Treatment of keloids through Runx2 siRNA‑induced inhibition of the PI3K/AKT signaling pathway

Lv,Wenchang; Wu,Min; Ren,Yuping; Luo,Xiao; Hu,Weijie; Zhang,Qi; Wu,Yiping

doi:10.3892/mmr.2020.11693

Treatment of keloids through Runx2 siRNA‑induced inhibition of the PI3K/AKT signaling pathway

Authors:
- Wenchang Lv
- Min Wu
- Yuping Ren
- Xiao Luo
- Weijie Hu
- Qi Zhang
- Yiping Wu
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Affiliations: Department of Plastic and Aesthetic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430000, P.R. China
Published online on: November 16, 2020 https://doi.org/10.3892/mmr.2020.11693
Article Number: 55
Copyright: © Lv et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Keloids are a skin fibroproliferative condition characterized by the hyperproliferation of fibroblasts and the excessive deposition of extracellular matrix (ECM) components. Previous studies have determined that Caveolin‑1 controlled hyperresponsiveness to mechanical stimuli through Runt‑related transcription factor 2 (Runx2) activation in keloids. However, the molecular mechanism of Runx2 regulating the pathological progression of keloids has not been elucidated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that most of the differentially expressed genes (DEGs), including Runx2, were significantly enriched in the biological processes ‘Positive regulation of cell proliferation’, in the cellular components ‘Extracellular matrix’, in the molecular functions ‘Extracellular matrix structural constituents’ and in the KEGG ‘PI3K‑Akt signaling pathway’. The aim of the present study was to investigate the expression levels of the Runx2 in human keloid tissues and primary human keloid fibroblasts (HKFs), and to determine the underlying molecular mechanisms involved in the fibrotic roles of Runx2 in keloid formation. Runx2 expression levels were analyzed in patient keloid tissues and HKFs using western blotting, reverse transcription‑quantitative PCR (RT‑qPCR) and immunofluorescence microscopy. Primary HKFs were transfected with a small interfering RNA (si) specifically targeting Runx2 (si‑Runx2). Subsequently, Cell Counting Kit‑8, wound healing and Transwell assays, flow cytometry, RT‑qPCR and western blotting were applied to evaluate the proliferation, migration, apoptosis, ECM deposition and PI3K/AKT signaling pathway of HKFs, respectively. In addition, western blotting was also used to determine the expression levels of phosphorylated AKT and PI3K in HKFs. The results revealed that Runx2 expression levels were upregulated in keloid tissues and primary HKFs compared with the normal skin tissues and human normal fibroblasts. Following the transfection with si‑Runx2, the proliferative and migratory abilities of HKFs were significantly reduced and the apoptotic rate was increased. The expression levels of type I, type III collagen, fibronectin, and α‑smooth muscle actin were downregulated in si‑Runx2‑transfected cells, which was hypothesized to occur through following the downregulation of the phosphorylation levels of PI3K and AKT. In conclusion, the findings of the present study indicated that Runx2 silencing in HKFs might significantly inhibit the cell proliferation, migration and the expression levels of ECM‑related proteins, and promote apoptosis via suppressing the PI3K/AKT signaling pathway. Thus, Runx2 siRNA treatment may reverse the pathological phenotype of keloids through the inhibition of PI3K/AKT signaling in patients.

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1	Ud-Din S and Bayat A: New insights on keloids, hypertrophic scars, and striae. Dermatol Clin. 32:193–209. 2014. View Article : Google Scholar : PubMed/NCBI
2	de Oliveira GV and Gold MH: Hydrocolloid dressings can be used to treat hypertrophic scars: An outpatient dermatology service devoted to treat keloids and challenging scars. J Cosmet Dermatol. Oct 26–2020.(Epub ahead of print). doi: 10.1111/jocd.13792 2020.
