Expression Of MT2-MMP In Clear Cell Renal Cell Carcinoma And Mechanism Of Action In Tumor Proliferation And Invasion

Renal cell carcinoma(RCC)is one of the most common cancer with more than 140,000 deaths per year and 350,000 new cases diagnosed in 2013 worldwide,and the clear cell renal cell carcinoma(ccRCC)is the most common type of renal cell carcinoma.The increased use of imaging techniques were helpful to finding renal masses incidentally,but more than 17%of all RCC have distant metastases at the time of diagnosis.There is less significant efficacy of chemotherapy in metastatic renal cell carcinoma.Recently,patients were treated with systemic therapy based on immune modulators,but the outcomes were only slightly improved.Therefore,novel therapeutic approaches are required to improve the prognosis of pancreatic cancer patients.Matrix metalloproteinases(MMPs)are a family of enzymes that involved in extracellular matrix(ECM)destruction,therefore contributing to tumor invasion and metastasis.MMPs consist of more than 25 different members,which can be classified into five subgroups.Among those subgroups,membrane-type MMPs(MT-MMPs)are a specific subtype,conducting pericellular proteolysis,which is supposed to be important step for the peri-cancerous tissue remodeling.MT2-MMP(MMP-15)was originally isolated from a human lung cDNA library,which composed of 669 amino acids that are encoded in 3530 bp of the full length gene.Recent studies indicated that MT2-MMPs were involved in the progression of multiple human cancers such as gastric cancer,liver cancer,lung cancer,ovarian cancer,esophageal cancer and breast cancer.However,the role of MT2-MMP in human RCC still remains unknown.In this study we tested the expression level of MT2-MMP in clear cell renal cell carcinoma tissues and cells,analyzed the correlation of MT2-MMP level and renal cell carcinoma pathology grade,clinical stage and prognosis.Then,we researched the effects of MT2-MMP in tumor cell invasion,metastasis and proliferation.Finally,we try to test the possible signal pathway to explore the role of the possible mechanism,which may provide a new marker and target for the diagnosis and treatment of renal cell carcinoma.Part 1 Expression of membrane type-2 matrix metalloproteinase in clear cell renal cell carcinoma and its clinical significanceObjective:To study the expression of membrane type-2 matrix metalloproteinase(MT2-MMP)in clear cell renal cell carcinoma and its relationship with the clinical features and prognosis of clear cell renal cell carcinoma.Methods:The expression of MT2-MMP in malignant tumors and its relationship with prognosis were analyzed by meta-analysis.Then the expression of MT2-MMP in tumors and peritumoral tissues of patients with clear cell renal cell carcinoma was detected by qRT-PCR.And it was detected by immunohistochemist in clear cell renal cell carcinoma tissues and peritumoral tissues in 90 cases of clear cell renal cell carcinoma microarrays.Chi-square test was utilized to analyze the expression differences of MT2-MMP in clear cell renal cell carcinoma and peritumoral tissues and the relationship with tumor size,stage,grade and age,gender.Kaplan-Meier method and Log-rank test and multivariate Cox model fitting method was used to test the correlation between the expression of MT2-MMP and prognosis of patients.Univariate and multivariate Cox regression models were used to analysis the the risk of death in patients with different clinical parameters including the expression of MT2-MMP.Pearson linear correlation analysis of MT2-MMP expression and microvessel density.Results:Meta-analysis showed that MT2-MMP was highly expressed in many tumors,and the expression of MT2-MMP was negatively correlated with prognosis(P<0.05).The expression of MT2-MMP mRNA in clear cell renal cell carcinoma was higher than that in adjacent tissues(P<0.05).The expression of MT2-MMP in clear cell renal cell carcinoma and peritumoral tissues were 217.4±24.8 and 153.6±30.7,the difference was statistically significant(P<0.05).There were significant differences in stages and histological differentiation between high MT2-MMP expression group and low MT2-MMP expression group(P<0.05).There were no significant differences in age,sex and location of tumors between different MT2-MMP expression