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Abstract：
OBJECTIVE: The study aims to elucidate the impact of LncRNA HULC in human skin fibroblasts (HSF) after burns in children. HULC might act as endogenous sponges for miR-663b to regulate the gene expression of TLR4.
METHODS: This study included 46 children with deep second-degree burns. On the 5th day after the injury, eligible samples from all patients were collected. HSF cells were selected to establish a thermal-injured model. qRT-PCR was applied to detect the expression of HULC, miR-663b, and TLR4 mRNA in burn wound and normal skin tissue. The dual-luciferase reporter and RIP assay were performed to explore a targeted binding relationship between HULC and miR-663b, or miR-663b and TLR4. Cell proliferation and invasion were evaluated through the assay of CCK-8 and transwell assay. The expression levels of α-SMA, Collagen I, MMP-1, and TIMP-1, which are associated with extracellular matrix (ECM) production, were examinated by western blot.
RESULTS: HULC and TLR4 mRNA expression were reduced on the 5th day after thermal injury in burn wounds, while miR-663b expression increased significantly (P<0.05), when compared to expression in the normal tissue. HULC and TLR4 mRNA concentration in HSF cells showed a transient increase after thermal injury, and a gradual decline with time was observed subsequently when compared to the control group. An inverse expression of miR-663b with the expression of HULC and TLR4 mRNA was observed simultaneously (P<0.05). A deficiency of HULC promotes the proliferation, invasion, and ECM synthesis of HSF cells with thermal injury; HULC functions as a ceRNA of miR-663b. Inhibitors of miR-663b partially rescued the effects on thermal-injured HSF cells induced by HULC deficiency (P<0.05). TLR4 is a target gene of miR-663b. The up-regulation of TLR4 also partially reversed the effect on the thermal-injury of HSF cells resulting from HULC deficiency (P<0.05).
CONCLUSIONS: LncRNA HULC may function as a molecular sponge to regulate the expression of the miR-663b/TLR4, and thereby inhibit the proliferation, invasion, and ECM synthesis of thermal-injured HSF cells. HULC knockdown might significantly promote wound healing in children after burns.
METHODS: This study included 46 children with deep second-degree burns. On the 5th day after the injury, eligible samples from all patients were collected. HSF cells were selected to establish a thermal-injured model. qRT-PCR was applied to detect the expression of HULC, miR-663b, and TLR4 mRNA in burn wound and normal skin tissue. The dual-luciferase reporter and RIP assay were performed to explore a targeted binding relationship between HULC and miR-663b, or miR-663b and TLR4. Cell proliferation and invasion were evaluated through the assay of CCK-8 and transwell assay. The expression levels of α-SMA, Collagen I, MMP-1, and TIMP-1, which are associated with extracellular matrix (ECM) production, were examinated by western blot.
RESULTS: HULC and TLR4 mRNA expression were reduced on the 5th day after thermal injury in burn wounds, while miR-663b expression increased significantly (P<0.05), when compared to expression in the normal tissue. HULC and TLR4 mRNA concentration in HSF cells showed a transient increase after thermal injury, and a gradual decline with time was observed subsequently when compared to the control group. An inverse expression of miR-663b with the expression of HULC and TLR4 mRNA was observed simultaneously (P<0.05). A deficiency of HULC promotes the proliferation, invasion, and ECM synthesis of HSF cells with thermal injury; HULC functions as a ceRNA of miR-663b. Inhibitors of miR-663b partially rescued the effects on thermal-injured HSF cells induced by HULC deficiency (P<0.05). TLR4 is a target gene of miR-663b. The up-regulation of TLR4 also partially reversed the effect on the thermal-injury of HSF cells resulting from HULC deficiency (P<0.05).
CONCLUSIONS: LncRNA HULC may function as a molecular sponge to regulate the expression of the miR-663b/TLR4, and thereby inhibit the proliferation, invasion, and ECM synthesis of thermal-injured HSF cells. HULC knockdown might significantly promote wound healing in children after burns.
摘要：
目的：该研究旨在阐明LncRNAHULC对儿童烧伤后人皮肤成纤维细胞（HSF）的影响。HULC可能作为miR-663b的内源性海绵调节TLR4的基因表达。
方法：本研究纳入46例深度二度烧伤患儿。受伤后的第5天,收集所有患者的合格样本.选择HSF细胞建立热损伤模型。qRT-PCR检测HULC的表达,miR-663b,和TLR4mRNA在烧伤创面和正常皮肤组织中的表达。进行双荧光素酶报告基因和RIP检测以探索HULC和miR-663b之间的靶向结合关系。或miR-663b和TLR4。通过CCK-8测定和transwell测定评估细胞增殖和侵袭。α-SMA的表达水平,胶原蛋白I,MMP-1和TIMP-1与细胞外基质（ECM）的产生有关，通过蛋白质印迹进行了检查。
结果：烧伤创面热损伤后第5天HULC和TLR4mRNA表达降低，miR-663b表达显著升高(P<0.05),当与正常组织中的表达相比较时。HSF细胞中的HULC和TLR4mRNA浓度在热损伤后出现短暂的增加，与对照组相比，随后观察到随时间的逐渐下降。miR-663b的表达与HULC和TLR4mRNA的表达同时相反(P<0.05)。HULC的缺乏促进增殖，入侵,热损伤HSF细胞的ECM合成；HULC作为miR-663b的ceRNA发挥作用。miR-663b抑制剂部分挽救了HULC缺陷诱导的热损伤HSF细胞的作用(P<0.05)。TLR4是miR-663b的靶基因。TLR4的上调也部分逆转了HULC缺陷对HSF细胞热损伤的影响(P<0.05)。
结论：LncRNAHULC可能作为一种分子海绵调节miR-663b/TLR4的表达，从而抑制细胞增殖，入侵,和热损伤HSF细胞的ECM合成。HULC敲除可能显著促进儿童烧伤后伤口愈合。
方法：本研究纳入46例深度二度烧伤患儿。受伤后的第5天,收集所有患者的合格样本.选择HSF细胞建立热损伤模型。qRT-PCR检测HULC的表达,miR-663b,和TLR4mRNA在烧伤创面和正常皮肤组织中的表达。进行双荧光素酶报告基因和RIP检测以探索HULC和miR-663b之间的靶向结合关系。或miR-663b和TLR4。通过CCK-8测定和transwell测定评估细胞增殖和侵袭。α-SMA的表达水平,胶原蛋白I,MMP-1和TIMP-1与细胞外基质（ECM）的产生有关，通过蛋白质印迹进行了检查。
结果：烧伤创面热损伤后第5天HULC和TLR4mRNA表达降低，miR-663b表达显著升高(P<0.05),当与正常组织中的表达相比较时。HSF细胞中的HULC和TLR4mRNA浓度在热损伤后出现短暂的增加，与对照组相比，随后观察到随时间的逐渐下降。miR-663b的表达与HULC和TLR4mRNA的表达同时相反(P<0.05)。HULC的缺乏促进增殖，入侵,热损伤HSF细胞的ECM合成；HULC作为miR-663b的ceRNA发挥作用。miR-663b抑制剂部分挽救了HULC缺陷诱导的热损伤HSF细胞的作用(P<0.05)。TLR4是miR-663b的靶基因。TLR4的上调也部分逆转了HULC缺陷对HSF细胞热损伤的影响(P<0.05)。
结论：LncRNAHULC可能作为一种分子海绵调节miR-663b/TLR4的表达，从而抑制细胞增殖，入侵,和热损伤HSF细胞的ECM合成。HULC敲除可能显著促进儿童烧伤后伤口愈合。
