Ponad 7000 publikacji medycznych!
Statystyki za 2021 rok:
odsłony: 8 805 378
Artykuły w Czytelni Medycznej o SARS-CoV-2/Covid-19

Poniżej zamieściliśmy fragment artykułu. Informacja nt. dostępu do pełnej treści artykułu tutaj
© Borgis - Postępy Nauk Medycznych 3/2015, s. 159-165
Mariola Wyględowska-Kania1, *Joanna Gola2, Anna Uttecht-Pudełko2, Dominika Wcisło-Dziadecka4, Małgorzata Kapral3, Barbara Strzałka-Mrozik2, Celina Kruszniewska-Rajs2, Magdalena Tkacz5, Urszula Mazurek2, Ligia Brzezińska-Wcisło1
Poziom ekspresji defensyny DEFB4A w różnicowaniu rogowiaka kolczystokomórkowego, raka kolczystokomórkowego i raka podstawnokomórkowego
Defensin DEFB4A transcript level in the differentiation of keratoacanthoma, squamous and basal cell carcinomas**
1School of Medicine in Katowice, Medical University of Silesia in Katowice, Department of Dermatology
Head of Department: prof. Ligia Brzezińska-Wcisło, MD, PhD
2School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia in Katowice, Department of Molecular Biology
Head of Department: prof. Urszula Mazurek, PhD
3School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia in Katowice, Department of Biochemistry
Head of Department: prof. Ludmiła Węglarz, PhD
4School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia in Katowice, Department of Skin Structural Studies
Head of Department: Associate Professor of Biology Krzysztof Jasik, PhD
5School of Computer Science and Material Science, University of Silesia in Katowice, Institute of Computer Science, Division of Information Systems
Head of Division: prof. Mariusz Boryczka, PhD
Streszczenie
Wstęp. Defensyny stanowią grupę peptydów o aktywności antybakteryjnej, antywirusowej i antygrzybiczej. Uczestniczą także w indukcji odpowiedzi immunologicznej i przeciwnowotworowej. Zmiany w poziomie ekspresji defensyn były badane w wielu patologiach skórnych, m.in. w łuszczycy, atopowym zapaleniu skóry, a także w niemelanotycznych nowotworach skóry (raku kolczystokomórkowym – SCC i raku podstawnokomórkowym – BCC).
Cel pracy. Celem tej pracy była ocena profilu mRNA genów kodujących defensyny oraz białka zaangażowane w indukcję ich ekspresji jako dodatkowego markera niemelanotycznych patologii skórnych: SCC, BCC i rogowiaka kolczystokomórkowego (KA).
Materiał i metody. Wycinki uzyskano z centralnej części guza (KA, SCC i BCC) i marginesów tkanki zdrowej. Profil mRNA genów kodujących defensyny oraz białka zaangażowane w indukcję ich ekspresji został wyznaczony techniką mikromacierzy oligonukleotydowych (Affymetrix). Walidację wyników przeprowadzono techniką QRT-PCR w czasie rzeczywistym.
Wyniki. Analiza techniką mikromacierzy wykazała zmiany profilu mRNA genów powiązanych z defensynami. We wszystkich guzach stwierdzono nadekspresję mRNA defensyny 2 (ang. defensin beta 2DEFB4A), w porównaniu do kontroli. Analiza techniką QRT-PCR w czasie rzeczywistym wykazała wzrost liczby kopii mRNA DEFB4A zarówno w KA, jak i SCC w porównaniu do BCC.
Wnioski. Poziom mRNA defensyny 2 jest przydatnym narzędziem w różnicowaniu KA i SCC od BCC. Rogowiak kolczystokomórkowy i rak kolczystokomórkowy nie mogą być różnicowane na podstawie liczby kopii mRNA DEFB4A.
Summary
Introduction. Defensins are peptide with antimicrobial, antiviral, antifungal activities and many other functions, such as induction of immunological response and antitumor activity. Changes in expression level of defensins was studied in many skin pathologies, including dermatological lesions such as psoriasis, atopic dermatitis and non-melanoma skin cancers (squamous cell carcinoma – SCC and basal cell carcinoma – BCC).
Aim. The objective of this study was to evaluate the mRNA profile of defensin-related genes’ transcripts as an additional molecular marker of non-melonoma skin pathologies: SCC, BCC and keratoacanthoma (KA).
Material and methods. Tissue samples were obtained from the central part of tumours (KA, SCC and BCC) and healthy margins. mRNA profile of genes coding defensins and proteins involved in their activation was determined using oligonucleotide microarrays (Affymetrix). Validation of the microarray analysis was performed using real-time QRT-PCR.
Results. Microarray analysis revealed changes in defensin-related genes’ profile. In all tumours DEFB4A (defensin beta 2) mRNA was up-regulated, compared with the healthy skin margins. Real-time QRT-PCR analysis showed increased DEFB4A transcript level both in KA and SCC comparing to BCC.
