cyclin-d1 and Keloid

Keloids have always been a difficult problem in the clinic. In our previous study, we demonstrated a Warburg effect in keloid fibroblasts (KFs), like tumors. In this study, we aimed to investigate the effects of the suppression of the Warburg effect on the biological activity and function of KFs.. KFs were isolated and cultured with different concentrations of oxamate, a classical competitive lactate dehydrogenase A (LDHA) inhibitor. First, the suppression effect of oxamate on the Warburg effect in KFs was verified. After treatment with oxamate, a scratch wound assay, real-time PCR, flow cytometry, CCK8 kit, and western blotting were used to detect the migration ability, collagen production, apoptosis, cell proliferation, cell cycle distribution, and related molecular mechanisms in KFs.. As expected, oxamate inhibited the Warburg effect in KFs in a dose-dependent manner. After the inhibition of the Warburg effect in KFs, the cell migration rate decreased significantly, the mRNA transcription levels of type I collagen and α-SMA were significantly lower, the cell apoptosis rate increased significantly, the cell proliferation activity decreased significantly, and G0/G1 phase cells in KFs increased significantly. The expression of cyclin D1 and its upstream regulatory factors, Akt protein and GSK3 β (phospho S9), decreased significantly.. Inhibiting the Warburg effect in KFs significantly suppressed cell proliferation, enhanced cell apoptosis, inhibited cell migration ability, reduced collagen secretion, and induced G0/G1 arrest through the Akt-GSK3β-Cyclin D1 pathway. Therefore, inhibiting the Warburg effect in KFs may provide a new option for the prevention and treatment of keloids.. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors   www.springer.com/00266 .

Topics: Cell Proliferation; Cells, Cultured; Collagen; Collagen Type I; Cyclin D1; Fibroblasts; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Keloid; Lactate Dehydrogenase 5; Proto-Oncogene Proteins c-akt; RNA, Messenger

JAK2/STAT3 signaling pathway plays an important role in keloid formation, but the upstream mechanism of their activation remains unclear.. This study aims to investigate the possible mechanism of lncRNA-ZNF252P-AS1 in keloid.. The differentially expressed genes in keloid and their upstream regulatory miRNAs and long non-coding RNAs (lncRNAs) were analyzed by bioinformatics database, and the targeting relationship was further verified by dual-luciferase reporter gene assay. LncRNA function as competitive endogenous RNA (ceRNA) in keloid was further verified by in keloid fibroblasts (KFs) and in nude mice with subcutaneous keloids.. BTF3 expression was up-regulated in keloid tissues. The targeting relationship between BTF3 and miR-15b-5p was confirmed by dual-luciferase reporter gene assay. miR-15b-5p overexpression inhibited BTF3, Bcl-2, Cyclin D1, C-myc, Collagen I, MMP2, MMP9, N-cadherin, and ZEB2 expressions in KFs, inhibited cell proliferation and migration, while promoted E-cadherin levels. BTF3 overexpression reversed miR-15b-5p effects on KFs. Bioinformatics analysis as well as clinical and cellular experiments confirmed that the lncRNA ZNF252P-AS1 was highly expressed in keloid/KFs. Dual-luciferase reporter gene assays confirmed the targeting relationship between lncRNA ZNF252P-AS1 and miR-15b-5p. LncRNA ZNF252P-AS1 overexpression inhibited miR-15b-5p and E-cadherin levels, upregulated BTF3, Bcl-2, Cyclin D1, C-myc, Collagen I, MMP2, MMP9, N-cadherin, and ZEB2 expressions, increased cell proliferation and migration, and activated JAK2/STAT3 pathway, while miR-15b-5p overexpression reversed this effect. The in vivo results were consistent with in vitro results. In vivo experiments further confirmed that lncRNA ZNF252P-AS1 reduced keloid volume and weight.. lncRNA ZNF252P-AS1 is a potential target for keloid treatment.

Topics: Animals; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Collagen; Cyclin D1; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Keloid; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Nude; MicroRNAs; RNA, Long Noncoding; Signal Transduction

Mounting evidence has reported that microRNAs (miRNAs) play irreplaceable roles in the development of keloid fibrosis. miR-4417 has been reported to contribute to nickel chloride-promoted lung epithelial cell fibrogenesis and tumorigenesis. However, whether miR-4417 is involved in keloid fibrogenesis as well as its underlying mechanisms remain largely elusive. In this study, the expression levels of miR-4417 and CyclinD1 in keloid tissues and fibroblasts were examined by qRT-PCR. Cell proliferation was determined by CCK assay. Western blot and flow cytometry were performed to evaluate cell apoptosis. Cell migration and invasion were measured by Transwell assay. Luciferase reporter assay was used to confirm the relationship between miR4417 and CyclinD1. As a result, we found that miR-4417 was significantly down-regulated in keloid tissues and fibroblasts. miR-4417 up-regulation led to the suppression of proliferation, migration, and invasion, while induced cell apoptosis in keloid fibroblasts. However, miR-4417 depletion exerted an opposite effect. CyclinD1 harbored the binding sites with miR-4417. Besides, the expression of CyclinD1 was evidently decreased in keloid tissues and fibroblasts. Meanwhile, miR-4417 was negatively correlated with CyclinD1 in keloid tissue. The effect of CyclinD1 knockdown on keloid fibroblasts was similar to that of miR-4417 overexpression. Furthermore, the elevated of CyclinD1 expression rescued the effect of miR-4417 up-regulation on keloid fibroblasts. miR-4417/CyclinD1 axis was required for cell proliferation, apoptosis, migration, and invasion in keloid fibroblasts. In conclusion, miR-4417 and CyclinD1 may be potential therapeutic targets for the treatment of keloid.

