1 and p-MAPK1 protein levels in the SGC7901 cells transfected with the miR-197 inhibitor or control, and SGC7901/5-FU cells transfected with the miR-197 mimics or control. p-MAPK1, phosphorylated mitogen-activated protein kinase 1; miR-197, microRNA-197; UTR, untranslated region; 5-FU, fluorouracil; WT, wild-type; mut, mutated; SGC7901/5-FU, SGC7901 cells resistant to 5-FU.
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f3-mmr-12-04-5019: MAPK1 is a direct target of miR-197. (A) Predicted binding sites of miR-197 on MAPK1 mRNA. The mutant UTR contains 5 bases in the complementary seed sequences. (B) miR-197 mimics transfected together with luciferase reporter vector pGL3-MAPK1 3′UTR or the mutant vector pGL3-MAPK1 3′UTR mut into SGC7901 cells. The relative luciferase intensity was significantly different between the miR-197 and miR-control group. Values are expressed as the mean ± standard deviation (n=3). *P<0.05. (C) Reverse transcription-quantitative polymerase chain reaction was conducted to detect the expression levels of MAPK1 mRNA in SGC7901 and SGC7901/5-FU cells which were transfected with the miR-197 mimics or control. Values are expressed as the mean ± standard deviation (n=3). (D) MAPK1 and p-MAPK1 protein levels in the SGC7901 cells transfected with the miR-197 inhibitor or control, and SGC7901/5-FU cells transfected with the miR-197 mimics or control. p-MAPK1, phosphorylated mitogen-activated protein kinase 1; miR-197, microRNA-197; UTR, untranslated region; 5-FU, fluorouracil; WT, wild-type; mut, mutated; SGC7901/5-FU, SGC7901 cells resistant to 5-FU.

Mentions: To determine the candidate target gene of miR-197, a bioinformatics analysis using Targetscan 6.2 (http://www.targetscan.org/) was conducted to predict potential target genes. It was observed that the miR-197 complementary binding sites were contained in the 3′UTR of MAPK1 mRNA (Fig. 3A). A luciferase reporter assay was performed to validate that MAPK1 can be directly targeted by miR-197 using engineered EGFP reporter vectors that had either the wild-type 3′UTR of MAPK1 or the mutant UTR with a 5-base mutation in the complementary seed sequence (Fig. 3A). pDsRed2-NI was also co-transfected for normalization. SGC7901 cells were co-transfected with MAPK1 3′UTR and miR-197 or the control vector. Compared with the control vector, over-expression of miR-197 was able to significantly inhibit EGFP expression (P<0.05) (Fig. 3B). By contrast, EGFP expression levels of cells transfected with a mutant of the MAPK1 3′UTR binding site were not influenced by overexpression of miR-197 (Fig. 3B), indicating that miR-197 was able to complementarily bind to the specific sites of the MAPK1 mRNA 3′UTR and negatively regulate the expression of the MAPK1 gene.


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 a direct target of miR-197. (A) Predicted binding sites of miR-197 on MAPK1 mRNA. The mutant UTR contains 5 bases in the complementary seed sequences. (B) miR-197 mimics transfected together with luciferase reporter vector pGL3-MAPK1 3′UTR or the mutant vector pGL3-MAPK1 3′UTR mut into SGC7901 cells. The relative luciferase intensity was significantly different between the miR-197 and miR-control group. Values are expressed as the mean ± standard deviation (n=3). *P<0.05. (C) Reverse transcription-quantitative polymerase chain reaction was conducted to detect the expression levels of MAPK1 mRNA in SGC7901 and SGC7901/5-FU cells which were transfected with the miR-197 mimics or control. Values are expressed as the mean ± standard deviation (n=3). (D) MAPK1 and p-MAPK1 protein levels in the SGC7901 cells transfected with the miR-197 inhibitor or control, and SGC7901/5-FU cells transfected with the miR-197 mimics or control. p-MAPK1, phosphorylated mitogen-activated protein kinase 1; miR-197, microRNA-197; UTR, untranslated region; 5-FU, fluorouracil; WT, wild-type; mut, mutated; SGC7901/5-FU, SGC7901 cells resistant to 5-FU.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-mmr-12-04-5019: MAPK1 is a direct target of miR-197. (A) Predicted binding sites of miR-197 on MAPK1 mRNA. The mutant UTR contains 5 bases in the complementary seed sequences. (B) miR-197 mimics transfected together with luciferase reporter vector pGL3-MAPK1 3′UTR or the mutant vector pGL3-MAPK1 3′UTR mut into SGC7901 cells. The relative luciferase intensity was significantly different between the miR-197 and miR-control group. Values are expressed as the mean ± standard deviation (n=3). *P<0.05. (C) Reverse transcription-quantitative polymerase chain reaction was conducted to detect the expression levels of MAPK1 mRNA in SGC7901 and SGC7901/5-FU cells which were transfected with the miR-197 mimics or control. Values are expressed as the mean ± standard deviation (n=3). (D) MAPK1 and p-MAPK1 protein levels in the SGC7901 cells transfected with the miR-197 inhibitor or control, and SGC7901/5-FU cells transfected with the miR-197 mimics or control. p-MAPK1, phosphorylated mitogen-activated protein kinase 1; miR-197, microRNA-197; UTR, untranslated region; 5-FU, fluorouracil; WT, wild-type; mut, mutated; SGC7901/5-FU, SGC7901 cells resistant to 5-FU.
Mentions: To determine the candidate target gene of miR-197, a bioinformatics analysis using Targetscan 6.2 (http://www.targetscan.org/) was conducted to predict potential target genes. It was observed that the miR-197 complementary binding sites were contained in the 3′UTR of MAPK1 mRNA (Fig. 3A). A luciferase reporter assay was performed to validate that MAPK1 can be directly targeted by miR-197 using engineered EGFP reporter vectors that had either the wild-type 3′UTR of MAPK1 or the mutant UTR with a 5-base mutation in the complementary seed sequence (Fig. 3A). pDsRed2-NI was also co-transfected for normalization. SGC7901 cells were co-transfected with MAPK1 3′UTR and miR-197 or the control vector. Compared with the control vector, over-expression of miR-197 was able to significantly inhibit EGFP expression (P<0.05) (Fig. 3B). By contrast, EGFP expression levels of cells transfected with a mutant of the MAPK1 3′UTR binding site were not influenced by overexpression of miR-197 (Fig. 3B), indicating that miR-197 was able to complementarily bind to the specific sites of the MAPK1 mRNA 3′UTR and negatively regulate the expression of the MAPK1 gene.

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