S100A4Expression In Human Colorectal Cancer And Its Effect And Mechanism On Proliferation And Migration Of Human Colorectal Cancer Cells

ObjectivesTo investigate the expression of S100A4in human colorectal cancerand its effect and mechanism on proliferation and migration of humancolorectal cancer cells.Methods1. Detection of S100A4expression in human colorectal cancer tissues:The tumor tissues and matched adjacent normal mucosa tissues of fivecolorectal cancer (CRC) patients were provided by the First AffiliatedHospital of Chongqing Medical University, Chongqing, China. S100A4expression was detected by RT-PCR, Western blot and IHC.2. Amplification and identification of Ad-S100A4: The recombinantadenoviruses carrying human S100A4gene (Ad-S100A4) and the controlrecombinant adenoviruses carrying GFP gene (Ad-GFP) were amplified inHEK293cells; Ad-S100A4was used to increase S100A4expression inCRC cells HCT116, SW480and LoVo while Ad-GFP was used as a negative control, then S100A4expression was identified by Western blot.3. The roles of S100A4on proliferation and migration of HCT116,SW480and LoVo cells: the MTT assay and the Transwell assay were usedto detect cell proliferation and migration of HCT116, SW480and LoVocells after infection with Ad-S100A4.4. The effect of S100A4on the PI3K/Akt/mTOR/p70S6K signalingpathway in SW480and LoVo cells: Western blot was used to detect thelevels of p-Akt, p-mTOR and p-p70S6K in SW480and LoVo cells treatedwith Ad-S100A4only or with Ad-S100A4and LY294002or Rapamycin.5. The effect of the PI3K/Akt/mTOR/p70S6K signaling pathway onS100A4-induced proliferation and migration of SW480and LoVo cells: theMTT assay and the Transwell assay were used to detect cell proliferationand migration in SW480and LoVo cells treated with Ad-S100A4andLY294002or Rapamycin.6. The effect of the PI3K/Akt/mTOR/p70S6K signaling pathway onS100A4-induced expression of VEGF and E-cadherin in SW480and LoVocells: Western blot was used to detect the levels of VEGF and E-cadherin inSW480and LoVo cells treated with Ad-S100A4only or with Ad-S100A4and LY294002or rapamycin.Results1. S100A4mRNA and its protein were found in the CRC tissues fromall CRC patients (5/5) by RT-PCR, Western blot and IHC, respectively. S100A4was mainly found in the cytoplasm and cell membrane of tumorcells. In contrast, S100A4mRNA and protein were not found in matchedadjacent normal tissues.2. After infection with Ad-S100A4or Ad-GFP for24,36and48h, theexpression of green flurescent protein (GFP) in HEK293cells weregradually increased and then Ad-S100A4or Ad-GFP was harvested andthen used in our study.The CRC cell lines HCT116, SW480and LoVo cells were infectedwith Ad-S100A4or Ad-GFP for24h and S100A4of their cell lysates weredetected by Western blot. The relative gray values of Ad-S100A4groupwere2.48times,2.07times and2.11times as much those of their Ad-GFPgroup, respectively (P<0.01), suggesting S100A4gene of Ad-S100A4wasexpressed in Ad-S100A4-infected CRC cells.3. MTT assay showed:1) In HCT116cells: The difference of the cellnumber between Ad-S100A4group and Ad-GFP group was not obviousfrom1d to3d (P>0.05);2) In SW480and LoVo cells: The cell number ofAd-S100A4group increased by43.1%and58.4%at2d, increased by56.2%and67.7%at3d, respectively (P<0.05).4. After infection for24h, the difference of the numbers of migratedHCT116cells between Ad-S100A4group and Ad-GFP group was notobvious, but the numbers of migrated SW480and LoVo cells inAd-S100A4group increased by152.7%and88.9%, respectively (P<0.05). 5. The levels of p-Akt, p-mTOR and p-p70S6K increased inAd-S100A4-infected SW480and LoVo cells detected by Western blot.1)In SW480cells: LY294002inhibited S100A4-induced p-Akt, p-mTOR andp-p70S6K as much as58.9%,76.7%and64.1%, respectively, andRapamycin inhibited S100A4-induced p-mTOR and p-p70S6K as much as74.2%and54.8%, respectively (P<0.05).2) In LoVo cells: LY294002inhibited S100A4-induced p-Akt, p-mTOR and p-p70S6K as much as64.8%,67.2%and49.1%, respectively, and Rapamycin inhibitedS100A4-induced p-mTOR and p-p70S6K as much as86.9%and48.3%,respectively (P<0.05).6. Both LY294002and Rapamycin partly suppressed S100A4-inducedproliferation of SW480and LoVo cells.1) In SW480cells:The inhibitionrates of LY294002and Rapamycin were47.8%and43.4%at2d,44.7%and43.1%at3d, respectively (P<0.05).2) In LoVo cells: The inhibitionrates of LY294002and Rapamycin were47.5%and48.2%at2d,50.7%and53.2%at3d, respectively (P<0.05).7. Both LY294002and Rapamycin partly suppressed S100A4-inducedmigration of SW480and LoVo cells.1) In SW480cells: The inhibitionrates of LY294002and Rapamycin were68.2%and63.1%, respectively(P<0.05).2) In LoVo cells: The inhibition rates of LY294002andRapamycin were50.1%and55.6%, respectively (P<0.05).8. The VEGF levels in Ad-S100A4-infected SW480and LoVo cells increased by60.4%and106.9%, E-cadherin levels decreased by72.5%and72.1%, respectively (P<0.01), which were partly abolished by treatmentwith either LY294002or Rapamycin.Conclusion1. S100A4expression is increased in CRC tissues.2. S100A4promotes proliferation and migration of SW480and LoVocells, but does not for HCT116cells.3. S100A4upregulates VEGF expression and downregulatesE-cadherin expression in SW480and LoVo cells.4. S100A4activates the PI3K/Akt/mTOR/p70S6K signaling pathway.5. The activation of the PI3K/Akt/mTOR/p70S6K signaling pathwayby S100A4is involved in the roles of S100A4on SW480and LoVo cells,including promoting cell proliferation and migration, upregulating VEGFand downregulating E-cadherin.