PI3K/AKT Pathway Promotes Keloid Fibroblasts Proliferation by Enhancing Glycolysis Under Hypoxia

Background Keloids are disfiguring fibro-proliferative disorders characterized by glucose metabolism reprogramming, namely elevated glycolysis and compromised oxidative phosphorylation. Our previous study demonstrated altered glucose metabolism and enhanced phosphorylation of the PI3K/AKT pathway in keloid fibroblasts (KFb) under hypoxic conditions. However, whether the PI3K/AKT pathway influences KFb cell function by regulating glucose metabolism under hypoxic conditions remains unclear. Results Our findings revealed that when the PI3K/AKT pathway was inactivated with LY294002 under hypoxia, the protein expression of glycolytic enzymes GLUT1, HK2, PFKFB3, PGK1, ENO1, PKM2, and LDHA decreased under hypoxia, while the amount of mitochondria and mitochondrial membrane potential (MMP) increased, and mitochondrial ultrastructure in KFb remained unchanged. The key parameters of extracellular acidification rate (ECAR) markedly diminished, and those of oxygen consumption rate (OCR) significantly increased after inhibition of the PI3K/AKT pathway. When the PI3K/AKT pathway was suppressed, the levels of ROS and mitochondrial ROS were significantly increased. Meanwhile, cell proliferation, migration, and invasion were inhibited, and apoptosis was increased when the PI3K/AKT pathway was blocked. Additionally, cell proliferation was compromised when KFb were treated with both SC79 (an activator of the PI3K/AKT pathway) and 2-DG (an inhibitor of glycolysis), compared to the SC79 group. Moreover, a positive feedback mechanism was demonstrated in the PI3K/AKT pathway and HIF-1α. Conclusions Our data collectively demonstrated that the PI3K/AKT pathway promotes proliferation and inhibits apoptosis in KFb under hypoxia by regulating glycolysis, indicating that the PI3K/AKT signaling pathway could be a therapeutic target for keloids.

Effect of 5-luorouracil Carboxymethyl Chitosan Nanoparticles on the Apoptosis Level of Keloid Fibroblasts

Objective to investigate the effect of 5-fluorouracil (5-FU) loaded carboxymethyl chitosan (CMC) nanoparticles on the apoptosis level of keloid fibroblasts. Methods keloid tissue (n = 80) was taken from inpatient and outpatient patients who were treated in our hospital from January 2020 to December 2020. The fresh keloid specimen tissues were washed with saline three times. After removing the epithelium, they were cut into 2mmx2mm tissue blocks for subculture. CMC nanoparticles loaded with 5-FU were prepared by ionically crosslinking CMC solution with calcium chloride. The viability of fibroblasts was determined by MTT assay. Transwell was used to assess fibroblast invasion. The wound healing test was used to evaluate the migration of fibroblasts. The apoptotic cells were analyzed by Annexin V-FITC and flow cytometry. The expression of apoptotic protein in fibroblasts was determined by Western blot analysis. The expression of transforming growth factor-β (TGF-β) and Smad2/3 were analyzed by immunohistochemistry. The mRNA expression of ERK1/2, protein kinase B (AKT) and nuclear transcription factor-κB (NF-κB) was analyzed by RT-qPCR. Results compared with the control group, there was no difference in the viability of fibroblasts in the nanoparticle group at 0h (P>0.05), and the viability of fibroblasts at 24h, 48h and 72h decreased by 37.29%, 29.58% and 28.38, respectively (P<0.05). Compared with the control group, the fiber cells composed of nanoparticles were less likely to pass through the pores of the basement membrane and their invasiveness decreased (P<0.05). Compared with the control group, the migration ability of fibroblasts in the nanoparticle group decreased (P<0.05). Compared with the control group, the apoptotic rate of fibers composed of nanoparticles increased (P<0.05). Compared with the control group, the expression levels of Bax, Caspase-3 and Caspase-9 apoptotic proteins in the nanoparticle group increased (P<0.05). Compared with the control group, the expression levels of TGF-β and Smad2/3 in the nanoparticle group decreased (P<0.05). Compared with the control group, the expression levels of ERK1/2, Akt and NF-κB mRNA in the nanoparticle group decreased (P<0.05). Conclusion 5-FU loaded CMC nanoparticles could reduce fibrosis in keloids, lower cell proliferation, migration and invasion, and increase cell apoptosis by inhibiting TGF-β related pathways.

Study on the Mechanism of miR-34a Affecting the Proliferation, Migration, and Invasion of Human Keloid Fibroblasts by Regulating the Expression of SATB1

Objectives. To explore the effect and mechanism of miR-34a on the proliferation, migration, and invasion of keloid fibroblasts (KFB). Methods. Isolate and culture KFB and normal skin fibroblast (NFB), detect the mRNA expression levels of miR-34a and integrin β5 (SATB1) in KFB and NFB by RT-qPCR, and detect SATB1 by western blot. The level of protein expression, MTT method, Transwell method, RT-qPCR, and western blot were used to detect the effects of overexpression of miR-34a or inhibition of SATB1 expression on the proliferation, migration, and invasion of KFB cells and the expression of related proteins. The dual luciferase reporter gene test verifies the targeting relationship between miR-34a and SATB1. Results. Compared with NFB, the expression of miR-34a was downregulated in KFB and the mRNA and protein expression levels of SATB1 were upregulated. Overexpression of miR-34a or inhibition of SATB1 expression inhibited the proliferation, migration, and invasion of KFB. miR-34a can negatively regulate the expression of SATB1, and overexpression of SATB1 reverses the effects of overexpression of miR-34a on the proliferation, migration, and invasion of KFB. Conclusions. miR-34a inhibits the proliferation, migration, and invasion of keloid fibroblasts by downregulating the expression of SATB1.

LncRNA HOXA11-AS Aggravates The Keloid Formation By Targeting miR-148b-3p/IGFBP5 Axis

Background: LncRNA homeobox (HOX) A11 antisense (HOXA11-AS) mediates cell-biological phenotypes of keloid fibroblasts and influence the keloid progression, yet the underlying mechanism is not fully understood.Methods: HOXA11-AS, miR-148b-3p and IGFBP5 expression were detected by RT-qPCR or western blots. CCK8 and colony formation assays were applied to examine the cell proliferation. The cell migration was determined via Transwell migration assays. The cell apoptosis was determined by western blots with anti-Bax antibodies and anti-Cleaved Caspase-3 antibodies. The interplay between miR-148b-3p HOXA11-AS and IGFBP5 was confirmed by luciferase reporter assays or RNA immunoprecipitation.Results: The amplification of HOXA11-AS and IGFBP5 was detected in keloid and keloid fibroblasts, while miR-148b-3p expression was reduced. Moreover, downregulation of HOXA11-AS in keloid fibroblasts inhibited cell proliferation, migration and triggered apoptosis. Mechanically, HOXA11-AS was proved to sponge miR-148b-3p and abrogate the inhibition on miR-148b-3p target, IGFBP5 mRNA, thus promoting keloid fibroblasts proliferation, migration and inhibiting apoptosis.Conclusion: These results find that HOXA11-AS promotes keloid progression by miR-148b-3p/IGFBP5 axis, suggesting the potential of targeting HOXA11-AS/miR-148b-3p/IGFBP5 axis to combat keloid.