Construction Of TIMP-3 Gene Eukaryotic Expression Vector And Study On Its Function In Human Prostate Cancer Cell Line

Tumor invasion and metastasis is an initiative process,and it needs topenetrate a series of natural barriers such as basement membrane andextracellular matrix(ECM). Matrix metalloproteinase (MMPs)is animportant matrix catabolic enzyme, and it plays great role in tumor growth,invasion , angiogenesis and metastasis. TIMPs family is coded proteinsby multigene family, and is a specific inhibitor of MMPs. TIMP-3 is a newmember of TIMPs family, existing in ECM, an important role in tumorgrowth, invasion and metastasis.ObjectiveTo construct an eukaryotic expression vector carrying TIMP-3 gene ,and transfect PC-3M cell line stably. Investigate the relationship betweenTIMP-3 and tumor adherence, immigration , invasion and apoptosis. Andlay a good foundation for the future study of TIMP-3 and tumor.Methods1.Acquired TIMP-3 gene from human placenta by RT-PCR, andcloned it into eukaryotic expression vector, pcDNA3.1(+).2. Transfectedinto PC-3M cells, and determined its expression in PC-3M cells byRT-PCR and Westenblot. 3.Observed the proliferation and invasion abilitiesof transfected PC-3M cells by following indexes: (1)growth curve and MTTchromatometry used to observe the effect of TIMP-3 on proliferation ability.(2)adherence assay used to observe the effect of TIMP-3 on adherenceability. (3)immigration assay used to observe the effect of TIMP-3 onimmigration ability. (4)monolayer cell invasion assay and Transwellinvasion assay used to o observe the effect of TIMP-3 on invasion ability.(5)AO/EB staining and flow cytometry used to observe the effect ofTIMP-3 on cell apoptosis.Results1. Amplificated TIMP-3 cDNA by TIMP-3 specific primer andelectrophoregram indicated an obvious and high-specific strap of 633 bp.2. pMD18-T-TIMP-3 recombinant plasmid released a TIMP-3specific strap of 633bp after enzyme cut by restriction enzyme EcoRI andXbaI. And it is identified by Shanghai Sangon bioengineering company.3. pcDNA3.1-TIMP-3 recombinant plasmid released a TIMP-3specific strap of 633bp after enzyme cut by restriction enzyme EcoRI andXbaI.4. Recombinant plasmid was transfected into PC-3M cells byliposome, and transfected PC-3M cells successfully. The transfecte cellswere screened by G418. We acquired stably transfected PC-3M cells withpcDNA3.1 empty plasmid, called empty plasmid group, and pcDNA3.1recombinant plasmid with TIMP-3, called TIMP-3 group. Assessed thehigh expression level of TIMP-3 in PC-3M cells by RT-PCR andWesternblot.5. Growth curve and MTT chromatometry indicated that theproliferation ability of TIMP-3 group cells were significantly lower thanthose of untransfected group and empty plasmid group(p<0.01).6. Adherence assay indicated that the adherence ability of TIMP-3group cells were significantly lower than those of untransfected group andempty plasmid group(p<0.01).7. Immigration assay indicated that the immigration ability ofTIMP-3 group cells were lower than those of untransfected group andempty plasmid group.8. Monolayer cell invasion assay and Transwell invasion assayindicated that the invasion cells number and invasion index of TIMP-3group cells were lower than those of untransfected group and emptyplasmid group(P<0.01).9. AO/EB staining and flow cytometry indicated that the apoptosiscells of TIMP-3 group increased, and its apoptosis rate was 17.91±2.09%.Discussion1. Construction of pcDNA3.1-TIMP-3 eukaryotic expression vectorTIMP-3 is a new member of TIMPs family, and its role in tumorgeneration and development is under further research. We extractedmRNA from placenta, and amplificated the TIMP-3 section by RT-PCR.We used highly efficient eukaryotic expression vector with CMVpromoter, pcDNA3.1 plasmid. Constructed pcDNA3.1-TIMP-3eukaryotic expression vector with TIMP-3. This construction is good forus to import the TIMP-3 gene into PC-3M cells by transfection method,and to let TIMP-3 protein express in PC-3M cells stably.2. TIMP-3 expression in PC-3M cellsTransfected plasmid with TIMP-3 gene into PC-3M cells, andtransfected empty plasmid as control. Transfected cells were screened byG418, so we acquired positive clones. Transferred these clones with trypsinasefilter paper, and continued to cultivate and passage. We determined itsexpression in PC-3M cells by RT-PCR and Westernblot. The PC-3M cellsthat transfected stably made for the future study on the effect of TIMP-3 ontumor invasion and metastasis.3. Effect of TIMP-3 on the invasion ability of PC-3M cellsFirst of all, we determined the cell activities by growth curve and MTTchromatometry. And studied the effect of TIMP-3 in the growth andproliferation process. MTT chromatometry is a more mature indexreflecting the cell proliferation ability. The consequence manifested thatthat TIMP-3 had inhibitory effect on tumor cells proliferation. Secondly,we determined the cell adherence and immigration activity by adherenceand immigration assay. The results indicated that the adherence andimmigration ability of TIMP-3 group was lower significantly thanuntransfected group and empty plasmid group. TIMP-3 can inhibited theadherence and immigration of tumor cells. TIMPs can inhibit the ability ofMMPs, so make the tumor cells lose the ability to degrade the ECM, andprotect EM and ECM, prevent endothelial cells from exgrafting and lumensforming. Thirdly, monolayer cell invasion assay and Transwell invasionassay were used to o observe the effect of TIMP-3 on invasion ability.Transwell invasion model in vitro is a more ideal experimental means toobserve the invasion behavior of tumor cells. The results indicated theinvasion cells and index of TIMP-3 group were lower than those of theother groups(P<0.01). These assays explained that TIMP-3 inhibited theinvasion character of tumor cells, and it took part in the invasion process oftumor.4. Effect of TIMP-3 on apoptosis of PC-3M cellsAO/EB staining showed that there were more apoptosis cells in TIMP-3group. Viable apoptotic cell (VA), nuclear chromatin is green and pyknosisstate or round bead in shape. Non-viable apoptotic cell (NVA), nuclearchromatin is orange and pyknosis state or round bead in shape. Flowcytometry manifested that TIMP-3 group cells appeared a hypodiploid peak,the apoptosis rate was 18.4%. The two experiments indicated that TIMP-3had a certain apoptosis inducing effect.Conclusions1. Amplified TIMP-3 cDNA from human placenta successfully byRT-PCR.2. Cloned TIMP-3 into pMD18-T plasmid, and constructed eukaryoticexpression vector with TIMP-3 successfully.3. pcDNA3.1 recombinant plasmid with TIMP-3 was transfected intoPC-3M cells by liposome, and the transfected cells were screened by G418.We acquired stably transfected PC-3M cells with pcDNA3.1 empty plasmidsuccessfully.4. RT-PCR and Western blot proved the high expression level ofTIMP-3 in PC-3M cells.5. The growth and proliferation ability of PC-3M cells with TIMP-3was inhibited. That indicated that TIMP-3 had the ability to inhibit theproliferation of PC-3M cells.6. TIMP-3 had a certain apoptosis inducing effect on PC-3M cells.7. The adherence, immigration and invasion abilities of PC-3M cellswith TIMP-3 cells lowered significantly. We concluded that TIMP-3 couldinhibit the invasion and metastasis of prostatic carcinoma.