Upregulation of TUG1 Expression in Hypertrophic Scars Facilitates Fibroblast Proliferation and Migration
To delineate the potential role of TUG1 in hypertrophic scars (HS), we first examined the expression levels of TUG1 in HS tissue. Gene chip analysis revealed a significant upregulation of TUG1 expression in HS tissue compared to control tissue (Fig. 1A). To validate these findings, we performed quantitative reverse transcription PCR (qRT-PCR) on HS and adjacent normal tissues, corroborating that TUG1 expression is markedly elevated in HS tissues (Fig. 1B).
We expanded our analysis by comparing TUG1 expression in adjacent normal tissue, normal scar tissue, and HS tissue. A consistent trend emerged, with TUG1 levels being the highest in HS tissue (Fig. 1C). These results confirm the specific upregulation of TUG1 in HS.
To explore the biological significance of TUG1 upregulation, we used an siRNA approach to knock down TUG1 in hypertrophic scar fibroblasts (HSFs). Downregulation of TUG1 significantly impeded both the proliferation and migration of HSFs, as evidenced by MTT assay (Fig. 1D) and Transwell migration assay (Fig. 1E).
Conversely, overexpression of TUG1 markedly enhanced the proliferative and migratory capabilities of HSFs. MTT assay revealed a substantial increase in HSF proliferation following TUG1 overexpression (Fig. 1F), which was corroborated by increased cell migration in Transwell assays (Fig. 1G).
Collectively, our results demonstrate that TUG1 is upregulated in HS tissue and its elevated expression significantly promotes the proliferation and migration of HSFs.
TUG1 Regulates Hypertrophic Scar Formation via miR-627 Targeting
The TargetScan database was employed to identify potential binding sites between TUG1 and miR-627, thereby predicting the molecular interaction between these two RNA molecules. As shown in Fig. 2A, the database reveals specific seed regions within TUG1 that are likely binding targets for miR-627.
The differential expression of miR-627 between hypertrophic scars (HS), adjacent normal tissue, and common scar tissue was investigated. Using quantitative real-time PCR (qRT-PCR), it was observed that miR-627 expression was significantly reduced in HS tissues compared to adjacent normal tissues (Fig. 2B). Further comparisons revealed that miR-627 levels were also substantially lower in HS tissues than in common scar tissues, further emphasizing the specific down-regulation of this miRNA in HS (Fig. 2C).
A regression analysis was performed to evaluate the correlation between TUG1 and miR-627 expression levels. Our data presented a negative correlation between the two, as illustrated by the regression equation and scatter plot in Fig. 2D. This suggests a potential regulatory role of TUG1 in controlling miR-627 expression.
To further substantiate the relationship between TUG1 and miR-627, a luciferase reporter assay was conducted. Human skin fibroblasts (HSFs) were co-transfected with TUG1 and a luciferase reporter gene linked to the miR-627 binding site. The results showed a significant decrease in luciferase activity in the HSFs compared to the control group, thus confirming the targeted regulatory relationship between TUG1 and miR-627 (Fig. 2E).
The collective data strongly suggest that TUG1 plays a pivotal role in the pathogenesis of hypertrophic scars by exerting a negative regulatory effect on miR-627. All data are presented as mean ± SD with n = 30, and statistical significance is indicated by ** (P < 0.05) when compared to the control group. The control group refers to the HSFs co-transfected with TUG1 and non-targeting control factors.
miR-627 Negatively Regulates IGFR1 in Hypertrophic Scars (HS)
To elucidate the mechanistic role of miR-627 in hypertrophic scars (HS), we first employed in silico analysis to identify its potential binding targets. The TargetScan database indicated that IGFR1 harbors a predicted binding site for miR-627 (Fig. 3A). To validate this interaction in a biological context, we analyzed IGFR1 expression in HS and adjacent normal tissues. Quantitative RT-PCR (qRT-PCR) analysis revealed that IGFR1 expression was significantly upregulated in HS tissues compared to adjacent normal tissues (Fig. 3B). To strengthen these findings, we extended our analysis to include normal scar tissue. Interestingly, the expression of IGFR1 in HS tissues was markedly elevated in comparison to both normal scar tissue and adjacent normal tissues, as detected by qRT-PCR (Fig. 3C). A regression analysis of miR-627 and IGFR1 expression levels further established an inverse relationship between the two molecules. The plotted regression equation demonstrated that increased miR-627 expression correlates with decreased IGFR1 expression (Fig. 3D). Finally, a luciferase reporter assay was conducted to confirm the physical interaction between miR-627 and IGFR1. Co-transfection with wild-type IGFR1 and miR-627 led to a significant reduction in luciferase activity, validating the binding specificity (Fig. 3E). All quantitative data are presented as mean ± SD(n = 30). Asterisks (**) indicate statistical significance compared to the control group (P < 0.05). The control group comprised cells co-transfected with IGFR1 and non-specific control RNA.