groups(P>0.05).MT2-MMP was a risk factor of poor overall-survival,and the H-score of MT2-MMP>216.0 was independent risk factor of clear cell renal cell carcinoma in patients with early death after surgery(P<0.05).Meanwhile,the expression of MT2-MMP was positively correlated with microvessel density(r=0.566,P<0.05).Conclusion:MT2-MMP plays an important role in the occurrence and development of clear cell renal cell carcinoma,and it can be used as prognostic indicators in patients with clear cell renal cell carcinoma.The higher the MT2-MMP expression is,the worse the prognosis is.Part 2 The role of MT2-MMP in the biological behavior of clear cell renal cell carcinomaObjective:To detect and compare the expression of MT2-MMP in three different kinds of clear cell renal cell carcinoma cells and normal renal cells,and to investigate the effect of MT2-MMP on proliferation and invasion of clear cell renal cell carcinoma.Methods:The real-time quantitative reverse transcriptase polymerase chain reaction(qRT-PCR)and Western blot was used to examine and compare the different expression level of MT2-MMP and MT1-MMP in different human renal cancer cells and normal renal cells.MT2-MMP downregulated lentiviral vector was constructed and transfected into ACHN and 786-0 cell lines.Cell invasion assay,CCK-8 cell proliferation assay,flow cytometry and cell adhesion assay were used to observe the function of tumor cells under the downregulated expression of MT2-MMP.Meanwhile,renal cancer cells with down-regulation of MT2-MMP expression and control cells were injected subcutaneously into nude mice.The effects of down-regulation of MT2-MMP expression on tumor growth and microangiogenesis were observed by measuring tumor size and immunohistochemical staining.Results:The MT1-MMP and MT2-MMP expression level were notably elevated in renal cancer cells(786-0,ACHN and OS-RC-2)versus human embryonic kidney cells(293T),and the expression level was different among these three kinds of cells(P<0.05).The expression of MT2-MMP and MT1-MMP in different renal cell lines was different,but there was no significant correlation(P<0.05).Adhesion and invasive activities of ACHN and 786-0 cells were suppressed with MT2-MMP shRNA transfection.MT2-MMP shRNA transfection significantly declined the proportion of cells in phase S and G2/M(P<0.05).Animal experiments showed that down-regulation of MT2-MMP expression could significantly inhibit the growth of tumor cells and tumor microangiogenesis(P<0.05).Conclusion:MT2-MMP is highly expressed in different clear cell renal cell carcinoma cell,which is independent of MT1-MMP.The decreased expression of MT2-MMP can inhibit the proliferation and invasion ability of clear cell renal cell carcinoma cells.Part 3 Screening differentially expressed genes related to proliferation and invasion of renal cell carcinoma with shMT2-MMP gene stable transfection by cDNA microarrayObjective:The differentially expressed genes related to proliferation and invasion of clear cell renal cell carcinoma cell lines stably transfected with shMT2-MMP gene were screened by cDNA microarray.Methods:Total RNA of clear cell renal cell carcinoma cell lines(ACHN and 786-0)stably transfected with Lv-shMT2-MMP gene and control group(ACHN and 786-0)stably transfected with Lv-NC were extracted and differentially expressed genes were screened by cDNA microarray.Results:Compared with the control group,3368 co-differentially expressed genes were screened out in clear cell renal cell carcinoma cell lines with down-regulated MT2-MMP expression,of which 1851 were co-expressing up-regulated genes and 1517 were co-expressing down-regulated genes.The analysis of gene function showed that these differentially expressed genes involved cell proliferation,differentiation,extracellular matrix tissue,cell adhesion,immune response,cell cycle and so on.It is related to many cancer-related signaling pathways.5 differentially expressed genes related to tumors were further screened out from all differentially expressed genes.Quantitative RT-PCR analysis showed that the variation trend of each gene was consistent with that of gene chipConclusion:MT2-MMP may promote the growth and metastasis of clear cell renal cell carcinoma by affecting a variety of genes related to the proliferation and invasion of tumors.