The Effects of LncRNA XIST on the Proliferation and Apoptosis of Human Keloid Fibroblasts by Regulating the miR-7-5p/ADAM10 Axis
DENG Jiajun, QIN Xiao, HE Ya, ZHENG Yan*
This study investigates the effect of LncRNA XIST (long non-coding RNA X inactive specific transcription factor) on the proliferation and apoptosis of HKF (human keloid fibroblast) by regulating the miR 7-5p/ADAM10 (a disintegrin and metalloproteinase 10) axis. qRT-PCR was used to analyze the expression levels of LncRNA XIST, miR-7-5p, and ADAM10 mRNA in normal skin tissues, keloid tissues, normal HFF-1 (human foreskin fibroblasts-1) and HKF. HKF were assigned into Control group, si-NC group, si-LncRNA XIST group, si-LncRNA XIST+anti-NC group, si-LncRNA XIST+anti-miR-7-5p group, mimic-NC group, miR-7-5p mimic group, miR-7-5p mimic+pcDNA-NC group, and miR-7-5p mimic+pcDNA-ADAM10 group. qRT-PCR was used to measure the expression of LncRNA XIST, miR-7-5p, and ADAM10 of cells in each group. Dual luciferase re porter assay was used to verify the relationship between LncRNA XIST and miR-7-5p, as well as between miR 7-5p and ADAM10. WST-1 and plate clone formation experiments were used to measure cell proliferation in each group. Flow cytometry was used to measure apoptosis of cells in each group. Western blot was used to measure the protein expression of ADAM10, cleaved-Caspase-3 and Caspase-3 of cells in each group. The results showed that LncRNA XIST and ADAM10 mRNA expressions were upregulated in keloid tissues and HKF, while miR-7-5p expression was downregulated. The dual luciferase reporter assay showed that LncRNA XIST had a targeted rela tionship with miR-7-5p, and miR-7-5p had a targeted relationship with ADAM10. The LncRNA XIST, ADAM10 mRNA and protein, survival rate, colony formation number of cells, and the expression level of Caspase-3 in the si LncRNA XIST group were lower than those in the si-NC group or Control group, whlie miR-7-5p, apoptosis rate, and the expression level of cleaved-Caspase-3 were higher (P<0.05). The effect of miR-7-5p mimic transfection on HKF was consistent with that of si-LncRNA XIST. Compared with the si-LncRNA XIST group or the si-LncRNA XIST+anti-NC group, the ADAM10 mRNA and protein, survival rate, clone formation number, and the expression level of Caspase-3 in the si-LncRNA XIST+anti-miR-7-5p group were higher, while miR-7-5p, apoptosis rate, and the expression level of cleaved-Caspase-3 were lower (P<0.05). Compared with miR-7-5p mimic group or miR-7 5p mimic+pcDNA-NC group, the mRNA and protein levels of ADAM10, survival rate, clone formation number, and the expression level of Caspase-3 in the miR-7-5p+pcDNA-ADAM10 group were increased, while the apop tosis rate and the expression level of cleaved-Caspase-3 were decreased (P<0.05). In conclusion, silencing LncRNA XIST may inhibit HKF proliferation and promote HKF apoptosis by regulating miR-7-5p/ADAM10 axis, providing a new idea for studying the pathogenesis and targeted therapy of keloid.
CN
EN