Inhibition Of CD147 Gene Expression Via RNA Interference Reduces Cell Proliferation, Activation, Adhesion, And Migration Activity In The Human Jurkat T-lymphoma Cell Line

ObjectiveTo investigate the effects of siRNA transfection on cell proliferation,cell cycle and the potential of activation, adhesion and transendothelialmigration in human Jurkat T-lymphoma cell line in vitro. To furtheridentify the roles and mechanism of CD147 in the human T-lymphomacell and investigate the feasibility of siRNA-based, CD147 targetingstrategy to treat human T-lymphoma.Methods1. The recombinant plasmid pSUPER/CD147siRNA was stablytransfected into Jurkat T cells applying electroporation, and the changesof the expression of CD147 mRNA and protein in transfected cells wereidentified by semi-quantitive reverse transcriptase-polymerase chainreaction (RT-PCR) and Western Blot, respectively;2. The change of proliferation level of Jurkat cells at 24-, 48- and72h after transfection was detected by MTT assay;3. Flow cytometry (FCM) was used to analyse the cell cycle andCD25 expression of the tranfected and control groups;4. The effects of CD147siRNA on Jurkat cells adhesion avtivity toextracelluar matrix(ECM) in vitro used the fibronectin-coatedflat-bottomed 96-well plates and the adherent cells were stained with0.5% crystal violet, and then the absorbance of the samples was measuredat 570 nm with an ELISA reader;5. The effects of CD147siRNA on transendothelial migration inJurkat cells in vitro were detected by Transwell chamber.Results1. While pSUPER/CD147siRNA1 exhibited no interference effecton the expression of CD147 mRNA and protein, pSUPER/CD147siRNA2resulted in the inhibition of CD147 in the human T-lymphoma cell line by67.12% in C2a and by 66.68% in C2b (p＜0.001). C2a and C2b were usedin the subsequent experiments; 2. Compared to untransfected Jurkat T-cells, at 24-, 48-, and 72hours, the proliferation of C2a- and C2b cells was inhibited to 47.07%and 55.27% (p＜0.01), 50.85% and 54.75% (p＜0.01), and 52.28% and57.21%, respectively (p＜0.01);3. FCM analysis showed that CD147-siRNA increased thefraction of Jurkat cells in S phase (p＜0.001), whereas the proportion ofcells in G1 phase decreased in comparison with untransfected JurkatT-cells (p＜0.001);4. The number of CD25-expressing cells was reduced in C2a- andC2b populations (5.06% and 5.57%, respectively) compared withuntransfected Jurkat T-cells (9.07%) (p＜0.05);5. CD147siRNA transfection significantly downregulatedadhesion activity of Jurkat cells, and binding by C2a- and C2b cellswas decreased by 29.74% and 32.17 % (p＜0.001);6. Compared with 64.5±7.26% of untransfected Jurkat T-cellstransmigrated to the lower chamber, only 14.72±3.74% of C2a- and15.39±5.75% of C2b cells did (p＜0.001).Conclusion1. Stably transfected human Jurkat T-lymphoma cell line withpSUPER/CD147siRNA was established;2. CD147 is correlated with cell proliferation, cell cycle,activation, adhesion and migration of Jurkat cells, and explore thepotential functions of CD147 in the tissue infiltration of human Tlymphoma;3. CD147 might be a new tumor therapy target molecule.