Liu Yuxin, Qi Xiaoliu, Zhou Yichong
Department of Burn and Plastic Surgery, The Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health Hangzhou, Zhejiang Province, China.
Am J Transl Res. 2021 Apr 15;13(4):2499-2510. eCollection 2021.
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.
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.
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).
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.
本研究旨在阐明长链非编码RNA HULC在儿童烧伤后人皮肤成纤维细胞（HSF）中的作用。HULC可能作为miR-663b的内源性海绵，调节Toll样受体4（TLR4）的基因表达。
本研究纳入46例深二度烧伤患儿。伤后第5天，收集所有患者的合格样本。选取HSF细胞建立热损伤模型。采用qRT-PCR检测烧伤创面及正常皮肤组织中HULC、miR-663b和TLR4 mRNA的表达。进行双荧光素酶报告基因实验和RNA免疫沉淀实验，以探究HULC与miR-663b，或miR-663b与TLR4之间的靶向结合关系。通过CCK-8实验和Transwell实验评估细胞增殖和侵袭能力。采用蛋白质免疫印迹法检测与细胞外基质（ECM）产生相关的α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原蛋白（Collagen I）、基质金属蛋白酶-1（MMP-1）和基质金属蛋白酶组织抑制因子-1（TIMP-1）的表达水平。
与正常组织相比，烧伤创面热损伤后第5天，HULC和TLR4 mRNA表达降低，而miR-663b表达显著增加（P<0.05）。热损伤后HSF细胞中HULC和TLR4 mRNA浓度短暂升高，随后与对照组相比随时间逐渐下降。同时观察到miR-663b与HULC和TLR4 mRNA表达呈负相关（P<0.05）。HULC缺失促进热损伤HSF细胞的增殖、侵袭及ECM合成；HULC作为miR-663b的竞争性内源RNA发挥作用（ceRNA）。miR-663b抑制剂部分挽救了HULC缺失对热损伤HSF细胞的影响（P<0.05）。TLR4是miR-663b的靶基因。TLR4上调也部分逆转了HULC缺失对HSF细胞热损伤的影响（P<0.05）。
长链非编码RNA HULC可能作为分子海绵调节miR-663b/TLR4的表达，从而抑制热损伤HSF细胞的增殖、侵袭及ECM合成。敲低HULC可能显著促进儿童烧伤后的伤口愈合。
