Impact Of CD147 And Its Inhibitors On The Invasiveness Of Non-Hodgkin's Lymphoma: Clinical Investigation And Experimental Study

The degradation of extracellular matrix (ECM) by matrix metalloproteinase (MMPs) is the key step for tumor cells' invasiveness and metastasis. Collagenase IV, including MMP-2 and MMP-9, are the most important MMPs and play a leading role in the process of ECM degradation. Extracellular matrix metalloproteinase inducer (EMMPRIN / CD147) is a newly-identified cell adhesion molecule and expresses on the membrane of different human tumor cells. As a hot-spot in the field of cancer research, EMMPRIN/CD147 induces fibroblast cells to generate MMPs, so as to promote metastasis of tumor.Despite of great improvement in the efficacy of treating childhood non-Hodgkin's lymphoma (NHL), there were still some cases suffering or even dying from early widespread extra-nodal invasion and early recurrence. To make a breakthrough at this point, focus has been placed on CD147 recently as it is a strong indicator for tumor invasion and metastasis. However, the expression of CD147 in malignant lymphoma cells, the relationship between CD147 and MMPs, and molecular pharmacological mechanism of CD147 agonist / antagonist have been less reported. The purpose of this study is to explore in which way CD147 and MMPs play a role in the process of NHL invasion and metastasis and what are the regulatory mechanisms, by investigating the correlation between CD147/MMP-9 expression in NHL cells and NHL patients' outcome; by testing the effects of CD147 agonist / antagonist on Jurkat cells, and by verifying the consequence of CD147 gene silencing. Results from present study are supposed to provide more experimental data supporting that short peptides might be used in clinical practice and CD147 might serve as a target of gene therapy in NHL. PartⅠ: Expression of CD147 and MMP-9 in NHL cells and its correlationto NHL prognosisObjective: To investigate the expression level CD147 and MMP-9 in NHL cells and explore its correlation to NHL clinical stage, tumor size, serum lactate dehydrogenase (LDH) concentration and NHL patients' outcome.Methods: Specimens excised from NHL patients were prepared. Expression of CD147 and MMP-9 in NHL cells were tested by utilizing SABC immunohistochemistry and RT-PCR techniques and correlated to patients' clinical characteristics and survival outcome.Results:1. The positive rate of CD147 expression was 73%(45/62), 17 cases (-), 11cases (+) , 34 cases (++) and 21 cases (+++); The positive rate of MMP-9 expression was 81%(50/62), 12 cases (-), 13cases (+), 18 cases (++) and 19 cases (+++).The Pearson rank correlation analysis indicated that there was a positive correlation between CD147 and MMP-9 expressions in NHL (r=0.763).2. Expression of CD147 determined in relation to something that includes clinical bone marrow invasion, tumor size, LDH values as well as the clinical stage; expression of MMP-9 has positive relation with bone marrow invasion, tumor size and clinical phases, but no relation with the age, sex, immune classification and LDH value.3. Survival curve analysis: 5-year survival rates were 78% ( 22/28 ) and 45% ( 15/34 ) in CD147 (-)～(+) and (++)～(+++) cases respectively, 5-year survival rates were 84% (21/25) and 43% ( 16/37 ) in MMP-9 (-)～(+) and (++)～(+++) cases respectively . the difference was significant. Cox multivariate analysis showed that the risk of CD147 and MMP-9 were an important prognostic factors.4. RT-PCR results showed that: CD147 mRNA expression ( 0.897±0.043) in high-risk groups (Ⅲ+Ⅳclinical phase) was higher than that in lower-risk group (Ⅰ+Ⅱclinical phase) (0. 