Conclusions. Defensin beta 2 mRNA level is a useful tool for the differentiation of KA and SCC from BCC. KA and SCC cannot be differentiated on the basis of the DEFB4A mRNA level.
Introduction
Defensins play a role as peptide antibiotics and constitute an important element of anti-infectious protection (1). Apart from a direct antimicrobial action, defensins also have many other functions, such as antifungal and antiviral activities, and chemotactic activity towards dendritic cells, T cells, basal cells and neutrophils. They may induce the production of certain chemokines and proinflammatory mediators; regulate complement activity; inhibit fibrinolysis and the production of certain glucocorticosteroids; intensify the proliferation of lymphocytes, endothelial cells and fibroblasts; bind and neutralize endotoxins; stimulate healing of sores; induce degranulation of basal cells and show antitumor activity (2). There are three groups of defensins – defensin α and β, and ? defensins – generated during the cyclisation of the two α defensins (1). In humans, six α (HNP 1-6 – human neutrophil peptide) and four β defensins (HBD 1-4) have been identified (3). Changed expression level of defensins was reported in many pathologies, including dermatological lesions such as psoriasis, acne vulgaris and atopic dermatitis (3-5). Examinations of cancer samples such as oesophageal squamous cell carcinoma, primary cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) also showed changes in defensins profile, suggesting their potential participation in these pathologies (6-8).
Knowledge of molecular markers of tumour transformation is essential for malignant diseases because it enables a diagnosis to be made of a disease already at the level of molecular changes, and it aids cause-directed and efficient therapy. Non-melanoma skin cancers are still problematic in diagnosis because of clinical features resembling other pathologies i.e. keratoacanthoma. Keratoacanthoma (KA) is a relatively low-grade malignancy that histologically and clinically resembles well-differentiated SCC (9, 10). It develops on unchanged skin in the form of a dome shaped, well-limited, hard nodule, usually with the same colour as the surrounding skin. The central part of the tumour has a crater-like depression with embanked margins, filled up with a keratin mass (11, 12). KA is characterised by sometimes fast growth and a tendency to regression with scarring (11). BCC is the most common type of epithelial cancer; it is characterised by low clinical and histological malignancy and slow growth and rarely metastasises (13, 14). It is usually found on unchanged skin of the face and head and spreads in multiple directions with edgings. The multiple and multifocal presence of BCC is classified as nevoid BCC syndrome (Gorlin-Goltz syndrome) (13, 14). Clinically, the changes can be highly differentiated, superficial, corneous, nevus-like, scleroderma-like, nodular or sore-like (15). SCC is the second most frequent (after BCC) skin cancer. It originates from squamous epithelium and develops from premalignant states (leucoplakia). It is characterised by fast growth and metastases to regional lymph nodes. Clinical forms of SCC (sore and papular) morphologically resemble KA and BCC (9, 16). Therefore additional markers could facilitate diagnosis of non-melanoma skin pathologies.
Significant risk factors for BCC, SCC and KA are: UV radiation, ionized radiation, post-infectious and post-drug immunosuppression, chemical carcinogens (arsenic, wood tars), old age, male gender, light skin type and human papilloma virus infection (8, 9, 16-20). Most of these factors influence immunological response. Due to its multidirectional activity, defensins may be involved not only in the molecular pathogenesis of non-melanoma skin cancers, but also in the immunological reaction leading to self-regression of KA. Understanding of the molecular mechanisms underlying KA regression may contribute to the development of an efficient treatment for skin cancers. Better knowledge about defensins level in non-melanoma skin pathologies could improve their diagnosis and could affect better understanding of molecular mechanism underlying these pathologies. Till this time there is no research comparing defensins profile in these three non-melanoma pathologies (KA, SCC and BCC) in one study.
Aim
