Zhonghua kou qiang yi xue za zhi = Zhonghua kouqiang yixue zazhi = Chinese journal of stomatology · 2016
[Target regulation of micro-RNA-203 to the expression of collagen type Ⅳ alpha 4 and its role in oral submucous fibrosis].
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Chen J, Liu B J, Du C, Cao Q, Li M, Feng H
Аннотация
OBJECTIVE: To explore the effect of microRNA-203(miR-203) on the expression of collagen type IV alpha 4(COL4A4) and its role in oral submucous fibrosis(OSF).
METHODS: It has been revealed that COL4A4 is the putative target of miR-203 by retrieving a variety of bioinformatics software tools. The expression of COL4A4 protein in the indicated tissues was examined by Western blotting analysis and the potential binding sites of miR-203 and COL4A4-3'UTR was analyzed by using bioinformatics. This study integrated a fragment of COL4A4 3'UTR containing the target sequence into the pMIR-REPORT luciferase vector. Then, the COL4A4 3'UTR-luciferase and miR-203 were contransfected into 293T cells. Beta-gal was used as a control to monitor transfection efficiency. Cells were harvested and luciferase activity was measured after 24 h of transfection.
RESULTS: The relative expression mean valves of COL4A4 were 2.46 ± 2.26 in OSF and 0.70 ± 0.49 in normal control. The expression of COL4A4 in OSF was higher than normal control(P<0.05) and the difference between groups was statistically significant. The relative expression mean valves of miR-203 were 2.57 ± 2.09 in OSF and 154.07 ± 191.11 in normal control. The expression of miR-203 in OSF was lower than in normal control(P<0.01) and the difference between groups was statistically significant. Bioinformatics analysis revealed that the COL4A4 3'UTR had a conserved site (site 1) and 3 non-conserved sites(site 2, site 3, site4) complementary to the miR-203. MiR-203 could significantly inhibit site 1-luciferase reporter activity, partially inhibited site 3- and site 4-luciferase reporter activities, while no significant effect on site 3-luciferase reporter activity.
CONCLUSIONS: MiR-203 was negatively correlated with the expression of COL4A4 protein in OSF. Moreover, miR-203 repressed the expression of COL4A4 by targeting 3'UTR site of COL4A4 mRNA.
METHODS: It has been revealed that COL4A4 is the putative target of miR-203 by retrieving a variety of bioinformatics software tools. The expression of COL4A4 protein in the indicated tissues was examined by Western blotting analysis and the potential binding sites of miR-203 and COL4A4-3'UTR was analyzed by using bioinformatics. This study integrated a fragment of COL4A4 3'UTR containing the target sequence into the pMIR-REPORT luciferase vector. Then, the COL4A4 3'UTR-luciferase and miR-203 were contransfected into 293T cells. Beta-gal was used as a control to monitor transfection efficiency. Cells were harvested and luciferase activity was measured after 24 h of transfection.
RESULTS: The relative expression mean valves of COL4A4 were 2.46 ± 2.26 in OSF and 0.70 ± 0.49 in normal control. The expression of COL4A4 in OSF was higher than normal control(P<0.05) and the difference between groups was statistically significant. The relative expression mean valves of miR-203 were 2.57 ± 2.09 in OSF and 154.07 ± 191.11 in normal control. The expression of miR-203 in OSF was lower than in normal control(P<0.01) and the difference between groups was statistically significant. Bioinformatics analysis revealed that the COL4A4 3'UTR had a conserved site (site 1) and 3 non-conserved sites(site 2, site 3, site4) complementary to the miR-203. MiR-203 could significantly inhibit site 1-luciferase reporter activity, partially inhibited site 3- and site 4-luciferase reporter activities, while no significant effect on site 3-luciferase reporter activity.
CONCLUSIONS: MiR-203 was negatively correlated with the expression of COL4A4 protein in OSF. Moreover, miR-203 repressed the expression of COL4A4 by targeting 3'UTR site of COL4A4 mRNA.