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MicroRNA-140 Inhibits Cell Proliferation in Gastric Cancer Cell Line HGC-27 by Suppressing SOX4.

Zou J, Xu Y - Med. Sci. Monit. (2016)

Bottom Line: SOX4, a predicted target of miR-140, was mutated to verify its regulation by miR-140, and was overexpressed to analyze its function in cell proliferation.Doxorubicin treatment was performed to investigate the effect of miR-140 on drug resistance.Overexpressing SOX4 led to promoted cell viability, colony formation, and cell cycle progress. miR-140 overexpression also improved the anti-viability effects of doxorubicin, suggesting its potential in reducing the drug resistance of gastric cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China (mainland).

ABSTRACT
BACKGROUND Gastric cancer is a malignant tumor with a high morbidity and mortality. MicroRNAs are important regulators of gene expression, influencing the progression of gastric cancer. This study aimed to reveal the role of microRNA-140 (miR-140) in gastric cancer cell proliferation and its potential mechanisms. MATERIAL AND METHODS Gastric cancer tissues and cell lines BGC-823, SGC-7901, and HGC-27 were used to analyze miR-140 levels compared to normal tissues and cell line GES-1. In HGC-27 cells transfected with miR-140 mimic, we performed MTT, colony formation assay, and cell cycle assay by flow cytometry. SOX4, a predicted target of miR-140, was mutated to verify its regulation by miR-140, and was overexpressed to analyze its function in cell proliferation. Doxorubicin treatment was performed to investigate the effect of miR-140 on drug resistance. RESULTS miR-140 was down-regulated in gastric cancer tissues and cell lines, with the lowest expression level in HGC-27. miR-140 overexpression inhibited HGC-27 cell viability and colony formation and resulted in G0/G1 arrest. miR-140 suppressed SOX4 expression via binding to the 3' untranslated region, while the mutant SOX4 could not be regulated. Overexpressing SOX4 led to promoted cell viability, colony formation, and cell cycle progress. miR-140 overexpression also improved the anti-viability effects of doxorubicin, suggesting its potential in reducing the drug resistance of gastric cells. CONCLUSIONS These findings suggest that miR-140 directly inhibits SOX4, which might be one of its mechanisms in suppressing gastric cancer cell proliferation. This study provides a promising therapeutic strategy for treating gastric cancer and facilitates microRNA research in various diseases.

No MeSH data available.


Related in: MedlinePlus

SOX4 is directly inhibited by miR-140. (A) SOX4 mRNA is predicted to be a target for miR-140, with the binding sequence AACCACU in the 3′UTR (position 2132 to 2138). Multi-site mutation is performed to mutate 3 nucleotides (underlined) in the binding site. Mut-SOX4 3′UTR, mutant type of SOX4 3′UTR. (B) Luciferase activity assay indicates that the binding sequence in SOX4 3′UTR is necessary for regulation by miR-140. HGC-27 cells overexpressing miR-140 are co-transfected with the reporter vectors of wild-type or mutant-type of SOX4 3′UTR (SOX4 3′UTR or mut-SOX4 3′UTR). The activity of mutant-type cannot be regulated by miR-140. (C) SOX4 mRNA level is down-regulated by miR-140 overexpression. (D) SOX4 protein level is suppressed by miR-140 overexpression. * P<0.05. SOX4 – sex-determining region Y (SRY)-box 4.
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f3-medscimonit-22-2243: SOX4 is directly inhibited by miR-140. (A) SOX4 mRNA is predicted to be a target for miR-140, with the binding sequence AACCACU in the 3′UTR (position 2132 to 2138). Multi-site mutation is performed to mutate 3 nucleotides (underlined) in the binding site. Mut-SOX4 3′UTR, mutant type of SOX4 3′UTR. (B) Luciferase activity assay indicates that the binding sequence in SOX4 3′UTR is necessary for regulation by miR-140. HGC-27 cells overexpressing miR-140 are co-transfected with the reporter vectors of wild-type or mutant-type of SOX4 3′UTR (SOX4 3′UTR or mut-SOX4 3′UTR). The activity of mutant-type cannot be regulated by miR-140. (C) SOX4 mRNA level is down-regulated by miR-140 overexpression. (D) SOX4 protein level is suppressed by miR-140 overexpression. * P<0.05. SOX4 – sex-determining region Y (SRY)-box 4.

Mentions: The potential regulatory mechanism of miR-140 was analyzed by investigating its target mRNA, SOX4, which was predicted to be a target of miR-140 by the online database TargetScanHuman 7.0, with the sequence AACCACU in SOX4 3′UTR being the potential binding site (Figure 3A). To verify whether SOX4 mRNA was a direct target of miR-140, we mutated 3 nucleotides in the binding site, and the generated mutant type was not supposed to be targeted by miR-140. Indeed, luciferase activity assay showed that the luciferase activity of vector containing wild-type SOX4 3′UTR could be suppressed by miR-140 overexpression (P<0.05, Figure 3B), while miR-140 failed to inhibit mutant type of SOX4 3′UTR (P>0.05), suggesting that this binding site in SOX4 3′UTR was essential for the regulation by miR-140. Together with the prediction results that SOX4 was a potential target for miR-140, it could be deduced that miR-140 can directly regulate SOX4 mRNA.