The aim of this work was to evaluate the mRNA profile of defensin family transcripts as an additional molecular marker of nonmelonoma skin pathologies.
Material and methods
The study included a group of 39 patient (16 females and 23 males; mean age 71.8 ± 9.5 years) diagnosed and treated in the Dermatology Clinics and Department of Medical University of Silesia in Katowice. Based on clinical and pathomorphological examination, 9 cases were kerathoacanthoma (KA), 11 were squamous cell carcinoma (SCC) and 19 were basal cell carcinoma (BCC). The changes were located on the skin of the face and head. The biopsies for the pathomorphological and molecular examination were obtained from the centre of the tumour and the margins of healthy tissues where no tumour cells were found. After surgical removal, the samples were immediately preserved in the RNA stabilisation reagent RNAlater (Qiagen GmbH, Hilden, Germany). For microarray analysis 19 samples (6 KA, 3 SCC, 7 BCC and 4 margins of healthy tissues) were selected. The study was approved by the Bioethical Commission of the Medical University of Silesia. All of the patients were informed about the research and signed an informed consent form.
RNA extraction
Total RNA was extracted from the tissue samples using TRIZOL® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA extracts were treated with DNase I (MBI Fermentas, Vilnius, Lithuania) and purified with a RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. The quality of RNA was estimated by electrophoresis on 1% agarose gel stained with ethidium bromide. The RNA concentration was determined on the basis of absorbance values at 260 nm using a Gene Quant Pro spectrophotometer (LKB Biochrom Ltd., Cambridge, UK).
Microarray analysis
The analysis of the expression profile of defensin-related genes was performed using commercially available oligonucleotide microarrays HG-U133A (Affymetrix, Santa Clara, CA) according to the manufacturer’s protocol. For finding significant genes comparative analysis was performed with the use of GeneSpring 12.6.1 platform (Agilent Technologies, Inc., Santa Clara, CA, USA) and PL-Grid Infrastructure. The differences were analysed using the Oneway ANOVA test with Benjamini-Hochberg Multiple Testing Correction and TukeyHSD Post Hoc test. Genes were considered as potentially differentiating when FC ≥ 1.1 (fold change) and the significance level was set at p < 0.05.
Real-time QRT-PCR
The levels of the DEFB4A and β-actin transcripts were evaluated with the use of the real-time QRT-PCR TaqMan technique. The quantitative analysis was carried out using a Sequence Detector ABI PRISM™ 7000 (Applied Biosystems, Foster, CA, USA). Amplification was performed with the use of commercially available oligonucleotide primers specific for DEFB4A and β-actin genes (DEFB4A: TaqMan Gene Expression Assay defensin, beta 4; β-actin: TaqMan B-actin Detection Reagents Kit, Applied Biosystems, Inc., Foster, CA, USA) and QuantiTect Probe RT-PCR Kit (Qiagen GmbH, Hilden, Germany). For the assay, positive (β-actin mRNA) and negative (no template) controls were used. The thermal profile for one-step RT-PCR was as follows: 50°C for 30 min for reverse transcription, 95°C for 15 min, 45 cycles at 94°C for 15 s and at 60°C for 60 s. The standard curve was created for β-actin cDNA (TaqMan DNA Template Reagents Kit, Applied Biosystems, Inc., Foster, CA, USA). A standard curve was then generated by plotting the Ct values against the log of the known amount of the β-actin cDNA copy number. The mRNA copy numbers of the gene examined were recalculated per 1 μg of the total RNA. The RT-PCR products and the molecular weight marker pBR 322/Hae III (Fermentas International Inc., Ontario, Canada) were separated on 8% polyacrylamide gel and visualised with silver salts. The length of the amplified fragments was assessed by analysis with GelScan v.1.45 software (Kucharczyk TE, Warsaw, Poland). The statistical analysis of the real-time QRT-PCR results was performed with the use of Statistica version 9.0 software (StatSoft Inc., Oklahoma, USA). The one-way ANOVA followed by Tukey posthoc analysis of the logarithmic parameters were used to assess differences in the expression of the studied genes between KA, SCC and BCC. The differences between the tumour and the margin of the normal tissue were analysed using the t-test. All of results were expressed as means ± SD. The significance level was set at p < 0.05.
Results
Microarray analysis