3	Li Y, Liu H, Liang Y, Peng P, Ma X and Zhang X: DKK3 regulates cell proliferation, apoptosis and collagen synthesis in keloid fibroblasts via TGF-β1/Smad signaling pathway. Biomed Pharmacother. 91:174–180. 2017. View Article : Google Scholar : PubMed/NCBI
4	Lee JY, Yang CC, Chao SC and Wong TW: Histopathological differential diagnosis of keloid and hypertrophic scar. Am J Dermatopathol. 26:379–384. 2004. View Article : Google Scholar : PubMed/NCBI
5	Lee YS, Liang YC, Wu P, Kulber DA, Tanabe K, Chuong CM, Widelitz R and Tuan TL: STAT3 signalling pathway is implicated in keloid pathogenesis by preliminary transcriptome and open chromatin analyses. Exp Dermatol. 28:480–484. 2019. View Article : Google Scholar : PubMed/NCBI
6	Zhang Q, Cai L, Wang M, Ke X, Zhao X and Huang Y: Identification of a novel mutation in the mechanoreceptor-encoding gene CXCR1 in patients with keloid. Arch Dermatol Res. 310:561–566. 2018. View Article : Google Scholar : PubMed/NCBI
7	Liu J, Zhu H, Wang H, Li J, Han F, Liu Y, Zhang W, He T, Li N, Zheng Z, et al: Methylation of secreted frizzled-related protein 1 (SFRP1) promoter downregulates Wnt/β-catenin activity in keloids. J Mol Histol. 49:185–193. 2018. View Article : Google Scholar : PubMed/NCBI
8	Bijlard E, Kouwenberg CA, Timman R, Hovius SE, Busschbach JJ and Mureau MA: Burden of Keloid Disease: A Cross-sectional Health-related Quality of Life Assessment. Acta Derm Venereol. 97:225–229. 2017. View Article : Google Scholar : PubMed/NCBI
9	Chen L, Zhang YH, Wang S, Zhang Y, Huang T and Cai YD: Prediction and analysis of essential genes using the enrichments of gene ontology and KEGG pathways. PLoS One. 12:e01841292017. View Article : Google Scholar : PubMed/NCBI
10	Liu W, Huang X, Liang X, Zhou Y, Li H, Yu Q and Li Q: Identification of Key Modules and Hub Genes of Keloids with Weighted Gene Coexpression Network Analysis. Plast Reconstr Surg. 139:376–390. 2017. View Article : Google Scholar : PubMed/NCBI
11	Fagone P, Mazzon E, Cavalli E, Bramanti A, Petralia MC, Mangano K, Al-Abed Y, Bramati P and Nicoletti F: Contribution of the macrophage migration inhibitory factor superfamily of cytokines in the pathogenesis of preclinical and human multiple sclerosis: In silico and in vivo evidences. J Neuroimmunol. 322:46–56. 2018. View Article : Google Scholar : PubMed/NCBI
12	Presti M, Mazzon E, Basile MS, Petralia MC, Bramanti A, Colletti G, Bramanti P, Nicoletti F and Fagone P: Overexpression of macrophage migration inhibitory factor and functionally-related genes, D-DT, CD74, CD44, CXCR2 and CXCR4, in glioblastoma. Oncol Lett. 16:2881–2886. 2018.PubMed/NCBI
13	Mevel R, Draper JE, Lie ALM, Kouskoff V and Lacaud G: RUNX transcription factors: orchestrators of development. Development. 146:dev1482962019. View Article : Google Scholar : PubMed/NCBI
14	Mümmler C, Burgy O, Hermann S, Mutze K, Günther A and Königshoff M: Cell-specific expression of runt-related transcription factor 2 contributes to pulmonary fibrosis. FASEB J. 32:703–716. 2018. View Article : Google Scholar : PubMed/NCBI
15	Herreño AM, Ramírez AC, Chaparro VP, Fernandez MJ, Cañas A, Morantes CF, Moreno OM, Brugés RE, Mejía JA, Bustos FJ, et al: Role of RUNX2 transcription factor in epithelial mesenchymal transition in non-small cell lung cancer lung cancer: Epigenetic control of the RUNX2 P1 promoter. Tumour Biol. 41:1010428319851014. 2019. View Article : Google Scholar : PubMed/NCBI
16	Fagone P, Mangano K, Pesce A, Portale TR, Puleo S and Nicoletti F: Emerging therapeutic targets for the treatment of hepatic fibrosis. Drug Discov Today. 21:369–375. 2016. View Article : Google Scholar : PubMed/NCBI
17	Fagone P, Mangano K, Mammana S, Pesce A, Pesce A, Caltabiano R, Giorlandino A, Portale TR, Cavalli E, Lombardo GA, et al: Identification of novel targets for the diagnosis and treatment of liver fibrosis. Int J Mol Med. 36:747–752. 2015. View Article : Google Scholar : PubMed/NCBI
