Influence Of Inhibiting MMP-26 Expression By RNA Interference In A549 Cell

Extracellular matrix is important as a scaffold for tissue structureand also as an environment regulating cell function in tissues. MatrixMetalloproteinases (MMPs) are a family of zincmetallo-endopeptidases secreted by cells, and are responsible for muchof the turnover of matrix components. The MMPs are involved in awide range of proteolytic events, in normal and pathologicalcircumstances. Normal physiological roles for the MMPs includeneurite growth, cell migration, bone elongation, wound healing,angiogenesis, ovulation, sperm maturation, uterine involution,menstruation, enamel formation, antigen processing and presentation,mammary gland development, hair follicle development, and embryoimplantation. Pathological processes involving MMPs include tumorgrowth and migration, fibrosis, arthritis, glaucoma, lupus scleroderma,cirrhosis, multiple sclerosis, aortic aneurysms, infertility, and manymore diseases. It is fair to say that a proteolytic event is a key to a widerange of biological processes, including tissue remodeling and alsomodification or release of biological factors. The MMPs areover-expressed during invasion and metastasis and degradeextracellular matrix, lead to invasion and metastasis of tumor. So, oneof the important future prospects is to address the inhibitors that actonly on specific MMPs for the treatment of cancer.The smallest MMP identified to date, MMP-26 is of261-amino-acid sequence. It includes only the minimal characteristicfeatures of the MMP family: a signal peptide, a prodomain and acatalytic domain. However, it lacks the hinge region andhemopexin-domain present in most MMPs. MMP-26 is not onlyexpressed in normal uterus, placenta, pellicula, glandular epithelialcells, vascular endothelial and endometrial stromal cells, but is widelyexpressed in malignant tumors from different sources as well as indiverse tumor cell lines.MMP-26 is a potent enzyme with a wider substrate specificitythan other MMPs. MMP-26 efficiently cleaves fibrinogen andextracellular matrix proteins, including fibronectin, vitronectin anddenatured collagen. MMP-26 is able to activate progelatinase B, TNF-αconvertase, estrogen receptor and inactive serpin. Matrilysin-2 wasexpressed in Escherichia coli, and, after purification and refolding, therecombinant protein was found to degrade synthetic substratescommonly used for assaying MMPs. Furthermore, this proteinhydrolyzed type IV collagen, fibronectin, fibrinogen, and gelatin.MMP-26 plays a role in endometrial and placental tissue remodeling,and promotes tumor invasion and metastasis.RNA interference (RNAi) refers to the introduction ofhomologous double stranded RNA (dsRNA) to specifically target agene's product, resulting in null or hypomorphic phenotypes. Thispowerful technology has been widely employed to manipulate geneexpression in mammalian and human cells, elucidate signal pathways,identify gene functions in a whole-genome scale and developRNAi-based drugs for various diseases, especially in cancers therapy.RNAi. Over-expression of oncogenes is involved in uncontrolledgrowth and proliferation of cells, the specific suppression of oncogeneactivities can reverse the phenotype of tumor cells and control theirgrowth and proliferation. It has been reported that RNAi-basedapproach is a very useful tool for inhibiting human breast cancer cellmigration in vitro and suppressing the invasion and metastasis ofgliomas in vivo. It is optimism the cure of cancer by RNAi seems notfar from us. In this study, we investigate influence of inhibitingMMP-26 expression by RNAi in A549 Cell to inspect the feasibility ofMMP-26 being a carcinoma metastasis marker and therapeutic targetgene.We have carried out these works:1. Following the principles of siRNA design, we designed 3 pairsof shRNAs to MMP-26 mRNA and constructedpSUPERIOR.puro-MMP-26 plasmids. We also designed andconstructed pSUPERIOR.puro-GFP and pSUPERIOR.puro-β-actin ascontrols.2. The combinant plasmids were transfected into A549 cells withcationic lipid complexes Lipofectamine and then stable transfectant ofA549 cells aquired using puromycin agent. Finally, blocked expressionof MMP-26 in the stable transfectant was detected by RT-PCR andwestern blot analysis.3. No difference was detected after MMP-26 has been silenced inA549 cells about morphology. However, Cells treated by RNAi showeda decreased cell migration by wound assay.Conclusion1. Significantly degression of MMP-26 mRNA and protein level inpSUPERIOR.puro-MMP-26-A,B,C vectors transfected A549 celldemonstrated MMP-26 shRNA were well desigened and could blockMMP-26 expression in A549.2. Tumor cell bionomics assay indicated silencing MMP-26 showlittle notable effect about morphology, but can significantly inhibitA549 migration in vitro. This also demonstrated MMP-26 wascorrelated with A549 invasion and metastasis.