Correlation Between β3GnT8 And The Transcription Factor C-jun And Their Function On The Migration And Invasion In Colon Cancer Cells

Objective:To investigate the transcriptional regulation of glycosyltransferase β3Gn T8 by transcription factor c-jun and the research on transcription factor c-jun and β3Gn T8 molecular mechanisms in colon cancer invasion and metastasis.Methods:(1)Western-blot analysis to examine the protein expression of c-jun and β3Gn T8 in the three colon cancer cells(SW480,SW620,Lo Vo),and Transwell analysis to examine the potential ability of migration and invasion in SW480,Lo Vo colon cancer cells on 24h;(2)chromatin immunoprecipitation assay(Ch IP) assessment to detect the transcriptional activation of β3Gn T8 by the transcription factor c-jun in SW480 and Lo Vo;(3)Exogenous c-jun sense plasmid vector and its empty vector were transfected into SW480 colon cancer cell and c-jun sh RNA interference vector and its control vector were transfected into Lo Vo colon cancer cell by lipofectamine;(4)The GFP fluorescence protein expressed in transfected cells were observed with fluorescence microscope.Real time-PCR and Western-blot were used to analysis the m RNA and protein expression level of c-jun,β3Gn T8,CD147 and some associated factors after upregulation c-jun in SW480 and knowdown c-jun in Lo Vo;(5)FCM was used to detect the effect of c-jun on the level of polylactosamine.(6)After c-jun was upregulated in SW480 cell and downregulated in Lo Vo cell,Transwell migration and invasion analysis and cell scratch repair assessment to examine the change of potential of migration and invasion in the two groups;(7)select the effective si RNA fragment of β3Gn T8 and β3Gn T2 with real time-PCR;(8)co-transfect c-jun sense vector and the effective si RNA fragment of β3Gn T8,β3Gn T2 in SW480 by lipofectamine,RT-PCR analysis the expression of β3Gn T8,and Transwell assay detect the migration ability of these groups.Results:(1)western-blot results indicated thatthe protein expression of c-jun and β3Gn T8 of these three colon cancer cells is SW480,SW620,Lo Vo in order from low to high,and transwell analysis showed that Lo Vo cellhas the highest invasive and migratory capabilities,SW480 cell was very low in 24 h,so in next experiment 48 h was selected;(2) Ch IP results shows that β3Gn T8 promoter has c-jun binding sites;(3)c-jun sense vector and silence vector and their empty vector groups were transfected into SW480 and Lo Vo successfully via observed through fluorescence microscope,Real time-PCR and Western-blot analysis show that the expression of β3Gn T8 and HG-CD147 were increased in SW480 cell while c-jun was up-regulated and decreased in Lo Vo while c-jun wan down-regulated compared with control groups;(4)flow cytometor(FCM) results showthe level of polylactosamine in cell surface is increased in SW480 cell transfected c-jun sense vector and decreased in Lo Vo while c-jun wan down-regulated compared with control groups;(5) the results of cells wound healing assay shows thatc-jun can promote theability of cell migration by modulating the N-glycosylated forms of HG-CD147 via β3Gn T8;(6)Transwell migration and invasion analysis shows that c-jun promote the cells invasion and migration ability by modulating the N-glycosylated forms of HG-CD147 via β3Gn T8;(7)the effective si RNA fragments(siβ3Gn T8-1754 and siβ3Gn T2-1336) were got by Real time-PCR,and co-transfected with c-jun to SW480 cell, RT-PCR analysis reflected that the expression of β3Gn T8 was increased compared to the si RNA groups and decreased compared to the c-jun over-expression group,and so as transwell results.Conclusion:(1)the protein expression of c-jun and β3Gn T8 of these three colon cancer cells is SW480,SW620,Lo Vo in order from low to high;(2) transcription factor c-jun may alter the glycosylation of CD147 by regulating the expression of glycosyltransferase β3Gn T8 and further to promote the potential of cell migration and invasion.