MEPE Protein Study On Prostate Cancer Cell Migration Mechanisms

Objective: The thesis is to study the effect of sugar-phosphate extracellular matrix protein MEPE expression levels in prostate cancer cells to bone metastasis of prostate cancer, and family of small G protein signaling pathway PI3 K / Rac1 / PAK1 involved in signal transduction mechanisms MEPE, providing experimental evidence for elucidating mechanisms of malignant tumor invasion and metastasis.Methods: Construct the adenovirus packaging plasmid p Yr-ads-4-m MEPE and p Yr-ads-4-si MEPE, packaged in HEK293 cells forming adenovirus particles. Transfect normal prostate epithelial cell line RWPE1 and prostate cancer cell lines PC3 separately, while transfect empty vector as a control cell lines. By classical scratch test, Transwell invasion / migration assay, Immunofluorescence experiments, compare the difference artificially upward or downward invasion / migration between MEPE protein expression levels of cell line and its corresponding empty vector cell lines. Further adoption of immunoblotting and fluorogenic Quantitative PCR test, we check MEPE protein expression levels of various cell lines of different signaling pathways PI3 K / Rac1 / PAK1 protein levels to see whether the signal has changed.Results: 1 After enzyme digestion and sequencing, pYr-ads-4-mMEPE and pYr-ads-4-siMEPE recombinant plasmid was constructed successfully, immunofluorescence and Western blot analysis of MEPE protein expression level increased in RWPE1 cells transfected with r Ad-4-m MEPE, the expression level decreased in MEPE protein of PC3 cells transfected with r Ad-4-si MEPE, and the expression level of MEPE protein was transfected into the corresponding empty vector cells and wild cell lines, indicating the successful construction of the expression of adenovirus plasmid in eukaryotic cells. After enzyme digestion and sequencing, p Yr-ads-4-m MEPE and p Yr-ads-4-si MEPE recombinant plasmid was constructed successfully, Transfected with r Ad-4-si MEPE of PC3 cell lines MEPE protein expression down-regulated, whereas the corresponding empty vector transfected cell lines MEPE protein levels consistent with the wild-type cell lines, indicating that adenovirus plasmid can be successfully expressed in eukaryotic cells. 2 Transfected with r Ad-4-m MEPE of RWPE1 cell lines, transfected with r Ad-4-si MEPE of PC3 cell lines as well as its control cell scratch test cell lines 0h, 48 h, respectively mobility are 83%、56%, vector control cell migration rate 68%、79%,high MEPE protein expression levels of cell lines showed the enhancement of the migration ability significantly. 3Transwell experiment showed that RWPE1 cells transfected with r Ad-4-m MEPE mobility than control cells increased by 15%, PC3 cells were transfected with r Ad-4-si MEPE mobility than control cells decreased by 23%.The experimental results showed that the expression of MEPE protein with high levels of cell migration ability was significantly enhanced.4 Western blot analysis showed that, PC3 cells transfected with r Ad-4-m MEPE RWPE1 was transfected into r Ad-4-si MEPE cell line, and the signal protein in PI3K/Rac1/PAK1 cell line compared with the expression level of MEPE expression level changed. With the higher level of MEPE protein expression, the expression level of PI3 K and Rac1 increased, while the expression level of PAK1 decreased; PC3 cells expressed lower levels of r Ad-4-si MEPE protein in MEPE transfected RWPE1 cells and empty vector transfected, PI3 K and the expression level of Rac1 was lower, and PAK1 and actin expression levels showed no significant change.Conclusion: 1. MEPE protein expression level affect the invasion/ migration ability of prostate cancer cell. 2.MEPE proteins involved in prostate cancer cell migration activity associated with PI3 K / Rac1 / PAK1 signaling pathways.