The Mechanism Of Membrane-Type 1 Matrix Metalloproteinase(MT1-MMP,MMP-14) In Invasive Pituitary Adenoma

Pituitary adenomas(PAs) are noncancerous tumors, but about 1/3 of PAs invading into the cavernous and sphenoid sinus and the clivus, which have been classified as “invasive pituitary adenomas(IPAs)”. Except for prolactinomas sensitive to bromocriptine treatment, the majority of IPAs mainly chiefly depends on surgical resection and postoperative radiotherapy. It is difficult to operate completely for pituitary adenomas invading adjacent tissues which lead tumor to relapse easily after surgery. The mechanism of PAs invasiveness is a complex process, including matrix metalloproteinases and their inhibitors, cytokines. Membrane type matrix metalloproteinase-1(MT1-MMP, MMP-14) is one of important members of the matrix metalloproteinase family. It can degrade extracellular matrix(ECM) and basement membrane. MT1-MMP(MMP-14) along with its inhibitors(tissue inhibitor of metalloproteinases, TIMPs) play a key role in maintaining the integrity of cell membrane. In order to understand the invasive behavior of PAs, the possible mechanism of MT1-MMP(MMP-14) in invasive pituitary adenomas was conduct in our study.PartⅠ Assessment of invasive pituitary adenoma ObjectiveTo establish the evaluation criteria of invasive pituitary adenomas(IPAs) and to obtain the specimen of invasive pituitary adenoma. Methods 1. According to the Hardy grading, staging method for evaluating pituitary adenoma invasive sphenoid sinus and Knosp classification method to define pituitary adenomas invading cavernous sinus. 2. Based on MRI findings, 125 cases of pituitary adenoma were divided into invasive pituitary adenoma(IPA,58 cases) and non invasive pituitary adenoma(NIPA,67 cases). According to surgery results, MRI determined the sensitivity (Sen), specificity(Spe), positive predictive value(PPV) and negative predictive value(NPV) of IPAs were analyzed.3. The clinical characteristics of invasive pituitary adenoma were investigated. Results 1. The sensitivity and the positive predictive value for MRI in invasion sphenoid sinus were 85.3% and 82.9% respectively. The sensitivity and the negative predictive value were 100% for NIPA confirmed by surgery with Knosp 0 to grade I, while the specificity and positive predictive value were 100%, the negative predictive values were 93.8% and 90.9% in Knosp III and Knosp IV confirmed as the invasive pituitary adenoma with operation 2. The clinical manifestations were as follows: There was a high incidence of macroadenomas and giant adenoma in IPAs. Complete resection rate was low and the pituitary apoplexy, recurrence were relatively high. 3. The incidence of invasive pituitary adenoma was similar both in functional pituitary adenoma and non functional pituitary adenoma. The incidence of invasive pituitary adenoma had no obvious statistical difference between functional pituitary adenoma. ConclusionThe accuracy of Knosp classification method in judging the invasive pituitary adenoma was confirmed in this study. Preoperative magnetic resonance combined with operation were the better assessment for invasive pituitary adenomaPartⅡ Expression and significance of MT1-MMP(MMP-14) in invasive pituitary adenomas ObjectiveTo detect the expressions of MT1-MMP(MMP-14), VEGF, TGF-beta, p53 and PTTG in invasive pituitary adenomas and the relationship among them were analyzed. Methods 1. According to Knosp classification, 82 cases of pituitary adenoma were divided into IPA group of 37 cases and NIPA group of 45 cases, and the peritumoral pituitary tissue of 3 cases as the control group. Expression profiles of MMPs gene were screened in invasive pituitary adenoma tissues using real-time quantitative PCR. The expression level of VEGF, TGF-β, p53 and PTTG m RNA were detected. 2. The expression level of MT1-MMP(MMP-14) and MMP-2 were measured with immunohistochemistry in same samples. 3. The mouse pituitary adenoma At T-20 cell was subcultured. The expressions of MMPs, VEGF, TGF-b, p53 and PTTG m RNA in At T-20 cell and normal mouse pituitary tissues were detected by real-time PCR. The correlation of MT1-MMP(MMP-14) and other factors are further verified. Results 1. Expression spectrum of MMPs gene and expression of related factor: The expression of MT1-MMP(MMP-14) and MMP-1, 2, 9 m RNA were high and MT1-MMP(MMP-14) m RNA expression level was significantly higher(P<0.001) in IPAs compared with NIPAs and peritumoral pituitary tissues. The level of VEGF, TGF- beta and PTTG m RNA in IPA tissues were higher than other two groups(P < 0.001), But no significant changes in MMP-15 and P53 m RNA(P > 0.05). MT1-MMP(MMP-14) was positively correlated with MMP-1, 2, 9, VEGF, TGF-β and PTTG(P<0.05). It is not associated with MMP-15 and P53(P > 0.05). 