Studies On The Function Of Matrix Metalloproteinase-2 From Basic To Clinical

The gene of human matrix metalloproteinase-2 (MMP-2) was cloned and expressed by Huhtala.P in 1990, and it contains signal sequence, propeptide, catalytic domain, fibronectin-like domain and hemopexin domain. MMP-2 is secreted as 72 kd proenzyme of 631 amino acids, hydrolyzed to a 65 kd active protein. MMP-2 degrades type IV collagen, the major component of basement membranes and gelatin (denatured collagen). It associates with healing, reproduction and involution of reproductive organs. It was found that MMP-2 was related with migration and metastasis of tumor, autoimmune diseases, dental diseases and tumor angiogenesis. In our research work, we expressed the specific domain of MMP-2, the fibronectin-like domain in vitro, and used the recombinant protein to immunize mice and then obtained the specific monoclonal antibodies against MMP-2. We established the immunization radiation metry analysis method (IRMA) for MMP-2 determination and compared the level of MMP-2 in solid tumor with healthy people. This affords a convenient means for determining the relative level of MMP-2. In addition, we used the new technique, RNA interference, to make the gene of MMP-2 silence and find more roles of MMP-2; thirdly, we studied the role of MMPs in chronic myeloid leukemia (CML). The preparation and function study of monoclonal antibodies to MMP-2 In the structure of all the MMPs members we known now, there are three common domain: catalytic domain, amino-terminal domain and C-terminal domain. Besides the three domains, there is a specific domain of MMP-2, the fibronectin-like domain. It was suspected to participate in the connection of MMP-2 with substrates, and this step was the premise for active role of enzyme. By blocking this link will help inhibit the invasion and metastasis of endothelial cells and tumor cells. We designed the primers of MMP-2 according to the sequence in gene bank. After extracting total RNA from Endothelial cells EAhy926 and reverse transcription-PCR (RT-PCR), the cDNA of fibronectin-like domain was got and then was recombined with expression vector PET20b(+).The recombined vector was transformed to E. coli BL21(DE3) plus. The positive clones were chosen and induced by IPTG. Before used to immune mice, the recombined protein was purified by Ni-NTA agarose column and Balb/c mice were immunized. We screened the positive clones by ELISA method and used the positive cells to prepare ascites. The size of the recombinant protein is 21kd. After the spleen cells of the mice and hybridoma cells SP2/0 were hybridized, we got 2 pieces of cells that can last secreting McAb against MMP-2 named SZ-117 and SZ-116 respectively. The titers of McAbs in ascites were 2×10―5and 1×10―5 respectively. The heavy chain of the McAbs both belonged to IgG1 subclass. The concentrations of ascites were 1.5mg/ml and 1.8mg/ml after purifying by proteinG-Sephrose4B affinity chromatography column. The antibodies could react with perforated platelets. They could not only identify 72 kd protein in broken EAhy926 cell liquid but also recombinant 69kd proenzyme. MMP-2 exists in the stromal tissue of stomach, cholecystis, spleen, ovarian, prostate, salping and lymph node but no in the thyroid, small intestine or liver. The MTT test showed that SZ-117 had no effect on the proliferation of EAhy926 or pancreatic carcinoma cells 1990.It could inhibit the migration ability of two kinds of cells induced by fibronectin and collagen IV and more powerful on collagen IV. There were similar results about invasion ability test.The ability of endothelial cells to form tubes in vitro was reduced 47.3% after reacting with SZ-117. At present, the inhibitors against MMP-2 are not very selective. We prepared the specific antibodies to MMP-2 and have verified that they have high affinity and specificity. All these help to study the function of MMP-2 and the role it plays in the occurrence and progression of solid tumor and blood tumor. The preliminary study on the function of MMP-2 RNA interference is a technique that uses the double single of RNA (dsRNA) to make gene silence for special sequence. Compared with other methods, RNAi has its distinct characters: 1.simple and easy to be done 2.experiment cycle shorter than gene knocking out 3.higher specificity and efficacy than other antisense technique. In order to verify the function of MMP-2 in affecting cell behaviors through degrading the extracellular matrix and reveal some new role of it, we made the gene of MMP-2 silence using RNAi technique.The endothelial cells EAhy926 were transformed by small double chains RNA (siRNA) of MMP-2 and were analyzed by flow cytometry and RT-PCR at protein and gene levels respectively. The result showed that MMP-2 gene level dropped to the lowest valley after being interfered for 48h, 18% to the control; protein level 60h, 40% to the control. On the basis of MMP-2 gene of EAhy926 was silenced, we studied the role of MMP-2 on endothelial cells proliferation, migration, invasion, tube formation and cells cycle. The proliferation ability of EAhy926 had no obvious difference between interference or not applying MTT method. The migration ability of EAhy926 induced by two kinds of factors, type IV collagen (COL IV) and fibronectin (Fn), was inhibited after interfered by siRNA for 48h, and stronger inhibition to COL IV than Fn.