18	Chen J, Lin Y and Sun Z: Deficiency in the anti-aging gene Klotho promotes aortic valve fibrosis through AMPKα-mediated activation of RUNX2. Aging Cell. 15:853–860. 2016. View Article : Google Scholar : PubMed/NCBI
19	Tchetina EV, Demidova NV, Markova GA, Taskina EA, Glukhova SI and Karateev DE: Increased baseline RUNX2, caspase 3 and p21 gene expressions in the peripheral blood of disease-modifying anti-rheumatic drug-naïve rheumatoid arthritis patients are associated with improved clinical response to methotrexate therapy. Int J Rheum Dis. 20:1468–1480. 2017. View Article : Google Scholar : PubMed/NCBI
20	Hahn JM, Glaser K, McFarland KL, Aronow BJ, Boyce ST and Supp DM: Keloid-derived keratinocytes exhibit an abnormal gene expression profile consistent with a distinct causal role in keloid pathology. Wound Repair Regen. 21:530–544. 2013. View Article : Google Scholar : PubMed/NCBI
21	Chen YT, Yao JN, Qin YT, Hu K, Wu F and Fang YY: Biological role and clinical value of miR-99a-5p in head and neck squamous cell carcinoma (HNSCC): A bioinformatics-based study. FEBS Open Bio. 8:1280–1298. 2018. View Article : Google Scholar : PubMed/NCBI
22	Kang SU, Kim YS, Kim YE, Park JK, Lee YS, Kang HY, Jang JW, Ryeo JB, Lee Y, Shin YS, et al: Opposite effects of non-thermal plasma on cell migration and collagen production in keloid and normal fibroblasts. PLoS One. 12:e01879782017. View Article : Google Scholar : PubMed/NCBI
23	Garcia-Morales V, Friedrich J, Jorna LM, Campos-Toimil M, Hammes HP, Schmidt M and Krenning G: The microRNA-7-mediated reduction in EPAC-1 contributes to vascular endothelial permeability and eNOS uncoupling in murine experimental retinopathy. Acta Diabetol. 54:581–591. 2017. View Article : Google Scholar : PubMed/NCBI
24	Fang B, Liu Y, Zheng D, Shan S, Wang C, Gao Y, Wang J, Xie Y, Zhang Y and Li Q: The effects of mechanical stretch on the biological characteristics of human adipose-derived stem cells. J Cell Mol Med. 23:4244–4255. 2019. View Article : Google Scholar : PubMed/NCBI
25	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
26	Zhang M, Zeng J, Zhao Z and Liu Z: Loss of MiR-424-3p, not miR-424-5p, confers chemoresistance through targeting YAP1 in non-small cell lung cancer. Mol Carcinog. 56:821–832. 2017. View Article : Google Scholar : PubMed/NCBI
27	Rotgers E, Nurmio M, Pietilä E, Cisneros-Montalvo S and Toppari J: E2F1 controls germ cell apoptosis during the first wave of spermatogenesis. Andrology. 3:1000–1014. 2015. View Article : Google Scholar : PubMed/NCBI
28	Gene Ontology C; Gene Ontology Consortium, : Gene Ontology Consortium: Going forward. Nucleic Acids Res. 43D:D1049–D1056. 2015. View Article : Google Scholar : PubMed/NCBI
29	Kanehisa M, Furumichi M, Tanabe M, Sato Y and Morishima K: KEGG: New perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res. 45D1:D353–D361. 2017. View Article : Google Scholar : PubMed/NCBI
30	Cavalli E, Battaglia G, Basile MS, Bruno V, Petralia MC, Lombardo SD, Pennisi M, Kalfin R, Tancheva L, Fagone P, et al: Exploratory Analysis of iPSCS-Derived Neuronal Cells as Predictors of Diagnosis and Treatment of Alzheimer Disease. Brain Sci. 10:1662020. View Article : Google Scholar : PubMed/NCBI
31	Zhang Y, Cheng C, Wang S, Xu M, Zhang D and Zeng W: Knockdown of FOXM1 inhibits activation of keloid fibroblasts and extracellular matrix production via inhibition of TGF-β1/Smad pathway. Life Sci. 232:1166372019. View Article : Google Scholar : PubMed/NCBI
32	Berman B, Maderal A and Raphael B: Keloids and Hypertrophic Scars: Pathophysiology, Classification, and Treatment. Dermatol Surg. 43 (Suppl 1):S3–S18. 2017. View Article : Google Scholar : PubMed/NCBI
33	Bock O, Schmid-Ott G, Malewski P and Mrowietz U: Quality of life of patients with keloid and hypertrophic scarring. Arch Dermatol Res. 297:433–438. 2006. View Article : Google Scholar : PubMed/NCBI