Topics: Apoptosis; Cell Movement; Cell Proliferation; Cells, Cultured; Cyclin D1; Down-Regulation; Fibroblasts; Fibrosis; Gene Expression Regulation; Humans; Keloid; MicroRNAs

To study the effect of knocking down wingless-related MMTV integration site 2 (Wnt2) expression by RNAi on the growth and signaling pathways of ex vitro-cultured keloid fibroblasts (KFB).Human KFB were isolated from 10 keloid patient specimens. The KFB cells were then transfected with 4 pairs of small interfering RNA (siRNA) targeting human Wnt2, respectively. Reverse transcriptase-polymerase chain reaction and Western blot analysis were conducted to verify the knock down of Wnt2, and the expression of β-catenin glycogen synthase kinase-3β (GSK-3β) and cyclin D1 were examined.siRNA Wnt2 transfection (siWnt2) resulted in the significant inhibition of Wnt2 expression at both the mRNA and protein levels. The expression of β-catenin, GSK-3β, p-GSK-3β, and cyclin D1 at the protein level also decreased in siWnt2 cells. siWnt2 resulted in a substantially slower growth and significant delay in cell doubling time of the KFB cells compared with control groups. Further, the siRNA knock down of GSK-3β and β-catenin resulted in slower proliferation rates, respectively.Wnt2 siRNA has an inhibitive effect on keloid fibroblast proliferation, which may be a potential therapeutic approach for keloid and other human fibrotic diseases.

Topics: Adolescent; Adult; Blotting, Western; Child; Child, Preschool; Cyclin D1; Female; Fibroblasts; Glycogen Synthase Kinases; Humans; Keloid; Male; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Signal Transduction; Wnt2 Protein; Young Adult

Keloid is a disorder of fibroproliferative diseases that occurs in wounds, characterized by an exaggerated response to injury. The key factor responsible for the disease process has not been identified. This study sought to elucidate the role of β-catenin in the regulation of keloid phenotypes and signaling. Expression of β-catenin in keloid and normal non-keloid samples was measured by real-time polymerase chain reaction. Knockdown of β-catenin was achieved by delivering small interfering RNA to target β-catenin. Cell proliferation, cell cycle progression, and apoptosis of keloid cells were measured by functional assays in vitro. The proteins related to keloid fibrosis were measured by Western blotting. β-catenin expression was significantly upregulated in keloid tissue samples compared with the normal non-keloid age-adjusted skin sample counterparts. Functionally, targeting β-catenin with lipofection-delivered small interfering RNA oligonucleotide inhibited the proliferation and cell cycle arrest in G0/G1 phase and increased apoptosis of fibroblast cells, accompanied by downregulation of Wnt2 and cyclin D1 as well as the phosphorylation level of glycogen synthase kinase 3 beta in the keloid fibrosis. Our study supports a crucial role of β-catenin in the regulation of fibroproliferation and extracellular matrix deposition. Targeting β-catenin using small interfering RNA oligonucleotide may be a promising approach for preventing excessive fibroproliferative development after wound healing and may lead to the development of novel strategies for restoring keloid diseases.

Topics: Apoptosis; beta Catenin; Cell Proliferation; Cyclin D1; Female; Fibroblasts; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Humans; Keloid; Phosphorylation; RNA, Small Interfering; Signal Transduction; Wnt2 Protein

To study the inhibition effect of CyclinD1 specific small interfering RNA(siRNA) on CyclinD1 gene expression in human keloid fibroblast, investigating the effect of CyclinD1 specific siRNA (siRNA-CyclinD1) on the cell cycle, multiplication and apoptosis.. According to the principle of siRNA design, siRNA-CyclinD1 was designed and the keloid fibroblast were transfected. RT-PCR was used to examine CyclinD1 mRNA expression. Flow cytometry was used to examine cell cycle. The apoptotic rate was analyzed by using Annexin V-FITC/PI kit. The DNA gragmentation were measured by DNA ladder analysis.. After transfection, the expression of CyclinD1 mRNA decreased remarkably. Twenty-four, forty-eight and seventy-two hours after transfection, the radio of G1 stage cell was (59.80 +/- 3.06)%, (66.01 +/- 4.03)% and (67.43 +/- 5.35)%, all significantly higher than in the control group (54.50 +/- 5.35)%; the radio of S stage cell was (18.40 +/- 1.42)%, (17.21 +/- 1.76)% and (11.07 +/- 1.00)%, significantly lower than in the control group (22.33 +/- 1.49)%; the proportion of the cells in G1 stage increased and those in the S stage decreased in the keloid fibroblast transfected with siRNA-CyclinD1. The apoptotic rate of the siRNA-CyclinD1 group was (7.82 +/- 0.45)%, (15.71 +/- 1.06)%, (18.32 +/- 1.08)%, all significantly higher than in the control group (0.68 +/- 0.12)%, and the DNA gragmentation can be seen remarkably.. Chemically synthesized siRNA- CyclinD1 effectively inhibits. The expression of CyclinD1 in keloid fibroblast thus arresting the cell cycle at G1 stage and enhancing cell apoptosis. Our study provided a preliminary results in seaching of a RNAi therapy of keloid.

Topics: Apoptosis; Cells, Cultured; Cyclin D1; Fibroblasts; Flow Cytometry; Gene Silencing; Humans; Keloid; RNA, Small Interfering; Transfection