The expressions of metadherin and LEF-1 in mucosaassociated lymphoid tissue lymphoma of ocular adnexal

INTRODUCTION

M ucosa-associated lymphoid tissue (MALT) lymphoma known as extranodal marginal zone B-cell lymphoma is among the commonest histological subset[1], cover around 35% to 90% of primary ocular adnexal lymphoma cases[2].MALT lymphoma, usually presenting an indolent course,frequently relaps in various extranodal sites[3]. Generally, the neoplasm can be found on different sites on ocular adnexal,ranging from eyelid, conjuctiva, lacrimal gland to orbita.This usually bring some bad effects to patients, such as mass nodular, painful swelling, dry eye symptoms, diplopia,strabism, even decreased vision to some extent. What’s more,with the incidence of this disease rising up recently, researchers are increasingly interested in fi guring out its pathogenesis. The former studies have found its connection with microorganism infection, such as helicobacter pylori (Hp) or Chlamydophila psittaci (Cp), and with autoimmunologically mediated diseases[4]. But it is fewer about oncogene and molecular mechanism.

Metadherin, also known as astrocyte elevated gene-1 (AEG-1),introduced in primary human fetal astrocytes initially[5-6], has been probed overexpression in many mankind malignancies,such as non-small lung cell cancer, breast cancer, esophageal cancer and so on[7-9]. Furthermore, researches support of AEG-1 association with neoplasm development, evolution,metastasis and invasion. As previous studies elucidated, AEG-1 contribute to tumor progression and evolution by activating abnormally various oncogenic signaling pathways such as phosphatidylinositol 3-kinase (PI3K)/Akt, nuclear factorkappaB (NF-κB), and wingless and INT-1 (Wnt)/ β-catenin pathways and so forth[10-11]. Ordinarily, the regulation of Wnt signaling pathway also works in physiological progression,including embryo development and keeping organs and tissues in adults. However, it appears to a driving force in many maliganancies[12-13]. It is supposed that AEG-1 brings about β-catenin the vital element of Wnt/β-catenin pathway nuclear translocation then upregulates different target gene expressions,by activating the Raf/MEK/mitogen-activated protein kinases(MAPK) signal pathway in hepatocellular carcinoma cells[14].Lymphoid enhancer-binding factor 1 (LEF-1), a major transcription factor of Wnt pathway, is one member of the LEF/T cell factors (TCFs) transcription factor family[15-16].LEF/TCFs are DNA binding transcription factors, functioning in Wnt signaling channel, by raising β-catenin to nucleus for target genes expression[17]. It acts as an executive of transcription. Specif i c inhibitor, AEG-1 siRNA, visibly downregulated LEF-1 expression. Researchers assumed that AEG-1 may be a participator in gastric carcinoma evolvement,which may depend on Wnt pathway[18] and this role of the oncogene has been studied in many tissues. However, little is known in ocular adnexal MALT lymphoma. Consequently,we hypothesize that the two factors, AEG-1 and LEF-1, may give play to crucial effect on MALT and participate in the pathogenesis of MALT.

The current study aims to detect the expression of AEG-1 and LEF-1 in ocular adnexal MALT lymphoma by immunohistochemistry and real-time polymerase chain reaction (RT-PCR)

SUBJECTS AND METHODS

Specimens’ Collection This study collected 30 specimens of ocular adnexal lymphoma (which has been diagnosed as MALT lymphoma by immumohistochemical in our institution)at the Pathologycal Lab of Ophthalmology Department,Aff i liated Hospital of Qingdao University from 2000 to 2015.There are No.5, No.7, No.4 and No.14 samples respectively taken from eyelid, conjunctiva, lacrimal gland and orbita. The total specimens include 19 males and 11 females, 16 beyond an age of 60, and 14 below 60. According to Ann Arbor clinical stage, the specimens from I to IV phase were 20,8, 1 and 1 respectively. Of 20 cases of reactive hyperplastic lymphadenopathy were used as negative control. The patients’agreement and favor from the Institutional Research Ethics Committee were acquired for research purposes.

Hematoxylin Eosin and Immumohistochemical Staining The tissues from various stages were made to paraffin section and hematoxylin eosin (HE) staining. Of 3-µm-thick paraffin sections were cut on a microtome (CM1900; Leica Microsystems, Deerfield, IL, USA) and then mounted onto glass slides with 100 g/L polylysine. Immunohistochemical staining proceeded in the light of the manufacturer’s specif i cation [SP kit (Biosynthesis Biotechnology)]. In short,after deparaffinising in xylene and dehydrating in ethanol,the sections were immersed in a citrate buffer and heated under high pressure steam for 15min to retrieve antigens, then immersed in 3% H2O2 for 15min to inactivate endogenous peroxidase. After washing three times with phosphate buffered saline (PBS), the sections were incubated with either rabbit anti-AEG-1 monoclonal antibody (1:400) or rabbit anti-LEF-1 monoclonal antibody (1:400) (Abcam Company) for 3h at room temperature before washing three times with PBS.After that, keeping these sections incubated with horseradish peroxidase labeled goat anti-rabbit polymer (Zhong Shan-Golden Bridge Biological Technology) for 20min at 37℃.After washing three times with PBS, the sections was stained by diaminobenzidine, and then dyed by hematoxylin counterstain. Neutral balsam was used for sealing all specimens for microscopy observation. Instead of the primary antibody,PBS was also used for negative control. Specif i city of primary antibody was also examined on reactive lymphoid hyperplasia.The positive expression appears as diffuse brown yellow particles located in nucleus or in cytoplasm.

