Effects of the circ_101238/miR-138-5p/CDK6 axis on proliferation and apoptosis keloid fibroblasts

Abstract

The formation of keloid scars is normally induced by cutaneous injuries, however, the detailed mechanisms underlying keloid formation remain largely unknown. The present study aimed to investigate the effects of circular RNA_101238 (circ_101238) on the proliferation and apoptosis of keloid fibroblasts and to identify the underlying molecular mechanisms of these effects. Reverse transcription-quantitative (RT-q)PCR was performed to determine the expression levels of circ_101238, microRNA (miRNA/miR)-138-5p and cyclin-dependent kinase 6 (CDK6) in keloids and normal skin tissues. Following transfection with short hairpin (sh)-circ_101238, LV-circ_101238, miR-138-5p mimics, miR-138-5p inhibitors and small interfering (si)-CDK6, cell proliferation was assessed using a cell counting kit-8 assay. Furthermore, cell apoptosis was evaluated via flow cytometric analysis, while a dual-luciferase assay was performed to confirm interactions between circ_101238, miR-138-5p and CDK6. The expression levels of the proliferation marker, CDK6 and apoptosis marker, caspase-3 were determined via RT-qPCR and western blot analyses. The results demonstrated that expression levels of circ_101238 and CDK6 were significantly increased in keloid samples, while miR-138-5p expression was reduced in comparison to normal skin. Furthermore, circ_101238 was demonstrated to bind miR-138-5p, which subsequently targeted CDK6. Proliferative activity and CDK6 expression were significantly decreased in keloid fibroblasts following transfection with sh-circ_101238 or miR-138-5p mimics, while cell apoptosis was markedly increased. Furthermore, co-transfection with miR-138-5p mimics reversed the effects caused by overexpression of circ_101238. Treatment of keloid fibroblasts with si-CDK6 counteracted the biological behavior changes induced by miR-138-5p inhibitors. Additionally, transfection with LV-CDK6 reversed the effects caused by miR-138-5p mimics. Taken together, the results of the present study demonstrated that circ_101238 was upregulated in keloid tissues in comparison with normal tissues and that circ_101238 knockdown inhibited cell proliferation, while promoting apoptosis of keloid fibroblasts via the miR-138-5p/CDK6 axis. These results suggest that circ_101238 may serve as a promising therapeutic candidate for keloid therapy and that circ_101238/miR-138-5p/CDK6 signaling has the potential to regulate the growth of keloid fibroblasts.

Keywords: apoptosis; circular RNA_101238; cyclin-dependent kinase 6; fibroblast; keloid; micoRNA-138-5p; proliferation.

Figure 1

Circ_101238 expression is significantly upregulated in keloid samples and fibroblasts, while circ_101238 knockdown suppressed the growth of keloid fibroblasts. (A) Circ_101238 expression levels were determined in 30 keloid tissues and matched adjacent normal controls via RT-qPCR analysis. (B) Circ_101238 expression levels were assessed in normal and keloid fibroblasts. The transfection efficiencies of (C) sh-circ_101238 and (D) LV-circ_101238 were confirmed via RT-qPCR analysis. (E) The proliferative ability of keloid fibroblasts transfected with sh-circ_101238 was significantly inhibited. (F) The mRNA levels of CDK6 were downregulated in cells transfected with sh-circ_101238. (G) The protein levels of CDK6 were reduced in transfected cells. (H) Cell apoptosis was significantly enhanced in keloid cells following transfection with sh-circ_101238. (I) Cell apoptotic rate was increased in transfected cells. (J) The mRNA levels of caspase-3 were elevated following the transfection with sh-circ_101238. (K) The protein levels of caspase-3 were enhanced in cells transfected with sh-circ_101238. (L) Western blotting of caspase-3 in transfected cells. All experiments were performed in triplicate. ^*P<0.05 vs. normal samples, ^#P<0.05 vs. LV-NC, ^@P<0.05 vs. sh-NC. Circ, circular RNA; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin; NC, negative control; OD, optical density.

Figure 2

miR-138-5p is a putative target of circ_101238 in keloid tissue and is involved in circ_101238-regulated cell growth of keloid fibroblasts. (A) The complementary binding sites between miR-138-5p and circ_101238 were predicted via bioinformatic analysis. (B) Treatment with miR-138-5p notably decreased luciferase activity of circ_101238-WT compared with the mutant. (C) miR-138-5p expression levels significantly increased in keloid cells transfected with sh-circ_101238 compared to sh-NC. (D) The levels of miR-138-5p were reduced following the transfection with LV-circ_101238. (E) miR-138-5p expression significantly decreased in keloid samples compared to normal skin. (F) The levels of circ_101238 and miR-138-5p were inversely correlated in keloid tissue (r=-0.2221; P=0.00622). (G) The transfection efficiencies of miR-138-5p mimics and inhibitors were confirmed via reverse transcription-quantitative PCR analysis. (H-J) The proliferative ability of keloid fibroblasts significantly enhanced following transfection with miR-138-5p inhibitors. (K-O) Cell apoptosis significantly decreased following transfection with miR-138-5p inhibitors compared to miR-NC. All experiments were performed in triplicate. ^*P<0.05 vs. normal samples, ^#P<0.05 vs. LV-NC, ^@P<0.05 vs. sh-NC, ^$P<0.05 vs. miR-NC. miR, microRNA; circ, circular RNA; sh, short hairpin; Mut, mutant; WT, wild type; NC, negative control; OD, optical density; CDK6, cyclin-dependent kinase 6.

Figure 3

CDK6 is a promising downstream molecule of miR-138-5p. (A) The binding sites of miR-138-5p and CDK6 transcripts were predicted via bioinformatic analysis. (B) The association between miR-138-5p and CDK6 was confirmed via the dual-luciferase activity assay. (C) The mRNA levels of CDK6 were downregulated in keloid fibroblasts following transfection with miR-138-5p mimics. (D) The protein levels of CDK6 were reduced in cells transfected with miR-138-5p mimics. (E) In comparison with normal skin, CDK6 expression was significantly upregulated in keloid tissues. (F) The levels of miR-138-5p and CDK6 were inversely correlated in keloid samples (r=-0.2193; P=0.00732). All experiments were performed in triplicate. ^*P<0.05 vs. normal samples, ^$P<0.05 vs. miR-NC. CDK6, cyclin-dependent kinase 6; miR, microRNA; WT, wild type; Mut, mutant; NC, negative control.

Figure 4

Circ_101238 influences the growth of keloid fibroblasts via the miR-138-5p/CDK6 signaling pathway. (A-C) The effects on cell proliferation and apoptosis induced by LV-circ_101238 were reversed following transfection with miR-138-5p mimics. (D-F) Enhanced cell proliferation caused by transfection with miR-138-5p inhibitors was reversed following CDK6 knockdown. (G-I) The effects induced by transfection with miR-138-5p mimics were counteracted following overexpression of CDK6. All experiments were performed in triplicate. ^#P<0.05 vs. LV-NC, ^$P<0.05 vs. miR-NC. Circ, circular RNA; miR, microRNA; CDK6, cyclin-dependent kinase 6; NC, negative control; OD, optical density.