Powyżej zamieściliśmy fragment artykułu, do którego możesz uzyskać pełny dostęp.
Mam kod dostępu
  • Aby uzyskać płatny dostęp do pełnej treści powyższego artykułu albo wszystkich artykułów (w zależności od wybranej opcji), należy wprowadzić kod.
  • Wprowadzając kod, akceptują Państwo treść Regulaminu oraz potwierdzają zapoznanie się z nim.
  • Aby kupić kod proszę skorzystać z jednej z poniższych opcji.

Opcja #1

19

Wybieram
  • dostęp do tego artykułu
  • dostęp na 7 dni

uzyskany kod musi być wprowadzony na stronie artykułu, do którego został wykupiony

Opcja #2

49

Wybieram
  • dostęp do tego i pozostałych ponad 7000 artykułów
  • dostęp na 30 dni
  • najpopularniejsza opcja

Opcja #3

119

Wybieram
  • dostęp do tego i pozostałych ponad 7000 artykułów
  • dostęp na 90 dni
  • oszczędzasz 28 zł
Piśmiennictwo
1. Niedźwiedzka-Rystwej P, Deptuła W: Defensins: an important innate element of the immune system in mammals. Postepy Hig Med Dosw 2008; 62: 524-529.
2. McDermott AM, Redfern RL, Zhang B et al.: Defensin expression by the cornea: multiple signalling pathways mediate IL-1beta stimulation of hBD-2 expression by human corneal epithelial cells. Invest Ophthalmol Vis Sci 2003; 44: 1859-1865.
3. Morizane S, Gallo RL: Antimicrobial peptides in the pathogenesis of psoriasis. J Dermatol 2012; 39(3): 225-230.
4. Tanghetti EA: The role of inflammation in the pathology of acne. J Clin Aesthet Dermatol 2013; 6(9): 27-35.
5. Harder J, Dressel S, Wittersheim M et al.: Enhanced expression and secretion of antimicrobial peptides in atopic dermatitis and after superficial skin injury. J Invest Dermatol 2010; 130(5): 1355-1364.
6. Shi N, Jin F, Zhang X et al.: Overexpression of human β-defensin 2 promotes growth and invasion during esophageal carcinogenesis. Oncotarget 2014; 5(22): 11333-11344.
7. Haider AS, Peters SB, Kaporis H et al.: Genomic analysis defines a cancer-specific gene expression signature for human squamous cell carcinoma and distinguishes malignant hyperproliferation from benign hyperplasia. J Invest Dermatol 2006; 126(4): 869-881.
8. Gambichler T, Skrygan M, Huyn J et al.: Pattern of mRNA expression of β-defensins in basal cell carcinoma. BMC Cancer 2006; 6: 163.
9. Zargaran M, Baghaei F: A clinical, histopathological and immunohistochemical approach to the bewildering diagnosis of Keratoacanthoma. J Dent Shiraz Univ Med Sci 2014; 15(3): 91-97.
10. Tan KB, Tan SH, Aw DC et al.: Simulators of Squamous Cell Carcinoma of the Skin: Diagnostic Challenges on Small Biopsies and Clinicopathological Correlation. J Skin Cancer 2013; 2013: 752864.
11. Ramos LM, Cardoso SV, Loyola AM et al.: Keratoacanthoma of the interior lip: review and report of case with spontaneous regression. J Appl Oral Sci 2009; 17: 262-265.
