[Target regulation of micro-RNA-203 to the expression of collagen type Ⅳ alpha 4 and its role in oral submucous fibrosis].
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.
Department of Oral Mucosa, Xiangya Stomatological Hospital, Central South University, Changsha 410000, China.
J
J
Chen
Department of Oral Mucosa, Xiangya Stomatological Hospital, Central South University, Changsha 410000, China.
B J
BJ
Liu
Department of Oral Mucosa, Xiangya Stomatological Hospital, Central South University, Changsha 410000, China.
C
C
Du
Department of Oral Mucosa, Xiangya Stomatological Hospital, Central South University, Changsha 410000, China.
Q
Q
Cao
Department of Oral Mucosa, Xiangya Stomatological Hospital, Central South University, Changsha 410000, China.
M
M
Li
Department of Oral Mucosa, Xiangya Stomatological Hospital, Central South University, Changsha 410000, China.
H
H
Feng
D020413:3' Untranslated Regions; D001665:Binding Sites; D024141:Collagen Type IV; D019295:Computational Biology; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases; D035683:MicroRNAs; D009914:Oral Submucous Fibrosis; D012333:RNA, Messenger; D014162:Transfection
D020413:3' Untranslated Regions; D024141:Collagen Type IV; D035683:MicroRNAs; D012333:RNA, Messenger; D008156:Luciferases