MOLECULAR MEDICINE REPORTS 14: 3735-3742, 2016 Haptoglobin protein and mRNA expression in psoriasis and its clinical significance FU-JUN TIAN1, YING-YING ZHANG2, LI-QIAN LIU1, YING XIONG1, ZONG-SHAN WANG1 and SHOU-ZHONG WANG1 1Department of Dermatology, Linyi People's Hospital; 2Department of Burns Surgery, Yishui Central Hospital, Linyi, Shandong 276003, P.R. China Received September 9, 2015; Accepted July 19, 2016 DOI: 10.3892/mmr.2016.5672 Abstract. The present study aimed to explore the associa- were closely associated with the occurrence of psoriasis; there- tion between haptoglobin protein and mRNA expression and fore, haptoglobin may be considered a promising novel clinical psoriasis. A total of 138 patients with psoriasis that were indicator for the diagnosis of psoriasis. undergoing therapy at Linyi People's Hospital (Linyi, China) between January 2011 and January 2015 were enrolled in the Introduction present study. The mRNA expression levels of haptoglobin were detected by in situ hybridization; immunohistochem- Psoriasis is a chronic, proliferative and inflammatory skin istry was used to detect haptoglobin protein expression; and disease, which typically affects individuals in their twenties, double‑labeling immunofluorescence was used to count Lang- yet can occur at any age (1). The incidence of psoriasis in erhans cells; western blotting was also conducted to determine the European population is 2‑3%, whereas it only accounts protein expression. A receiver operating characteristic (ROC) for ≤0.1% in the Asian population, and is exceedingly rare curve was generated to assess the diagnostic value of hapto- in Africa (2). This long lasting disease is typically character- globin for psoriasis. Compared with the normal and negative ized by recurrent episodes of red, itchy and scaly patches of control (NC) groups, the mRNA expression levels of hapto- abnormal skin, which may also be sharply demarcated from the globin were markedly increased in the experimental group adjacent normal skin (3). There are five main types of psoriasis: (P<0.05). Haptoglobin protein expression was also markedly Plaque, inverse, guttate, pustular and erythrodermic (2,4). In increased in the experimental group compared with in the particular, plaque psoriasis, which is also known as psoriasis normal and NC groups (P<0.05). Conversely, there was no vulgaris, makes up ~90% of cases. It typically presents as red significant difference in haptoglobin expression between the patches with white scales on top (2,5,6). At present, psoriasis NC group and the normal group (P>0.05). The critical value of remains a serious, poorly understood disease. One hypothesis haptoglobin mRNA in the diagnosis of psoriasis was 2.93, and suggests that the occurrence of psoriasis is due to functional sensitivity and specificity were 91.3 and 73.6%, respectively. immaturity of keratinocytes induced by an inflammatory The area under the ROC curve was 0.883 [95% confidence cascade in the dermis resulting from various immune cells (6). interval (CI)=0.837-0.929]. The critical value of haptoglobin These immune cells, including dendritic cells, macrophages protein in the diagnosis of psoriasis was 0.995, and sensi- and T cells, move from the dermis to the epidermis and secrete tivity and specificity were 76.1 and 99.9%, respectively. The inflammatory cytokines (7). Previous studies have reported that area under the ROC curve was 0.926 (95% CI=0.837‑0.929). a defect in regulatory T cells may underlie this disease (7,8). The present study demonstrated that the mRNA and protein Therefore, T lymphocytes may have an important role in the expression levels of haptoglobin were increased in patients occurrence of psoriasis. with psoriasis. Haptoglobin mRNA and protein expression Haptoglobin is a positive acute-phase reactant and plasma glycoprotein, which binds to free hemoglobin to form the hemoglobin-haptoglobin complex. Haptoglobin is known to be predominantly produced in the liver (9). Haptoglobin exerts anti-infectious effects, prevents iron loss and renal damage, Correspondence to: Dr Li-Qian Liu, Department of Dermatology, regulates immune function, and defends against the oxida- Linyi People's Hospital, 27 Jiefang Road, Lanshan, Linyi, Shandong 276003, P.R. China tive effects of hemoglobin/iron (10,11). Several studies have E-mail: [email protected] demonstrated that haptoglobin engages in various disease states, including lung cancer, pancreatic cancer, liver cancer, Key words: haptoglobin, psoriasis, in situ hybridization, Langerhans diabetes and prostate cancer (12-14). Condyloma acuminatum cells, immunohistochemistry, double-labeling immunofluorescence, is one of the most common anogenital skin diseases (15). western blotting Previous studies have reported that haptoglobin may inhibit the functional maturation of Langerhans cell, and may there- fore be involved in the immunoregulatory process (16,17). 