PRL-3 Up-regulates VEGF,VEGF-C Expression And Has A Positive Correlation With MVD,LVD In Lung Cancer

IntroductionPhosphatase of regenerating liver-3 (PRL-3) is a tyrosine phosphatase. Among normal adult human tissues, PRL-3 mRNA is expressed primarily in skeletal muscle and heart, with lower expression levels in pancreas, spleen, lung and testis[1,2] but little is currently known about its physiological role in these tissues. A role in cardiac functions is indicated by the observation that PRL-3 appears to be involved in the modulation of intracellular calcium mobilization in response to AngiotensinⅡ[1]. In the last few years, evidence has accumulated for the association of PRL-3 with oncogenic states and several indications have linked its expression to human colon cancer progression and metastasis. The first data came from the analysis of global gene-expression profiling of metastatic colorectal cancers (CRC) which showed that PRL-3 is selectively over- -expressed in metastatic CRC cells with lower levels in nonmetastatic tumors and normal colorectal epithelium [3]. Subsequently, many scholars found that PRL-3 was high expression of in many tumors, such as breast cancer, ovarian cancer, intestinal cancer, gliomas and stomach cancer, and was closely related to the metastasis [4-8].PRL-3 expression has also been reported in a Hodgkin's lymphoma cell line[9], melanomas, liver, ovarian, breast and gastric cancers where its presence seems to have an important role in the acquisition of metastatic potential of tumor cells [10,11]. The substrates and molecular mechanisms of action of the PRLs have remained elusive. Recent findings indicate that PRLs may function in regulating cell adhesion structures to effect epithelial-mesenchymal transition. The identification of PRL substrates is key to understanding their roles in cancer progression and exploiting their potential as exciting new therapeutic targets for cancer treatment.VEGF is an important mediator that promotes blood vessel formation and increases the proliferation and migration of vascular endothelial cells. VEGF-C, which belongs to the VEGF family, can induce lymphatic endothelial cell proliferation after binding with its receptor VEGFR-3, and this stimulates lymphatic vessel formation. VEGF and VEGF-C play important roles in angiogenesis and lymphangiogenesis in tumors.Our previous study showed that high PRL-3 expression was associated with high expression of VEGF and VEGF-C. We presume that PRL-3 may up-regulates the expression of VEGF and VEGF-C by ERK phosphorylation.Materials and Methods1,Cell cultureThe cell line used in this study was A549 cell (adenocarcinoma of lung). They were cultured in the Dulbcco's Modifed Eagle Medium (DMEM) containing 10% FBS (Gibco, USA), 100U/ml penicillin, and 100U/ml streptomycin at 37℃with 5% CO2 until 75% confluent. Cells were incubated with DMEM (include repectively anti-PRL antibody 100ng/ml, ERK1/2 inhibitor PD98059 10μmol/ml Cell signaling USA) for 1, 2, 4, 8, 24h.2,Methods2. 1 Western blottingThe cells or tissues were extracted with lysis buffer. The supernatants were centrifuged. The supernatant containing total protein was harvested. Aliquots containing 60μg of proteins were separated on a 12% SDS-polyacrylamide gel and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk, respectively incubated with primary antibodies at 4℃overnight, and followed by each corresponding second antibody at room temperature for 2h. Next, DAB visualization and then densitometric analysis of the bands was performed.2. 2Reverse Transcription-Polymerase Chain Reaction (RT-PCR)Total RNA was isolated from cells in the logarithmic growth phase using TRIZOL (Invitrogen). RT-PCR procedures followed the protocol (takara).2. 3 Immunofluorescence stainingThe cells grown on coverslips were fixed with 4% paraformaldehyde followed by 0.2% Triton X-100, PBS washing, and blocking with nonimmune animal sera at 37℃for 30 min. The cells were incubated with primary antibodies respectively at 4℃overnight, followed by incubation with FITC-labeled secondary antibodies or 0.1μg/μl TRITC-labeled phalloidin at 37℃for 30 min, washing with PBS, incubation with 1μg/μl Hoechst 33342 at 37℃for 30 min, washing with PBS, mounting with 50% glycerine, and observation under a fluorescence microscope.2. 4 In vitro cell growth assays103 cells in 200μl of medium were plated in a 96-well plate and incubated for 24h, 48 h, 72h, 96h. 20μl of 5 mg/ml MTT was added to each well. After a 4 h incubation, the medium containing MTT was removed and replaced with 150μl of dimethyl sulfoxide (DMSO, Sigma) for further incubation for 10 min till the formazan was dissolved. The OD value of each well was measured using a microplate reader (SPECTRA Thermo, USA) with a test wavelength of 490 nm. The wells containing only the medium were used as controls. The proliferation curve was plotted.2.5 Cell migration and invasion assays5×104 cells were trpsinized, washed, resuspended in serum-free DMEM, and placed in the top portion of the chamber.The lower portion of the chamber contained 10% FBS as a chemoattractant. The chambers were incubated at 37℃, 5% CO2 for 6h, followed by washing with PBS, and fixing in 100% methanol, stained with Hematoxylin, photographed, and counted. For the invasion ability assay, precooled serum-free DMEM was mixed with Matrigel (BD Biosciences, USA) (1:7 dilutions). The upper compartments were filled with 100μl of mixture, and the Matrigel was allowed to solidify at room temperature for 3h. Other procedures followed the migration protocol (BD Biosciences, USA). The chambers were incubated for 24h. Assays in which cells were exposed to anti-PRL antibody, PD98059 were done as decribed that both the top and bottom chambers contained anti-PRL antibody and PD98059 throughout the assay.3,Statistical analysisThe statistical package SPSS 13.0 (SPSS incorporated, Chicago) was used for all analyses. Values of P < 0.05 were considered statistically significant.Results1,Blocking the expression PRL-3 on the impact of ERK1/2,p-ERK, VEGF in A549 cells.Blocking PRL-3 expression with anti- PRL-3 antibody down-regulated the expression of VEGF and VEGF-C mRNA and decreased levels of p-ERK, VEGF and VEGF-C proteins. RT-PCR results showed that the expression of mRNA of VEGF-C and VEGF was downregulated. Western results showed that the levels of proteins of VEGF and VEGF-C was decreased after incubation for 4h.However, Blocking PRL-3 expression did not affect the expression of ERK1/2 proteins.2. The effect of inhibiting PRL-3, ERK on the proliferative, migratory, and invasive abilities of A549 cellThe results of the MTT assay showed that the Blocking PRL-3 did not affect A549 cell proliferation in the migration assay, the number of A549 cell in control group that migrate through microporous membrane was 37.47±10.32. Blocking PRL-3 decreased significantly the number of the cells that migrate through microporous membrane. Inhibiting the activity of ERK reduced obviously cells that migrate through microporous membrane. In the invasion assay, the number of A549 cell in control group that invade through Matrigel was. Blocking PRL-3 decreased significantly the number of the cells that invade through Matrigel. Inhibiting the activity of ERK reduced obviously cells.Conclusion1,PRL-3 up-regulations VEGF expression via ERK1/2 phosphoylation in A549 cell; PRL-3 promote migratory and metastasis ability via ERK1/2 phosphoylation .2,PRL-3 can up-regulation VEGF-C expression, but the machanisium is still elusive needing further study。...