TUG1 Modulates the Bioactivity of Hypertrophic Scar Fibroblasts (HSFs) by Regulating miR-627 and IGFR1 Expression
To further explore the specific mechanism of TUG1 regulating the development of HS. We performed a regression curve equation of the expression level of TUG1 and IGFR1 showed that there was a positive correlation between TUG1 and IGFR1 (Fig. 4A). To study the mechanism of TUG1 regulating IGFR1 and HSFs through downstream miR-627, we repressed the expression of TUG1 in HSFs and determine the miR-627 expression in HSFs. Unsurprisingly, knockdown of TUG1 promoted miR-627 expression and upregulation of TUG1 reduce miR-627 expression (Fig. 4B and 4C). Similarly, reduced HGFR1 after knockdown of TUG1 in HSFs, while the overexpression of TUG1 significantly promoted IGFR1 protein in HSFs (Fig. 4D and 4E). HSFs was divided into four groups and transfected with control and scramble, control and TUG1, miR-627 and scramble or miR-627 and TUG1 respectively. The results showed that the expression level of IGFR1 protein in HSFs with overexpression of TUG1 was significantly higher than that in control group, while the expression level of IGFR1 in miR-627 overexpression group was significantly lower than that in control group. The co-transfection of miR-627 and TUG1 can counteract the effect on the expression of IGFR1 in HSFs (Fig. 4F). Compared with the control group, the expression of miR-627 was inhibited and the expression of IGFR1 was promoted in the TUG1 overexpression group, while the expression of TUG1 and IGFR1 was down-regulated in the miR-627 overexpression group. The expression of miR-627 and TUG1 co-transfection group was similarly to which of the control group (Fig. 4G). TUG1 overexpression substantially promoted proliferation and miR-627 overexpression decreased proliferation. The proliferation rate of HSFs in miR-627 and TUG1 co-transfection group was similarly to that in control group (Fig. 4H). The results showed that compared with the control group, the migration ability of HSFs in TUG1 overexpression group increased, while that in miR-627 overexpression group decreased, and the HSFs migration ability in miR-627 and TUG1 co-transfection group was similarly to that in control group (Fig. 4I). The above results suggested that TUG1 promotes the proliferation and migration of HSFs by attenuate the inhibition of miR-627 on IGFR1.
Characterization of Hypertrophic Scar (HS) Tissue under Various Transfection Conditions
HS tissues collected after model establishment and histological result showed different appearance in different transfection group. Proliferation range of HS tissue was larger under TUG1 overexpression. Meanwhile, the color was darker and the surface was more protruding after 3 weeks of modeling. On the contrary, proliferation range of HS tissue after 3 weeks of up-regulation of miR-627 was smaller than tissues of 1 week group, featured with lighter color, closer to normal tissue and flatter surface (Fig. 5A). After 3 weeks of modeling, HS tissues showed different HE staining and Masson staining under different transfection group. The results showed that the morphological structure of HS tissue after up-regulation of TUG1 was significantly thicker and more irregular than that of the control group, while the proliferation of miR-627 up-regulation group was slighter than that of the control group (Fig. 5B). Three weeks after modeling, the HS tissue showed different immunohistochemical results under different transfection factors. The results showed that compared with the control group, the brown granules in the HS model tissue up-regulated TUG1 were significantly increased, while the brown granules in the up-regulated miR-627 group were decreased (Fig. 5C). Three weeks after the establishment of the model, the expression of IGFR1 in HS tissues in different transfection group showed that the expression of IGFR1 in HS tissues up-regulated TUG1 was higher than that in the control group, while the expression of IGFR1 in HS tissues up-regulated miR-627 was lower than that in the control group (Fig. 5D). Three weeks after modeling, the expression of miR-627 was inhibited and the expression of IGFR1 was promoted in HS tissue after up-regulation of TUG1, while the expression of TUG1 and IGFR1 was inhibited in HS tissue after up-regulation of miR-627. The expression of co-transfection TUG1 and miR-627 showed similar pattern with the control group (Fig. 5E). Collectively, these data indicated that TUG1 promotes proliferation and migration of HSFs via TUG1/miR-627/IGFR1 modulation axis.