Circ_0008450 downregulates Runx3 to promote the proliferation and epithelial-mesenchymal transition of human keratinized epithelial cells

Abstract

Keloid is an extremely common and often overlooked benign neoplastic disease, but its consequences should not be underestimated. Therefore, a deep exploration of the pathological mechanism of keloid becomes very essential. After 22 samples were collected from each patient's keloid tissues and normal skin tissues, circ_0008450 and Runx3 expression was tested by qRT-PCR. When primary human keratinized epithelial cells were transfected by sh-circ_0008450 or sh-Runx3, cell proliferation, apoptosis, migration, and EMT process were assessed by CCK-8, BrdU assay, apoptosis assay, migration assay, and Western blot. Finally, transfection was performed to explore the effect of circ_0008450 on the TGF-β/Smad signal pathway by adopting western blot. Circ_0008450 was highly expressed in keratinized epithelial tissues. After the transfection of sh-circ_0008450 into primary human keratinized epithelial cells, cell proliferation, migration, and EMT process were inhibited, and apoptosis was stimulated. Moreover, circ_0008450 silence-induced above changes were partly reversed by transfecting sh-Runx3. In addition, transfecting sh-circ_0008450 could repress TGF-β/Smad pathway, while transfecting sh-Runx3 activated the above pathway. Circ_0008450 down-regulated Runx3 to promote the proliferation and EMT process of human keratinized epithelial cells. This discovery may be related to the activation of the TGF-β/Smad pathway.

Keywords: Keloid; Runx3; TGF-β/Smad pathway; circ_0008450.

Figure 1.

Circ_0008450 was highly expressed in keratinized epithelial tissues. Keratinized epithelial tissues and normal epithelial tissues were obtained for qRT-PCR experiment. The expression of circ_0008450 was assessed. *** p < 0.001

Figure 2.

Circ_0008450 silence inhibited the growth of keratinized epithelial cell. Keratinized epithelial cells transfected with sh-circ_0008450 (sh-circ_0008450) or sh-NC were applied for the detection of cell proliferation and apoptosis. (a) The expression of circ_0008450 was assessed by qRT-PCR. *** p < 0.001. Proliferation marker (b) viability and (c) BrdU⁺ cells were assessed by CCK-8 assay and BrdU incorporation labeling respectively. *** p < 0.001. Apoptosis marker (d) apoptotic rate and (e-f) CyclinD1, CDK4, cleaved caspase 3, and cleaved caspase 9 proteins were assessed by apoptosis assay and western blot respectively. *** p < 0.001

Figure 3.

Circ_0008450 silence prevented the migration of keratinized epithelial cell and EMT process. Keratinized epithelial cells transfected with sh-circ_0008450 (sh-circ_0008450) or sh-NC were applied for the detection of migration and EMT process. (a) Relative migratory rate was assessed by migration assay. ** p < 0.01 or *** p < 0.001. (b-c) EMT marker E-cadherin, Vimentin, Fibronectin and ZO-1 were assessed by western blot. ** p < 0.01 or *** p < 0.001

Figure 4.

Runx3 was negatively regulated by circ_0008450. Keratinized epithelial cells transfected with sh-circ_0008450 (sh-circ_0008450) or sh-NC were applied for the detection of Runx3 (a) mRNA and (b and c) protein by qRT-PCR and Western blot respectively. *** p < 0.001. (d) Keratinized epithelial tissues and normal epithelial tissues were obtained for qRT-PCR experiment. The expression of Runx3 was assessed by qRT-PCR. *** p < 0.001. (e) Keratinized epithelial tissues were obtained for qRT-PCR experiment. The expression of circ_0008450 and Runx3 was assessed by qRT-PCR. R² = 0.6909, p < 0.001

Figure 5.

Circ_0008450 silence inhibited the growth of keratinized epithelial cell via the up-regulation of Runx3. (a) Keratinized epithelial cells transfected with sh-Runx3 (sh-Runx3) or sh-control were applied for the detection of Runx3 mRNA by qRT-PCR. *** p < 0.001. Keratinized epithelial cells transfected with sh-circ_0008450 (sh-circ_0008450) or sh-Runx3 (sh-Runx3) or both were applied for the detection of cell proliferation and apoptosis. Proliferation marker (b) viability and (c) BrdU⁺ cells were assessed by CCK-8 assay and BrdU incorporation labeling respectively. ** p < 0.01 or *** p < 0.001. Apoptosis marker (d) apoptotic rate and (e and f) CyclinD1, CDK4, cleaved caspase 3 and cleaved caspase 9 proteins were assessed by apoptosis assay and western blot respectively. * p < 0.05, ** p < 0.01 or *** p < 0.001

Figure 6.

Circ_0008450 silence prevented the migration of keratinized epithelial cell and EMT process via the up-regulation of Runx3. Keratinized epithelial cells transfected with sh-circ_0008450 (sh-circ_0008450) or sh-Runx3 (sh-Runx3) or both were applied for the detection of migration and EMT process. (a) Relative migratory rate was assessed by migration assay. * p < 0.05, ** p < 0.01 or *** p < 0.001. (b-c) EMT marker E-cadherin, Vimentin, Fibronectin, and ZO-1 were assessed by Western blot. ** p < 0.01 or *** p < 0.001

Figure 7.

Circ_0008450 silence repressed the TGF-β/Smad signal pathway via the up-regulation of Runx3. (a and b) Keratinized epithelial cells treated with LY2109761 were applied for the detection of phosphorylation of Smads (Smad2 and Smad3) by Western blot. *** p < 0.001. (c and d) Keratinized epithelial cells treated with LY2109761, sh-circ_0008450 (sh-circ_0008450), sh-Runx3 (sh-Runx3) or TGF-β1 were applied for the detection of phosphorylation of Smads (Smad2 and Smad3) by western blot. * p < 0.05, ** p < 0.01 or *** p < 0.001