281±0.051), the difference was significant (t = 29.76, P <0.01). MMP-9mRNA expression in High-risk group was 1.176±0.081, which was higher than that in lower risk group ( 0.363±0.073), the difference was significant (t = 10.04, P <0.01).Conclusion:CD147 and MMP-9 expression in NHL cells are closed associataed with the prognosis of NHL patients—the higher the expression level, the poorer the outcome.PartⅡ: Impact of CD147 agonist and antagonist on Jurkat cell proliferation and invasivenessObjective: To study the impact of CD147 agonist (cyclophilin A, CyPA) and CD147 antagonist (antagonistic peptide 9, AP-9 ) on the expression of CD147 and MMP-9 and on the proliferation and invasiveness of Jurkat cells.Methods:Jurkat cells treated with drugs were used in the follow study.1. MTT assay was performed to evaluate the proliferation of cell treated with CyPA ( 50 ng/ml, 100 ng/ml, 200 ng/ml), AP-9 ( 50μg/ml, 100μg/ml, 200μg/ml) and CyPA + AP-9 (AP-9 200μg/ml + CyPA50 ng/ml, 100 ng/ml, 200 ng/ml respectively) for 6h, 12h, 24h. 2. Western blot was applied to detect the protein expression of CD147 in Jurkat cells treated with CyPA ( 50 ng/ml, 200 ng/ml), AP-9(50μg/ml, 200μg/ml) and CyPA + AP-9 (AP-9 200μg/ml + CyPA50 ng/ml and 200 ng / ml respectively) for 12h, 24h, 36h.3. Gelatin zymography assay was utilized to detect the activity of MMP-9 in cells treated with CyPA ( 50 ng/ml, 200 ng/ml), AP-9 ( 50μg/ml, 200μg/ml) and CyPA + AP-9 (AP-9 200μg/ml + CyPA 50 ng/ml, 200 ng/ml) for 48h.4. Transwell chamber invasion test was been to calculate the cell migration by treating with CyPA ( 200 ng/ml), AP-9 ( 200μg/ml) and CyPA + AP-9 ( AP-9 200μg/ml and CyPA 200 ng/ml) for 48h.Results:The proliferation of Jurkat cell treated with CyPA was enhanced in time and dose-dependent manner, what's more, the expression of CD147, the activity of MMP9 and the migration number of cells were increased remarkably. While the role was opposite as CyPA treated with AP-9.There was no significant change in proliferation or levels of expression of CD147, MMP-9 and the number of cell migration treated with CyPA and AP-9.Conclusion:A time- and dose-dependent enhancement of Jurkat cells proliferation, upgrading of CD147 / MMP-9 expression, and incensement of tumor cell invasion are demonstrated after CyPA pre-treating. However, AP-9 inhibits the expression of CD147 and secretion of MMP-9, thus prohibits tumor cells from invasion. PartⅢ: Silencing CD147 gene by RNA interference and its impact onJurkat cell proliferation and invasivenessObjective:1. To construct eukaryotic expression vector interfered by CD147 RNA.2. To evaluate CD147 expression level and potential of invasiveness in Jurkat cells once the CD147 gene was silenced.Methods:1.The shRNA- pGE-I-CD147 vector was constructed and tranfected into Jurkat cells by electroporation, the stable tranfected cells of expression shRNA-CD147 recombinant plasmid was received by G418-selecting.2 The expression of CD147 mRNA in transfected cells was determined by RT-PCR.3 Flow cytometry assay was performed to evaluate change of cell cycle and proliferation in transfected cell.4. Matrix adhesion assay was applied to detect the change of transfected stromal cell adhesion.Results:1. DNA sequencing confirmed that the recombinant plasmid was successfully constructed, and pGE-I-CD147shRNA1-Jurkat cells and pGE-I-CD147shRNA2-Jurkat cells were constructed by G418-selecting.2.RT-PCR testing, CD147 mRNA expression in pGE-I-CD147shRNA1-Jurkat cells was decreased (CD147/β-actin gray : 0.12±0.03 vs 0.92±0.03 ); flow cytometry analysis indicated the cells in G0 + G1 phase in pGE-I-CD147shRNA1-Jurkat cells was more than that in the control group ( 89.85±7.85% vs 57.55±5.64%, P <0.05 ); matrix adhesion showed cell adhesion rate in pGE-I-CD147shRNA1-Jurkat cells was higher than that in the control group (47.12±4.55% vs 21.74±7.65, P<0.05).Conclusion:shRNA-CD147 recombinant plasmid silences CD147 gene in Jurkat cells. Subsequently, CD147 expression is downgraded; proliferation and invasiveness of Jurkat cells are inhibited. Different segments of RNA produces different interfering consequence, which further pronounces the role of CD147 expression in the development of NHL and provides experimental clues supporting use CD147 as a target of gene therapy for NHL.Summary1. The expression of CD147 and MMP-9 in NHL cells are closely associated with and can predict NHL patient's prognosis—the higher the expression, the worse the outcome.2. Downgrading CD147 expression either by antagonist (AP-9) application or by silencing CD147 gene leads to an inhibition of tumor cells proliferation, and subsequently decreases invasiveness potential of tumor cells. CD147 may become a target of gene therapy for NHL.