Selection of five typical regions at random, with 1000 cells in all (200 for each field), were observed under a highpower optical microscope for 400. The status was evaluated independently by at least two researchers. Moreover,immunostaining categories based on the positive cells percent,grade score was as follows: 0: ＜5%; 1: 5%-25%; 2: 25%-50%;3: 50%-75%; and 4: ＞75%; the staining intensity was classif i ed into the following status: 1=weak staining; 2=moderate staining; 3=strong staining. Multiply the grade score of positive tumor cells percent by staining intensity, a value was got for every case, of which value ＜1 was determined negative otherwise positive[19].

Real-time Polymerase Chain Reaction RNA was extracted from dissolved aim tissues, with the TRIzol reagent,according to the manufacturer’s recommendations. Of 1 μL RNA is used for reverse transcriptase cDNA (single chain cDNA synthesis kit), which conduct in the total volume of 20 μL system. During this procedure, cDNA acts as a template, and specific primers involved. RT-PCR analysis the expression of AEG-1 and LEF-1 mRNA, and β-actin acted as an internal control. Breif l y, SybrGreen qPCR Master Mix (2×) 10 μL, 0.5 μL primer F, 0.5 μL primer R, 7 μL dH2O, 2 μL cDNA. PCR amplification conditions: 95℃denaturation for 15s, 60℃ annealing and extension for 1min,40 cycles. Calculation of the expression of the sample by standard curve compares to internal reference β-actin. β-actin primer sequence: F-GATTACTGCTCTGGCTCCTAGC,R-GACTCATCGTACTCCTGCTTGC; AEG-1 primer sequence: F-TTACCACCGAGCAACTTACAAC,R-ATTCCAGCCTCCTCCATTGAC; LEF-1 primer sequence: F-GACGAGATGATCCCCTTCAA,R-AGGGCTCCTGAGAGGTTTGT, these sequences were referenced to literature[20] .

Statistical Analysis SPSS17.0 software was used for statistical analysis (SPSS, Chicago, IL, USA). The statistically different expressions of AEG-1 and LEF-1 among groups were determined by χ2 tests, among the stages were determined by Fishers’ exact probabilities. The mutual relation between AEG-1 and LEF-1 was analyzed by Spearman correlation test. PCR result analysis was manifested by one-way ANOVA and both mutual compares in LSD test. P value of ＜0.05 was considered statistically signif i cant.

RESULTS

Real-time Polymerase Chain Reaction Our current result indicated that AEG-1 and LEF-1 mRNA was distinctly higher in most MALT lymphoma tissues than in the reactive lymphoid hyperplasia (P＜0.001; Figure 1A, 1B).

Hematoxylin Eosin Staining There are normal follicular structures in reactive lymphoid hyperplasia tissues which consisted of different patterns of mature lymphocytes and is scattered with plasmocytes, histocytes and immunoblasts(Figure 2A). The samples of MALT lymphoma was composed of diffuse, similar and small lymphocytes which appeared as irregular nucleus, deep stain. Considering as high-differentiated and moderately-differentiated lymphoma (Figure 2B).

Immumohistochemical Staining

Expression of astrocyte elevated gene-1 in mucosa-associated lymphoid tissue lymphoma The result shows that a positive staining, brown yellow particles accumulating, mainly appears much membrane, less cytoplasm (Figure 3B, 3C). Furthermore,difference of AEG-1 protein expressions in various Ann Arbor clinical stage shows a statistical meaning (P＜0.05; Table 1),but no relation when it comes to the age, gender, or occurrence site. We examined higher proportion of AEG-1 positive staining in MALT lymphoma (73.3%) than that in reactive lymphoid hyperplasia (20%) significantly (P＜0.05; Table 2).While there was almost no positive staining in control group(Figure 3A).

Expression of lymphoid enhancer-binding factor-1 in mucosa-associated lymphoid tissue lymphoma As the result shows, the positive staining primarily was apparently detected in nucleus (Figure 4B, 4C). We examined a higher rate of expression of LEF-1 in MALT lymphoma (53.3%) than in reactive lymphoid hyperplasia (10%) signif i cantly (P＜0.05;Table 2). In addition, LEF-1 protein high expression represent remarkable diversity among different Ann Arbor clinical stage(P＜0.05; Table 1). However, the relationship cannot be found,when involving the age, gender, and occurrence site. However no positive staining detected in control group (Figure 4A).

