The Effects And Mechanisms Of Cytoplasmic Macrophage Colony-Stimulating Factor On The Proliferation, Migration And Invasion Of HeLa Cells

OBJECTIVES The aim is to establish a cell line that stably expresses cytoplasmic M-CSF, and to explore the effects and mechanisms of cytoplasmic M-CSF on the proliferation, migration and invasion of HeLa cells.METHODS: Both pCMV/cyto/myc vector and pCMV/cyto/myc-M-CSF vector was trasfected into HeLa-cell by trasfectaimine. After screening by G418, the positive clones were amplified and comfirmed by RT-PCR, Western blot and immunocytochemistry. The effect of cytoplasmic M-CSF on the proliferation of HeLa cells were analyzed by cell conuting, MTT and antisense oligonucleotides. The migration and invasion of cell was measured by in vitro Transwell assay and Matrigel-coated polycarbonate filters. The expression of cyclinE, cyclinD1/2 /3, CDK2/4 /6, Rac1, and matrix metalloproteinase 2 and 9 (MMP2/9) were assayed by semiquantitative RT-PCR. And expression of bothα-tubulin and cdc42 were displayed by immunofluorescence. The activity of MMP2 was detected by gelatin zymography.RESULTS The results from RT-PCR, Western blot and immunocytochemistry showed that high levels of both M-CSF mRNA and M-CSF protein were expressed and localized to the cytosol in M-CSF-transfected HeLa cells(referred as to HeLa-M cell) , Which suggest that a cell line that highly expresses cytoplasmic M-CSF was successfully established. Our result indicated that HeLa-M cell had a larger volumn, faster growth rate and shorter doubling time than either pCMV/cyto/myc transfected HeLa cells (referred as to HeLa-C cell) or untransfected HeLa cells (referred as to HeLa cell). M-CSF-specific antisense oligonucleoside significantly inhibited HeLa-M cell proliferation and had little effect on either HeLa-C cell or HeLa-C cell growth, which suggest that the faster growth of HeLa-M cell is associated in the expression of cytosolic M-CSF. Cytoplasmic M-CSFs up-regulated both the expression of cyclinE, cyclinD1 and cyclinD3, CDK2, CDK 4 and CDK6, aRho GTPase ralative protein (Rac1), cdc42 and MMP2, but had little effect on expression of MMP9 and cyclin D2. Forthermore, Cytoplasmic M-CSFs induced the rearrangement of theα-tubulin in HeLa cells and significantly promoted the migration and invasion of HeLa cells in vitro.CONCLUSION A cell line that highly expresses cytoplasmic M-CSF was established. Cytoplasmic M-CSFs up-regulate the expression of cyclinE, cyclinD1 and cyclinD3, CDK2, CDK 4 and CDK6 and induces the proliferation of HeLa cells. Cytoplasmic M-CSFs up-regulate the expression of Rac1 and cdc42 and cause the rearrangement of theα-tubulin in HeLa cells. Furthermore Cytoplasmic M-CSFs increase both the expression and activity of MMP2 and promote the migration and invasion of HeLa cell in vitro. But cytoplasmic M-CSFs have little effect on expression of cyclin D2 and MMP9.