12. Lanssens S, Ongenae K: Dermatologic lesions and risk for cancer. Acta Clin Belg 2011; 66: 177-185.
13. Archontaki M, Korkolis DP, Arnoglannaki N et al.: Giant Basal cell carcinoma: clinicopathological analysis of 51 cases and review of the literature. Anticancer Res 2009; 29: 2655-2663.
14. Van der Geer S, Ostertag JU, Krekels GA: Treatment of basal cell carcinomas in patients with nevoid basal cell carcinoma syndrome. J Eur Acad Dermatol Venereol 2009; 23: 308-313.
15. Buljan M, Bulat V, Situm M et al.: Variations in clinical presentation of basal cell carcinoma. Acta Clin Croat 2008; 47: 25-30.
16. Sari Aslani F, Akbarzadeh-Jahromi M, Jowkar F: Value of CD10 Expression in Differentiating Cutaneous Basal from Squamous Cell Carcinomas and Basal Cell Carcinoma from Trichoepithelioma. Iran J Med Sci 2013; 38(2): 100-106.
17. Koyuncuer A: Histopathological evaluation of non-melanoma skin cancer. World J Surg Oncol 2014; 12: 159.
18. Andersson K, Michael KM, Luostarinen T et al.: Prospective study of human papillomavirus seropositivity and risk of nonmelanoma skin cancer. Am J Epidemiol 2012; 175(7): 685-695.
19. Situm M, Buljan M, Bulat V et al.: The role of radiation in the development of basal cell carcinoma. Coll Antropol 2008; 32: 167-170.
20. Bzhalava D, Johansson H, Ekstrom J et al.: Unbiased Approach for Virus Detection in Skin Lesions. PLoS ONE 2013; 8(6): e65953. doi:10.1371/journal.pone.0065953.
21. Elston DM: Mechanisms of Regression. Clin Med Res 2004; 2: 85-88.
22. Pyne JH, Windrum G, Sapkota D, Wong JC: Keratoacanthoma versus invasive squamous cell carcinoma: a comparison of dermatoscopic vascular features in 510 cases. Dermatol Pract Concept 2014; 4(3): 6.
23. Lin MJ, Pan Y, Jalilian C, Kelly JW: Dermoscopic characteristics of nodular squamous cell carcinoma and keratoacanthoma. Dermatol Pract Concept 2014; 4(2): 2.
24. Kambayashi Y, Fujimura T, Aiba S: Comparison of Immunosuppressive and Immunomodulatory Cells in Keratoacanthoma and Cutaneous Squamous Cell Carcinoma. Acta Derm Venereol 2013; 93: 663-668.
25. Muehleisen B, Jiang SB, Gladsjo JA et al.: Distinct Innate Immune Gene Expression Profiles in Non-Melanoma Skin Cancer of Immunocompetent and Immunosuppressed Patients. PLoS ONE 2012; 7(7): e40754. doi:10.1371/journal.pone.0040754.
26. Carlos AR, Escandell JM, Kotsantis P et al.: ARF triggers senescence in Brca2-deficient cells by altering the spectrum of p53 transcriptional targets. Nat Commun 2013; 4: 2697.
27. Toriseva M, Ala-aho R, Peltonen S et al.: Keratinocyte Growth Factor Induces Gene Expression Signature Associated with Suppression of Malignant Phenotype of Cutaneous Squamous Carcinoma Cells. PLoS ONE 2012; 7(3): e33041. doi:10.1371/journal.pone.0033041.