MicroRNA-140 Inhibits Cell Proliferation in Gastric Cancer Cell Line HGC-27 by Suppressing SOX4.

Zou J, Xu Y - Med. Sci. Monit. (2016)

SOX4 is directly inhibited by miR-140. (A) SOX4 mRNA is predicted to be a target for miR-140, with the binding sequence AACCACU in the 3′UTR (position 2132 to 2138). Multi-site mutation is performed to mutate 3 nucleotides (underlined) in the binding site. Mut-SOX4 3′UTR, mutant type of SOX4 3′UTR. (B) Luciferase activity assay indicates that the binding sequence in SOX4 3′UTR is necessary for regulation by miR-140. HGC-27 cells overexpressing miR-140 are co-transfected with the reporter vectors of wild-type or mutant-type of SOX4 3′UTR (SOX4 3′UTR or mut-SOX4 3′UTR). The activity of mutant-type cannot be regulated by miR-140. (C) SOX4 mRNA level is down-regulated by miR-140 overexpression. (D) SOX4 protein level is suppressed by miR-140 overexpression. * P<0.05. SOX4 – sex-determining region Y (SRY)-box 4.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4930272&req=5

f3-medscimonit-22-2243: SOX4 is directly inhibited by miR-140. (A) SOX4 mRNA is predicted to be a target for miR-140, with the binding sequence AACCACU in the 3′UTR (position 2132 to 2138). Multi-site mutation is performed to mutate 3 nucleotides (underlined) in the binding site. Mut-SOX4 3′UTR, mutant type of SOX4 3′UTR. (B) Luciferase activity assay indicates that the binding sequence in SOX4 3′UTR is necessary for regulation by miR-140. HGC-27 cells overexpressing miR-140 are co-transfected with the reporter vectors of wild-type or mutant-type of SOX4 3′UTR (SOX4 3′UTR or mut-SOX4 3′UTR). The activity of mutant-type cannot be regulated by miR-140. (C) SOX4 mRNA level is down-regulated by miR-140 overexpression. (D) SOX4 protein level is suppressed by miR-140 overexpression. * P<0.05. SOX4 – sex-determining region Y (SRY)-box 4.
Mentions: The potential regulatory mechanism of miR-140 was analyzed by investigating its target mRNA, SOX4, which was predicted to be a target of miR-140 by the online database TargetScanHuman 7.0, with the sequence AACCACU in SOX4 3′UTR being the potential binding site (Figure 3A). To verify whether SOX4 mRNA was a direct target of miR-140, we mutated 3 nucleotides in the binding site, and the generated mutant type was not supposed to be targeted by miR-140. Indeed, luciferase activity assay showed that the luciferase activity of vector containing wild-type SOX4 3′UTR could be suppressed by miR-140 overexpression (P<0.05, Figure 3B), while miR-140 failed to inhibit mutant type of SOX4 3′UTR (P>0.05), suggesting that this binding site in SOX4 3′UTR was essential for the regulation by miR-140. Together with the prediction results that SOX4 was a potential target for miR-140, it could be deduced that miR-140 can directly regulate SOX4 mRNA.

Bottom Line: SOX4, a predicted target of miR-140, was mutated to verify its regulation by miR-140, and was overexpressed to analyze its function in cell proliferation.Doxorubicin treatment was performed to investigate the effect of miR-140 on drug resistance.Overexpressing SOX4 led to promoted cell viability, colony formation, and cell cycle progress. miR-140 overexpression also improved the anti-viability effects of doxorubicin, suggesting its potential in reducing the drug resistance of gastric cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China (mainland).

ABSTRACT
BACKGROUND Gastric cancer is a malignant tumor with a high morbidity and mortality. MicroRNAs are important regulators of gene expression, influencing the progression of gastric cancer. This study aimed to reveal the role of microRNA-140 (miR-140) in gastric cancer cell proliferation and its potential mechanisms. MATERIAL AND METHODS Gastric cancer tissues and cell lines BGC-823, SGC-7901, and HGC-27 were used to analyze miR-140 levels compared to normal tissues and cell line GES-1. In HGC-27 cells transfected with miR-140 mimic, we performed MTT, colony formation assay, and cell cycle assay by flow cytometry. SOX4, a predicted target of miR-140, was mutated to verify its regulation by miR-140, and was overexpressed to analyze its function in cell proliferation. Doxorubicin treatment was performed to investigate the effect of miR-140 on drug resistance. RESULTS miR-140 was down-regulated in gastric cancer tissues and cell lines, with the lowest expression level in HGC-27. miR-140 overexpression inhibited HGC-27 cell viability and colony formation and resulted in G0/G1 arrest. miR-140 suppressed SOX4 expression via binding to the 3' untranslated region, while the mutant SOX4 could not be regulated. Overexpressing SOX4 led to promoted cell viability, colony formation, and cell cycle progress. miR-140 overexpression also improved the anti-viability effects of doxorubicin, suggesting its potential in reducing the drug resistance of gastric cells. CONCLUSIONS These findings suggest that miR-140 directly inhibits SOX4, which might be one of its mechanisms in suppressing gastric cancer cell proliferation. This study provides a promising therapeutic strategy for treating gastric cancer and facilitates microRNA research in various diseases.

No MeSH data available.


Related in: MedlinePlus