3736 TIAN et al: HAPTOGLOBIN AND PSORIASIS Abnormal immune function is crucial for the development of samples were washed with DEPC solution for 6 min and were psoriasis; therefore, haptoglobin expression and its regulatory digested in proteinase K solution for 30 min at 37˚C to remove mechanisms in psoriasis tissues may be of great significance. In surface proteins that could impede the combination of probes. addition, due to haptoglobin gene polymorphisms, haptoglobin The samples were then rinsed in phosphate-buffered saline has been investigated as a biomarker with regards to its corre- (PBS) and DEPC solution for 6 min, respectively. The slides lation with disease (18). The present study aimed to examine were treated with 20 µl pre-hybridization solution and were the mRNA and protein expression levels of haptoglobin in incubated at 42˚C for 2 h. The sequence of the synthesized psoriasis tissues, and to determine its clinical significance in haptoglobin oligonucleotide probes was: 5'-TTC TAC TGA psoriasis. TGT TAT CTC CAG GAT GCT GTA-3' (3'-marked by digoxin). Subsequently, the samples were hybridized at 42˚C for 22 h, Materials and methods and were washed with 2xSS and 1xSSC for 15 min, separately. The samples were further incubated in sheep serum blocking Patients. A total of 138 patients with psoriasis (76 men and buffer at 37˚C for 30 min, biotin‑anti‑digoxin antibody at 37˚C 62 women; mean age, 44.39±18.22 years; age range, 13-75 years) for 1 h, and horseradish peroxidase-conjugated streptomycin that were undergoing therapy at Linyi People's Hospital (Linyi, at 37˚C for 30 min. Finally, the samples were stained with China) between January 2011 and January 2015 were enrolled diaminobenzidine and were counterstained with nuclear fast in the present study. The duration of disease was 1-240 months red. Negative control samples were treated with PBS, rather (mean, 113.25±69.89 months). Lesional skin specimens from than hybridization solution, and normal liver tissue sections the patients with psoriasis were selected as experimental group were used as positive controls. Bright orange-red staining in samples, whereas normal-looking skin specimens from the the cytoplasm was considered to be positive staining. The experimental group within 2 cm of psoriasis were selected as staining was analyzed by MetaMorph Microscopy Automa- negative control (NC) group samples. The inclusion and exclu- tion & Image Analysis Software (V4.6; Molecular Devices, sion criteria for the present study were as follows: i) Patients LLC, Sunnyvale, CA, USA). According to the image gray with typical clinical manifestations of psoriasis vulgaris that scale results, there were five staining grades: i) Negative (‑), was diagnosed by histopathology; ii) no systemic application no staining, gray value >130; ii) weak (±), slight staining, gray of vitamin A acid and glucocorticoid within 4 weeks, and no value 125-130; iii) positive (+), shallow staining, gray value local treatment within 2 weeks; iii) all individuals were unre- l15-125; moderately strong (++), deep staining, gray value lated; iv) patients with localized, pustular, erythrodermic and 105-115; very strong (+++), deeper staining, gray value <105. psoriatic arthritis were excluded; v) patients with atypical early clinical performance, those that failed to be definitively diag- Reverse transcription‑polymerase chain reaction (RT‑PCR). nosed, and those without sufficient diagnostic evidence were Tissues were weighed and then put into a mortar (precooled excluded; vi) patients with severe disorders, including cardio- with liquid nitrogen) for homogenization. Tissues (100 mg) vascular diseases and cancer were excluded; vii) patients who were added to 1 ml TRIzol reagent (Invitrogen; Thermo were pregnant or breastfeeding were excluded; viii) patients Fisher Scientific, Inc., Waltham, MA, USA) for the extrac- who were reluctant to cooperate, or for whom there was tion of total RNA. The RT system consisted of the following incomplete clinical data were excluded. The normal group reagents: 10X RT buffer (1 µl), MgCl2 (25 mM), 2 µl dNTP consisted of 106 normal human skin samples obtained from complex (10 mM, 1 µl), reverse transcriptase (0.5 µl), non-psoriatic patients (63 males and 43 females; mean age, RNasin Inhibitor (0.25 µl), Random 9 mers (0.5 µl), total 42.15±15.62 years; age range, 21-70 years) from the orthopedic RNA (≤500 ng, 2 µl); the system was diluted with 0.1% surgery unit of the hospital, none of which suffered from DEPC-treated water to a final volume of 10 µl. RT was immunological diseases or other systematic diseases. There conducted under the following conditions: 30˚C for 10 min, were no statistical differences in age and gender between the 42˚C for 30 min and 99˚C for 5 min. The PCR system was experimental group and the normal group. The skin tissues 5X PCR buffer (10 µl), Taq DNA polymerase (0.25 µl), were immediately embedded with Optimal Cutting Tempera- forward primer and reverse primer (both 20 pmol, 0.5 µl) ture (OCT) compound (Hede Biological Technology Co. Ltd., with β-actin as an internal control (Table I); the system was Beijing, China), and were maintained at ‑70˚C until further diluted with 0.1% DEPC‑treated water to a final volume of use.