28. Zhang X, Makino T, Muchemwa FC et al.: Activation of the extracellular signal-regulated kinases signaling pathway in squamous cell carcinoma of the skin. Biosci Trends 2007; 1(3): 156-160.
29. Muro I, Fang G, Gardella KA et al.: The TRAF3 adaptor protein drives proliferation of anaplastic large cell lymphoma cells by regulating multiple signaling pathways. Cell Cycle 2014; 13(12): 1918-1927.
30. Schonthaler HB, Guinea-Viniegra J, Wculek SK et al.: S100A8-S100A9 protein complex mediates psoriasis by regulating the expression of complement factor C3. Immunity 2013; 39(6): 1171-1181.
31. Kim TW, Yu M, Zhou H et al.: Pellino 2 is critical for Toll-like receptor/interleukin-1 receptor (TLR/IL-1R)-mediated post-transcriptional control. J Biol Chem 2012; 287(30): 25686-25695.
32. Du B, Luo W, Li R et al.: Lgr4/Gpr48 negatively regulates TLR2/4-associated pattern recognition and innate immunity by targeting CD14 expression. J Biol Chem 2013; 288(21): 15131-15141.
33. Johansen C, Vestergaard C, Kragballe K et al.: MK2 regulates the early stages of skin tumor promotion. Carcinogenesis 2009; 30(12): 2100-2108.
34. Oren A, Ganz T, Liu L, Meerloo T: In human epidermis, beta-defensin 2 is packaged in lamellar bodies. Exp Mol Pathol 2003; 74: 180-182.
35. Dinulos JG, Mentele L, Fredericks LP et al.: Keratinocyte expression of human beta defensin 2 following bacterial infection: role in cutaneous host defense. Clin Diagn Lab Immunol 2003; 10: 161-166.
36. Jansen PA, Rodijk-Olthuis D, Hollox EJ et al.: Beta-defensin-2 protein is a serum biomarker for disease activity in psoriasis and reaches biologically relevant concentrations in lesional skin. PLoS One 2009; 4: e4725.
37. Langhorst J, Junge A, Rueffer A et al.: Elevated human beta-defensin-2 levels indicate an activation of the innate immune system in patients with irritable bowel syndrome. Am J Gastroenterol 2009; 104: 404-410.
38. Shestakova T, Zhuravel E, Bolgova L et al.: Expression of human beta-defensins-1, 2 and 4 mRNA in human lung tumor tissue: a pilot study. Exp Oncol 2008; 30: 153-156.
39. Shestakova T, Zhuravel E, Bolgova L et al.: Immunohistochemical analysis of beta-defensin-2 expression in human lung tumors. Exp Oncol 2010; 32: 273-276.
40. Han S, Bishop BM, van Hoek ML: Antimicrobial activity of human beta-defensins and induction by Francisella. Biochem Biophys Res Commun 2008; 371: 670-674.
41. Yoshimoto T, Yamaai T, Mizukawa N et al.: Different expression patterns of beta-defensins in human squamous cell carcinomas. Anticancer Res 2003; 23: 4629-4633.
otrzymano: 2015-02-02
zaakceptowano do druku: 2015-02-26

Adres do korespondencji:
*Joanna Gola
Department of Molecular Biology
SPLMS SUM
ul. Jedności 8, 41-100 Sosnowiec
tel. +48 (32) 364-10-27
fax +48 (32) 364-10-20
jgola@sum.edu.pl

Postępy Nauk Medycznych 3/2015
Strona internetowa czasopisma Postępy Nauk Medycznych