circ-0030042通过miR-145/PTEN轴调控对增生性瘢痕成纤维细胞增殖及迁移的影响

Effect of circ-0030042 on Proliferation and Migration of Fibroblasts in Hypertrophic Scar by Regulation of miR-145/PTEN Axis

目的:探讨circ-0030042与人第10号染色体缺失的磷酸酶(Phosphatase and tensin homolog deleted on chromosome ten,PTEN)的相互作用关系,并分析其在增生性瘢痕(Hypertrophic scar,HS)患者中对成纤维细胞增殖与迁移的影响及作用机制。方法:通过circRNA序列和定量聚合酶链反应(PCR技术)检测正常皮肤成纤维细胞(NSFBs)和增生性瘢痕患者成纤维细胞(HSFBs)中circ-0030042的表达。用CCK8检测法检测转染48 h后的HSFBs细胞增殖情况。利用stubRFP-sensGFP-LC3基因转染、流式细胞仪及电子显微镜观察circ-0030042对miR-145/PTEN轴调控VEGF水平的表达。利用生物信息学分析、RNA免疫沉淀、免疫荧光检测等方法,揭示circ-0030042介导HS患者成纤维细胞增殖与迁移的作用机制。结果:circ-0030042在增生性瘢痕中显著上调,过表达时作为VEGF海绵抑制miR-145诱导的成纤维细胞,维持体内稳定性。此外,circ-0030042通过海绵化VEGF水平并阻断其miR-145捕获转录因子(FOXO1)mRNA来影响自噬,而circ-0030042诱导FOXO1的抑制被VEGF水平过表达或circ-0030042结合减少所抵消。过表达circ-0030042对成纤维细胞的增殖抑制与VEGF表达的抑制作用被过表达miR-145部分抵消。结论:干扰circ-0030042通过靶向下调miR-145/PTEN轴进而抑制HSFBs细胞的增殖与迁移,进一步诱导恶性细胞凋亡。

Objective To investigate the interaction relationship between circ-0030042 and Phosphatase and tensin homolog deleted on chromosome ten(PTEN),and to analyze its effect and mechanism on fibroblast proliferation and migration in patients with proliferative scar(HS).Methods The expression of circ-0030042 in normal skin fibroblasts(NSFBs)and fibroblasts(HSFBs)of patients with proliferative scars was detected by circRNA sequence and quantitative polymerase chain reaction(PCR).The proliferation of HSFBs cells after transfection 48 h was detected by CCK8 assay.The expression of circ-0030042 on the miR-145/PTEN axis to regulate VEGF levels was observed by stubRFP-sensGFP-LC3 gene transfection,flow cytometry and electron microscopy.Bioinformatics analysis,RNA immunoprecipitation,immunofluorescence and other methods were used to reveal the mechanism of CIRC-0030042 mediating fibroblast proliferation and migration in HS patients.Results Circ-0030042 is significantly up-regulated in hypertrophic scars.Overexpression of circ-0030042 acts as a VEGF sponge to inhibit miR-145-induced fibroblasts and maintain stability in vivo.In addition,circ-0030042 affects autophagy by spongeizing VEGF levels and blocking its miR-145-capturing transcription factor(FOXO1)mRNA,while circ-0030042-induced inhibition of FOXO1 is offset by overexpression of VEGF levels or decreased binding of circ-0030042.The proliferation inhibition of fibroblasts and VEGF expression by overexpression circ-0030042 were partially offset by overexpression miR-145.Conclusion Interference with circ-0030042 inhibits the proliferation and migration of HSFBs cells by targeting down-regulating the miR-145/PTEN axis,and further induces apoptosis in malignant cells.