| Background:Keloids are benign fibrous tumors resulting from an abnormally healed skin wound, characterized by an excessive accumulation of extracellular matrix. There is currently no universally effective treatment method for keloids. Radiation therapy is considered as a adjuvant treatment for keloid scarring, however radioresistance has been shown to be a serious impediment to treatment efficacy. There is therefore a need for the discovery of novel critical molecular targets whose inhibition might enhance the radiotherapeutic response. Hypoxia is thought to be a factor limiting the efficacy of radiation therapy in tumors. To date, the most important mediator identified of the cell’s response to reduced oxygen availability is the hypoxia inducible factor-1. Higher level of HIF-1α expression after radiation therapy in keloid fibroblasts has been demonstrated in our recent experiments. Based on the findings of our previous study and tumor research, we hypothesized a possible close relationship between HIF-1α and keloid radioresistance. The current study aimed to investigate whether targeting HIF-1α can enhance the radiotherapeutic efficacy of keloids.Objective:1.To identify the protein expression of HIF-1α in keloid fibroblasts before and after radiation therapy.2. To observe the silencing effect of HIF-la on keloid fibroblasts radiosensitivity and proliferation.3.To investigate the efficacy of 2ME2 on keloid fibroblasts radiosensitivity and proliferation.4.To preliminarily study the mechanism of 2ME2 on keloid fibroblasts.Methods:1.Western blot analysis was used to detect protein expression of HIF-la in the keloid fibroblasts under normoxic or hypoxic conditions with or without radiation therapy.2.Lentivirus-mediated siRNA transduction method was used to block the expression of HIF-1α gene. CCK-8 assay was used to detect keloid fibroblasts proliferation after LeshHIF-1α transduction. Radiation-induced apoptosis was then analyzed by flow cytometry using propidium iodide (PI)/annexin V-fluorescein isothiocyanate staining.3.Radiation therapy was applied after treatment with different concentrations of 2ME2 for 24h. The radiation-induced apoptosis was analyzed by flow cytometry and the proliferation of keloid fibroblasts was analyzed by CCK-8 assays.4.Keloid fibroblasts were treated with different concentrations of 2ME2 for 24h under hypoxic condition. HIF-la and HIF-1β protein level were determined by western blot and HIF-la mRNA levels were assessed by quantitative RT-PCR. The conditioned media from keloid fibroblasts treated with 2ME2 for 24h were analyzed for VEGF expressed as pg/ml using ELISA assay.Results:1.Upregulation of HIF-la protein level in keloid fibroblasts after radiation therapy.2.Lentivirus-mediated siRNA targeting HIF-la exerted a radiosensitizing effect on keloid fibroblasts. Significantly increased apoptosis was observed in LeshHIF-la cells compared with LeshNC cells after radiation therapy. The CCK results showed that the growth of keloid fibroblasts was not inhibited by transfection of HIF-la shRNA plasmids.3.Significantly increased apoptosis rates were observed in 2ME2-treated cells when compared to vehicle-treated cells, especially for the late stages of apoptosis. CCK-8 assays, analyzing the proliferation of keloid fibroblasts, showed a dose-dependent inhibition of keloid fibroblasts in the 2ME2-treated groups4.2ME2 reduces the protein expression of HIF-la and VEGF in keloid fibroblasts. Treatment with 2ME2 was specific for the regulation of HIF-la protein expression, but had no measurable effect on the protein levels of HIF-1β.Besides the mRNA levels of HIF-1α were not significantly changed by 2ME2.Conclusion:1.Knockdown of HIF-la could enhance the radiosensitivity of keloid fibroblasts.2.2ME2 could effectively inhibit the protein expression of HIF-la, but had no measurable effect on mRNA levels of HIF-1α.3.2ME2 showed an anti-proliferation effect and enhanced the radiosensitivity of keloid fibroblasts.4.HIF-1α could be a potential therapeutic target for keloids treatment. |
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