Objective: To investigate the expression of microRNA-296 (miR-296) in rabbit
hypertrophic scars and its role in human fibroblasts (HFbs). Methods: The experimental method was used. Twelve healthy adult New Zealand long-eared rabbits regardless gender were randomly divided into normal control group and
scar group, with 6 rabbits in each group. The rabbit ear
hypertrophic scar model was created in
scar group according to the literature, and the rabbits in normal control group did not receive any treatment. On 60 days after setting up the models in
scar group,
hematoxylin-
eosin staining was performed to observe the growth and arrangement of fibroblasts (Fbs) in the ear
scars and skin tissue of rabbits in the two groups. The
mRNA expressions of miR-296 and transforming growth factor-β1 (TGF-β1) in ear
scars and skin tissue of rabbits in the two groups were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction, and the correlation of
mRNA between miR-296 and TGF-β1 was performed with Pearson regression analysis. Two batches of HFbs were used and transfected respectively with corresponding sequences, with the 1st batch being divided into TGF-β1 wild type+miR-296 negative control group and TGF-β1 wild type+miR-296 mimic group and the 2nd batch being divided into TGF-β1 mutant type+miR-296 negative control group and TGF-β1 mutant type+miR-296 mimic group. At 48 h after transfection,
luciferase reporter gene detection kit was used to detect the
luciferase and renal
luciferase expression of TGF-β1 in the cells of each group, with their ratio being used to reflect the gene expression level. Two batches of HFbs were used, and each batch of cells were divided into miR-296 negative control group and miR-296 mimic group, being transfected with the corresponding sequences. At 0 (immediately), 12, 24, 36, and 48 h after transfecting the first batch of cells, the cell proliferation was detected by
thiazolyl blue method. At 24 h after transfecting the second batch of cells, the expression of TGF-β1 and
collagen type Ⅰ was detected by Western blotting. The number of samples in cell experiments was 3. Data were statistically analyzed with analysis of variance for factorial design, independent sample t test. Results: On 60 days after setting up the models in
scar group, the Fbs of rabbit ear
scar tissue in
scar group proliferated and arranged disorderly, while the growth and arrangement of Fbs in rabbit ear skin tissue in normal control group were normal. The
mRNA expression of miR-296 of rabbit
scar tissue in
scar group (0.65±0.11) was significantly lower than 1.19±0.12 of rabbit ear skin tissue in normal control group (t=5.175, P<0.01). The
mRNA expression of TGF-β1 of rabbit ear
scar tissue in
scar group (1.47±0.06) was significantly higher than 1.10±0.03 of rabbit ear skin tissue in normal control group (t=12.410, P<0.01). Pearson regression analysis showed that there was a negative correlation between the
mRNA expression of miR-296 and TGF-β1 in the ear
scars and skin tissue of 12 rabbits (F=7.278, P<0.05). At 48 h after transfection, the gene expression of TGF-β1 of cells in TGF-β1 wild type+miR-296 mimic group was significantly lower than that in TGF-β1 wild type+miR-296 negative control group (t=35.190, P<0.01), while the gene expression of TGF-β1 of cells in the two TGF-β1 mutant type groups were close (P>0.05). The HFbs proliferation ability in miR-296 mimic group was significantly lower than that in miR-296 negative control group at 12, 24, 36, and 48 h after transfection(t=3.275, 11.980, 10.460, 17.260, P<0.05 or P<0.01). At 24 h after transfection, the
protein expressions of TGF-β1 and type Ⅰ
collagen of cells in miR-296 negative control group were significantly higher than those in miR-296 mimic group (t=3.758, 29.390, P<0.05 or P<0.01). Conclusions: The miR-296 expression in rabbit
hypertrophic scars is down-regulated; miR-296 can inhibit the proliferation of HFbs and the expression of type Ⅰ
collagen by down regulating the expression of TGF-β1.