| Objective:To construct the eukaryotic expression vector encoding protein of rat transient receptor potential M8 and investigate its effect on proliferation and migration of prostate cancer PC-3 cells.Methods:The full-length open read frame of rat TRPM8 was obtained and amplified by means of PCR using the rat cDNA library as the template. Target gene was amplified and digested by restricted enzyme BamH I and Xho I, then ligated into eukaryotic expression vector pcDNA3. The pcDNA3-based expression vector encoding TRPM8 (pcDNA3/TRPM8) was analysised by sequencing, RT-PCR using the expressing vector, and Western blot after transiently transfected into COS-7. Then pcDNA3/TRPM8 and pcDNA3 were transfected into androgen-independent prostate cancer PC-3 cells respectively. Cells stably expressing TRPM8 (PC-3-TRPM8) and empty vector (PC-3-Vector) were selected with G418 at the concentration of 500mg/L. The expression and location of TRPM8 in PC-3 cells was investigated by immunofluo-rescence stain and Western blot, while the function of which was evaluated through Ca2+ imaging. Afterwards, the cell cycle distribution of both cells was analysised by flowcytometry for the effect of TRPM8 on proliferation of PC-3 cell. Furthermore, the effects of TRPM8 on apoptosis-resistant ability and migration of PC-3 cells were also analysised by flowcytometry for apoptosis rate and scratching assay. Results:The eukaryotic expression vector of pcDNA3/TRPM8 was successfully constructed and the TRPM8 mRNA and protein were detected in COS-7 cells transiently transfected with pcDNA3/TRPM8 by RT-PCR and Western blot, respectively. We also can detect the expression of TRPM8 in PC-3-TRPM8 cells through immunofluo-rescence staining and Western blot using specific anti-TRPM8 antibody while there were no expression of TRPM8 in PC-3-Vector cells. In Ca2+ imagining, menthol at the concentration of 100uM can induce increased concentration of cytoplasmic Ca2+in PC-3-TRPM8 cells in both Ca2+-present solution and Ca2+-free solution, with the former higher than the latter. In PC-3-Vector cells the same results were obtained, whereas the cytoplasmic concentrations in both solutions were significantly lower than the corresponding ones in PC-3-TRPM8 cells. Immunofluo-rescence staining suggested that TRPM8 was expressed both on cell membrane and intracellularly. Cell cycle analysis indicated that percent of cell in G0/G1 stage of PC-3,PC-3-Vector, and PC-3-TRPM8 were 52.62%,56.98%, and 71.99% respectively (p<0.05). After incubation in RPMI-1640 containing 1% FBS for 48h, apoptosis rate of PC-3, PC-3-Vector, and PC-3-TRPM8 were 5.25%,5.51%, and 12.4% respectively (p<0.01). In the scratch motility assay,24h after scratching, the migration rate of PC-3, PC-3-Vector, an PC-3-TRPM8 were 100%,92.59%, and 58.73%(p<0.01). Western blot analysis indicated that compared with PC-3-Vector, protein expression of Cdk4, Cdk6, and activation of FAK were significantly decreased in PC-3-TRPM8.Conclusion:The recombinant vector pcDNA3/TRPM8 was successfully constructed and could be used for the expression of rat TRPM8 protein in eukaryotic cells, providing a research foundation for physiologic and pathobiologic role of TRPM8 channel in prostate cancer. Immunocytochemistry and Ca2+ imaging analysis revealed the overexpression of functional TRPM8 channel on both endoplasmic reticulum and plasma membrane of PC-3-TRPM8 cells. Menthol at the concentration of 100uM could activate these channels and induce the increased Ca2+ concerntration through both Ca2+ influx and Ca2+ release from intracellular Ca2+ pool. In PC-3 cells, TRPM8 showed negtive effects on cell proliferation, migration, as well as ability of anti-apoptosis, rather than a pro-proliferation effect. These effects might be caused by the disturbance of intracellular Ca2+ homeostasis. Hence TRPM8 channel might act as a potential target for dealing with androgen-independent prostate cancer in the future. |
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