Mutual relation between AEG-1 and LEF-1 in mucosaassociated lymphoid tissue lymphoma Our datas revealed a positive relation between the expression of AEG-1 and that of LEF-1 in MALT lymphoma (r=0.435, P＜0.05; Table 3).

DISCUSSION

Ocular adnexal lymphomas (OAL) are histological heterogeneous malignancies, reaching up to 55% of all orbital tumors[21].MALT was recognized as the most common histological subtype. However, we did know little about the pathogenesis of this disease at present. There remains much more to be explored for the future therapy. Reviewing the numerously previous papers, AEG-1, an oncogene, have involvement in tumor generation and evolution, irregularly expressivein diversified human malignancies, containing nervous[22],urogenital[23], respiratory[7], digestive[9] and Hemic and Lymphatic Systems[20]. LEF-1 broadly positive emerging in growth tissues during embryogenesis, but is limited to hair follicle bulbs, pre-B and pre-T lymphocytes in adulthood[24-25].In addition, LEF-1 was observed an elevation in some blood system malignances. More evidences have been found the two factors playing a role in hematological malignancies.We determined to detect their expression situation in ocular adnexal lymphoma.

The current exploration manifested that AEG-1, highly expressive in the cytoplasm, correlated signif i cantly with the development and progress of ocular adnexal MALT. Our study discovered AEG-1 expressive elevation either at mRNA orat protein in ocular adnexal MALT tissues. Besides, 73.3%(22/30) of MALT samples positively express AEG-1 protein presented by immunohistochemical staining, while the control group detected only little expression. Corresponding with outcome of immunohistochemical staining, the mount of AEG-1 mRNA is as much again as its counterparts. Dong et al[19] demonstrated that overexpression of AEG-1 at the level of protein and mRNA is relation to metastasis, Ann Arbor clinical stage, and poor prognosis in gastric cancer. Integrated the clinical characters of MALT patients, the different ratio of highly expressive AEG-1 along with the Ann Arbor clinical stage indicated a statistical signif i cance in MALT, nevertheless this is not exist in age, gender of patients or tumorous location.Consequently, the state of our protein expression was in consistent with the former researches.

LEF-1, the core factor of Wnt pathway, participated in transcription of tumor cell by conjunction to DNA combanding region. The outcome of our study indicated that 16/30 (53.3%)MALT samples showed a positive nuclear staining, with little expression of LEF-1 in its counterparts. Simultaneously,high expression of LEF-1 mRNA in tumor tissue had been examined compared with reactive lymphoid hyperplasia. This high expression of protein and mRNA is accordance with the accumulating evidences demonstrated in several malignancies,including colorectal[26], prostate[27], and leukemia[28]. Moreover,some researchers found that LEF-1 is linked with tumor migration and invasion. Liang et al[29] illustrated LEF1-miR-181 engaging in prostate cancer epithelial-mesenchymal transition process which specifically modulate subsequent migration and invasion.

Additionally, Walther et al[30] detected devoid of expression of LEF-1 in (0/6) marginal zone B-cell lymphoma by Immunohistochemistry, when studying the mechanism of LEF-1 in Burkitt’s lymphoma. It’s opposite to our result.Our expression of LEF-1 mRNA in test group is five times more than that in control group. This finding corresponds to the demonstration of Kühnl et al[31]. Together the clinical characters of MALT patients, the expression of LEF-1 displays a strongly correlation to the Ann Arbor clinical stage of patients with MALT lymphoma. However, this relationship has not been seen in age, gender or occurrence site. This indicated that expression of LEF-1 increases along with the grade of the tumor stage.

For the present, we found a positive relation between AEG-1 and LEF-1 expression in MALT lymphoma, and their high expression in association with tumor differentiation. Giving the limited samples, we only got the evidence of AEG-1 and LEF-1 involving the development of ocular adnexal MALT lymphoma. AEG-1 overexpression, testif i ed in many mankind malignancies, promoted tumor formation through NF-κB,PI3K/Akt and Wnt/β-catenin pathways[10-11]. As is known, Wnt signaling pathway regulate either of B-cell development or self-renewal of hematopoietic stem cells[32]. By implication,AEG-1 may have effect on Wnt signal pathway. However, to fi gure out the two factors’ pattern of interaction remains further researches.

To summarize, these research findings herein delivered that AEG-1 and LEF-1 overexpression is probably connected with the tumorigenesis of MALT. There is a positive correlation between AEG-1 and LEF-1 in ocular adnexal MALT.

ACKNOWLEDGEMENTS

Foundation: Supported by Public Domain of Science and Technology Project of Qingdao, Shandong Province, China(NO.2012-1-3-2-14-snh).

Conflicts of Interest: Qi D, None; Lin H, None; Gao Y,None; Lin J, None; Hu LT, None; Zhao GQ, None.

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