34	Liu Q, Wang X and Jia Y: Heat Shock Protein 90 Inhibitor Decreases Collagen Synthesis of Keloid Fibroblasts and Attenuates the Extracellular Matrix on the Keloid Spheroid Model. Plast Reconstr Surg. 137:759e–760e. 2016. View Article : Google Scholar : PubMed/NCBI
35	Philandrianos C, Kerfant N, Jaloux C Jr, Martinet L, Bertrand B and Casanova D: Keloid scars (part I): Clinical presentation, epidemiology, histology and pathogenesis. Ann Chir Plast Esthet. 61:128–135. 2016.(In French). View Article : Google Scholar : PubMed/NCBI
36	Chen J, Liu K, Liu Y, Wang X and Zhang Z: Targeting mTORC1/2 with OSI-027 inhibits proliferation and migration of keloid keratinocytes. Exp Dermatol. 28:270–275. 2019. View Article : Google Scholar : PubMed/NCBI
37	Ogawa R: Keloid and Hypertrophic Scars Are the Result of Chronic Inflammation in the Reticular Dermis. Int J Mol Sci. 18:6062017. View Article : Google Scholar : PubMed/NCBI
38	Komori T: Regulation of Proliferation, Differentiation and Functions of Osteoblasts by Runx2. Int J Mol Sci. 20:16942019. View Article : Google Scholar : PubMed/NCBI
39	Li G, Li YY, Sun JE, Lin WH and Zhou RX: ILK-PI3K/AKT pathway participates in cutaneous wound contraction by regulating fibroblast migration and differentiation to myofibroblast. Lab Invest. 96:741–751. 2016. View Article : Google Scholar : PubMed/NCBI
40	Evangelisti C, Evangelisti C, Chiarini F, Lonetti A, Buontempo F, Bressanin D, Cappellini A, Orsini E, McCubrey JA and Martelli AM: Therapeutic potential of targeting mTOR in T-cell acute lymphoblastic leukemia (review). Int J Oncol. 45:909–918. 2014. View Article : Google Scholar : PubMed/NCBI
41	Sharma VR, Gupta GK and Sharma AK, Batra N, Sharma DK, Joshi A and Sharma AK: PI3K/Akt/mTOR Intracellular Pathway and Breast Cancer: Factors, Mechanism and Regulation. Curr Pharm Des. 23:1633–1638. 2017. View Article : Google Scholar : PubMed/NCBI
42	Steelman LS, Martelli AM, Cocco L, Libra M, Nicoletti F, Abrams SL and McCubrey JA: The therapeutic potential of mTOR inhibitors in breast cancer. Br J Clin Pharmacol. 82:1189–1212. 2016. View Article : Google Scholar : PubMed/NCBI
43	Ge F, Wang F, Yan X, Li Z and Wang X: Association of BAFF with PI3K/Akt/mTOR signaling in lupus nephritis. Mol Med Rep. 16:5793–5798. 2017. View Article : Google Scholar : PubMed/NCBI
44	Fagone P, Ciurleo R, Lombardo SD, Iacobello C, Palermo CI, Shoenfeld Y, Bendtzen K, Bramanti P and Nicoletti F: Transcriptional landscape of SARS-CoV-2 infection dismantles pathogenic pathways activated by the virus, proposes unique sex-specific differences and predicts tailored therapeutic strategies. Autoimmun Rev. 19:1025712020. View Article : Google Scholar : PubMed/NCBI
45	Nicoletti F, Fagone P, Meroni P, McCubrey J and Bendtzen K: mTOR as a multifunctional therapeutic target in HIV infection. Drug Discov Today. 16:715–721. 2011. View Article : Google Scholar : PubMed/NCBI
46	Tu T, Huang J, Lin M, Gao Z, Wu X, Zhang W, Zhou G, Wang W and Liu W: CUDC 907 reverses pathological phenotype of keloid fibroblasts in vitro and in vivo via dual inhibition of PI3K/Akt/mTOR signaling and HDAC2. Int J Mol Med. 44:1789–1800. 2019.PubMed/NCBI
47	Aragona M and Blanpain C: Gene therapy: Transgenic stem cells replace skin. Nature. 551:306–307. 2017. View Article : Google Scholar : PubMed/NCBI
48	Mavilio F, Pellegrini G, Ferrari S, Di Nunzio F, Di Iorio E, Recchia A, Maruggi G, Ferrari G, Provasi E, Bonini C, et al: Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. Nat Med. 12:1397–1402. 2006. View Article : Google Scholar : PubMed/NCBI
49	Wang W, Qu M, Xu L, Wu X, Gao Z, Gu T, Zhang W, Ding X, Liu W and Chen YL: Sorafenib exerts an anti-keloid activity by antagonizing TGF-β/Smad and MAPK/ERK signaling pathways. J Mol Med (Berl). 94:1181–1194. 2016. View Article : Google Scholar : PubMed/NCBI
50	Tian Y, Jin L, Zhang W, Ya Z, Cheng Y and Zhao H: AMF siRNA treatment of keloid through inhibition signaling pathway of RhoA/ROCK1. Genes Dis. 6:185–192. 2018. View Article : Google Scholar : PubMed/NCBI