( Fn ctrl 0.581+0.012 vs 0.261+0.002; COL IV0.467+0.009 vs 0.110+0.010 P<0.01).The invasion test had the similar result as migration test.(Fn Ctrl 0.365+0.012 vs 0.101+0.002; COL IV 0.317+0.009 vs 0.102+0.010 P<0.01). The tube formation ability of endothelial cells dropped to 58.9% of control. In the cells cycle experiments , after RNAi for 48h and 72h, the ratio of G1 dropped from 65.9%±2.53%, 32.7±1.91 to 83.9±2.53,18.1±1.49 (P<0.01);S and G2 rose from 63.2±1.89,37.1±2.65 to 89.2±1.24,10.2±0.85 (P<0.01)。The changes of Rb,cyclinD1 and PCNA genes were analyzed by RT-PCR between pre-and post-interference, we observed that the relative quantities were 35%,51% and 22% to normal control respectively. It was shown that the three genes expression of endothelial cells interfered by MMP-2 siRNA were inhibited at different degree, but there were no changes of MMP-14 or TIMP-2. All these hint that there are no direct relationships between the change of cell cycle and MMP-14 and TIMP-2. Signal transduction is a very complex network, and we still not know how MMP-2 affects the expressions of Rb, cyclin D1 and PCNA. The clinical significance of matrix metalloproteinases production by bone marrow mononuclear cells from patients with chronic myeloid leukemia The major component of extracellular matrix is collage type IV, while MMP-2 and MMP-9 are the major two enzymes to degrade it. A lot of research work has verified that they play a very important role in angiogenesis and tumor metastasis. The role of MMPs in leukemia, a particular type of cancer, is not paid attention to until recently. Almost all work was related with acute leukemia, so we have a preliminary investigation on the role ofMMPs in CML. Bone marrows from normal adults and CML patients were collected. Mononuclear cells were isolated from bone marrow aspirates and used for RNA isolation. The cDNA was obtained after reverse transcriptase-PCR (RT-PCR), and then five kinds of gene were amplified. It was found that the mRNA of MMP-9 and TIMP-1 was presented in all of the 12 adult healthy donors, TIMP-2 was 50% (6/12) and MMP-2, MMP-14 were absent in all the healthy donors, and that the mRNA of MMP-9 and TIMP-1 was presented in all of the 30 CML patients. In contrast, TIMP-2 was detected in 26 of 30 samples and MMP-2, MMP-14 in 11 of 30 samples. In the 11 cases detected MMP-2, 3 cases had been in blast crisis, 6 cases in accelerated phase and 2 cases undergone progression within 12 months. It can be concluded that the expression of MMP-2 and MMP-14 is consistent and is related with the progression of disease, so MMP-2 and MMP-14 present a potential marker for deteriorating of CML. The expression difference and clinical significance of matrix metalloproteinase-2 in the serum of solid tumor patients Recently, MMP-2 level in serum/plasma has been an important marker for monitoring some diseases progress. We labeled SZ-116 with 125I by chloraseptine T method and established the immunization radiation metry analysis method (IRMA) for MMP-2 determination. There were two peaks after 125I -SZ-116 complex flowing through SephadexG-25 column. The liquid of the first peak was collected and stored in –20℃mixed with 100% glycerine. There were no changes of the standard curve of 125I -SZ-116 markers after stored for 3 months. We took two times the value of the zero standard tube as the minimum sensitive value of this method, and in theory, minimum sensitive value is 0.476 nmol/L (10 times), the assay range was 1.19 nmol/L~38.09 nmol/L. The variation coefficients (CV) of the 5 standard points (2.38 nmol/L, 4.76 nmol/L, 9.52 nmol/L, 19.04 nmol/L, 38.09 nmol/L) in 20 pieces of continuous curves were 1.78%~9.61%. The CV in one time test was 4.98% ±2.57% (n=10). The CV among different tests was 8.97% ±3.01% (n=5). We added different doses of recombinant protein and 95.2%~105.8% of them were retrieved, the average value was 100.9%.We assayed 38 normal adult, the average MMP-2 level in serum was 1.43 nmol/L ±0.584 nmol/L, and the range was 0.238 nmol/L ~ 2.14 nmol/L. There was no obvious discrepancy between difference ages or difference sexes. We collected 33 samples of non-malignancy tumor and 129 samples of malignancy tumor. The MMP-2 level in 33 samples of non-malignancy tumor serum had no obvious discrepancy compared with healthy people (1.45 ±0.391 vs 1.43 ±0.584 P>0.05), but the level in malignancy tumor was far higher than that in healthy people (2.38 ±0.771 vs 1.43 ±0.584 P<0.01). In the group of malignancy tumor, the MMP-2 level in patients with metastasis was higher than those without metastasis (4.88 ±0.873 vs 1.95 ±0.491 P<0.01). There was no obvious discrepancy between the patients that were before and after treated with chemistry drugs or radiation method (2.08 ±0.357 vs 2.15 ±0.359 P>0.05). It was shown that the level of MMP-2 related with the migration and metastasis of malignant tumor closely, so it may be a new mark for tumor. Monitoring the level of MMP-2 helps to learn the progression of diseases and judge curative effect.