2. The results of immunohistochemistry: MT1-MMP(MMP-14) and MMP-2 were higher expression level in invasive pituitary adenoma than that of non invasive pituitary adenoma group. MT1-MMP(MMP-14) staining results show its mainly located on the cell membrane. But the staining of MMP-2 was mainly expressed in stromal cells and in the cytoplasm. 3. To verify the correlation between the expression of MT1-MMP(MMP-14) and other factors: There were higher expression level in MT1-MMP(MMP-14), VEGF, TGF- beta and PTTG m RNA in At T-20 cell than that of the control group(P < 0.001), but no significant changes in MMP-2, 9, 15 and p53 m RNA(P > 0.05). And the MT1-MMP (MMP-14) associated with VEGF, TGF-β and PTTG positively. The results of our study suggested that MT1-MMP(MMP-14) might potential participate in pituitary adenoma invading process. ConclusionMT1-MMP(MMP-14) was highly expressed in invasive pituitary adenoma. There was a positive correlation VEGF, TGF-β with PTTG. It is suggested that MT1-MMP(MMP-14) may play an important role in the invasiveness and angiogenesis of pituitary adenoma. PartⅢ Effects on invasiveness behaviors of mouse pituitary adenomas after si RNA targeting MT1-MMP(MMP-14) ObjectiveTo explore the possible mechanism of MT1-MMP(MMP-14) contributes to invasiveness on mouse pituitary adenomas by transient transfected At T-20 cell line with MT1-MMP(MMP-14) interference vector. Methods 1. The most effective si RNA expression vector with MT1-MMP(MMP-14) was constructed and transfected At T-20 cells: According to design principle, a total of four possible sequence(MMP14-mus-1050, 1246, 1358 and 537) in Gen Bank as the target sequence. The recombined plasmid p GPU6/GFP/Neo-Mmp14 of four groups containing the interference fragment was constructed. At T-20 cells(1.5 ′ 104) were transiently transfected with recombinant plasmids using Lipofectamine? 2000. The most effective interference plasmid was selected using real-time PCR compared to those in untreated groups. Transfection efficiency was detected with fluorescent microscopy after 48 hours. 2. MT1-MMP(MMP-14) gene silencing: To use of real-time PCR in detecting the influence of VEGF, TGF-b, p53 and PTTG m RNA expression after MT1-MMP(MMP-14) was silenced. 3. TIMP-1 effect on At T-20 cells: At T-20 cells of cultivation of good condition were divided into three groups as followed: control group(cells cultured with conditioned medium), A group cultured with conditioned medium with TIMP1 protein(10 μmol/m L), and B group cultured with conditioned medium with TIMP1 protein(50 μmol/m L). The m RNA expression levels of MMPs, vascular factors and tumor associated genes were detected according to different concentrations of TIMP-1 protein on At T-20 cells. The proteins of MT1-MMP(MMP-14) and MMP-2 from different groups were measured with Western blot. 4. The change of invasion capacity of At T-20 cells treated with TIMP-1 proteins: Two groups of At T-20 cells treated with TIMP-1 proteins(10 μmol/m L、50 μmol/m L) and the control group were analyzed. The numbers of penetrated through transwell chambers coated with Matrigel of the At T-20 cells were calculated to judge the effects of different concentrations of TIMP-1 on the invasion ability of At T-20 cells. Results 1. Successful construction of MT1-MMP(MMP-14) si RNA expression vector and detection of four kinds of interference vector MMP14-mus-1050, 1246, 1358 and 537 compared with the control group, the inhibition efficiency of the MMP14-mus-1246 group was the most obvious, and the expression of green fluorescent protein p GPU6/GFP/ of Neo-si T1-1246 cell rate 70% and confirmed by repeated experiments. 2. The construction of transient silencing cell model: After transient-transfection of the MT1-MMP-si RNA expression vector into At T-20 cells, we observed that m RNA expression of PTTG, VEGF, TGF-β and MMP-9 were significantly suppressed in interference groups. There was no statistical significance in MMP-2, MMP-15 and p53. 3. At T-20 cells treated with TIMP-1 proteins: Higher concentration of TIMP-1 not only can inhibit MT1-MMP(MMP-14) expression in At T-20 cells but also down-regulatedexpression of VEGF, TGF-β and PTTG in m RNA level. Low concentration of TIMP-1can inhibit the expression of MT1-MMP(MMP-14) protein, but there were no significant changes in MMP-2. while the high concentration of TIMP-1 could inhibit MMP-2, but MT1-MMP(MMP-14) inhibitory effect is dramatic. 4. Invasion capacity assay: At T-20 Cells were treated with 10 μmol/m L and 50 μmol/m L TIMP-1 resptectively. After treatment, we found that with the increasing of TIMP-1 concentration and prolonged the time, the invasion capacity decreased obviously. ConclusionMT1-MMP(MMP-14) was involved in the regulation of PTTG and VEGF and play an important role in invasiveness of pituitary adenoma. TIMPs can inhibit the MT1-MMP(MMP-14) regulation of PTTG and VEGF. The results showed that MT1-MMP(MMP-14) may be a potential targeted molecular for the treatment of invasive pituitary adenomas.