The Effect Of RKIP On Migration Of Cholangiocarcinoma Cell

Cholangiocarcinoma (CCA), a cancer originating from the neoplastictransformation of the biliary epithelium, occurs frequently in hepatobiliarysystem tumor except of liver cancer. CCA is particularly common in themiddle-aged and old people, especially in male. CCA lacked specificsymptoms and signs at early stage and only at later stage extrahepaticcholangiocarcinoma shows typical jaundice. The early visiting rate inintrahepatic and extrahepatic cholangiocarcinoma patients are lower, so theefficacy of the therapy is not satisfacted, that affects the human health and lifeseriously. Such as other tumors, cholangiocarcinoma formation is also need amulti-step processes. It depends on the interaction of the genetic andenvironmental factors. During the tumorigenesis in cholangiocarcinoma,epithelial cells of human bile duct suffer from chronic injury which activatesthe proliferation signal, inactives the anti-proliferation signal, escapes fromthe apoptosis, replicates endless, promotes capillary aberrant clones and so on.At last, this leads to occurrence, invasion and metastasis of CCA.Raf kinase inhibitory protein (RKIP), a member of thephosphatidylethanolamine binding protein (PEBP) family, widely exists indifferent species. RKIP involves in regulation of several signal transductionpathways. It inhibits MAP kinase (Raf-MEK-ERK), G protein-coupledreceptor (GPCR) kinase and NFκB signaling cascades. Recently, severalresearches have displayed that RKIP is related closely to many tumors. Itparticipates in the occurrence and development of tumor. RKIP expresses lowin many tumors and regulates the growth, apoptosis, invasion and metastasisof tumor cells. But the effect of RKIP on different tumor biologic behaviorsand its mechanism is not very clear. We study the effect of RKIP on thebiologic behaviors of the growth, apoptosis, invasion and metastasis and its mechanism of cholangiocarcinoma.Objective: We investigated the RBE cell proliferation, apoptosis, cellinvasion and migration through the infection of over-expression of RKIPadenovirus and RNA interference restructuring adenovirus targeting RKIP. Wealso investigated the relevant mechanism of RKIP on RBE cell for the furtherresearch. We also want to know whether RKIP plays this roles byRaf/MEK/ERK pathway.Methods: Proliferation was evaluated by3-(4,5-dimethyl)2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and BrdU assay. Cellapoptosis was detected by flow cytometry, TUNEL assay and transmissionelectron microscopy (TEM). Cell invasion was examined through cellinvasion assay in vitro. Cell migration was detected by the wound closureassay. The RKIP, p-RKIP, Raf, p-Raf, ERK, p-ERK, GRK2and GAPDHprotein expressions were investigated by Western blot.Results:(1) The result of MTT showed that there was no differencebetween either of the four groups compared with the control group at differenttimes.(2) The result of BrdU assay showed that there was no differencebetween any of the four groups compared with the control group.(3) At48hpost-transfection, the combined marked Annexin-V/PE showed that theapoptotic rates of RBE cell had no difference among the five groups.(4) Theresult of TUNEL assay showed the same tendency, no difference wasobserved in the five groups.(5) TEM revealed that there was no apoptotic cellin RKIP-AD group and control group.(6) The cell invasion assay revealedthat compared with control group there was no difference between GFP-ADand NC-RNAi-GFP-AD group (60.67±22.02,52.67±16.62vs54.33±16.21).We also observed that the invasive cells in the RKIP-RNAi group wasincreased compared with the NC-RNAi-GFP-AD group (115.00±18.30vs52.67±16.62) P<0.05, and inversely the invasive cells in RKIP-AD group wasdecreased compared with the GFP-AD group (17.00±6.48vs60.67±22.02)P<0.05.(7) The wound closure assay showed that at12hours,RKIP-RNAi-AD group cells grew to86%while cells of NC-RNAi-GFP-AD grew only to71.6%, there was statistical difference between the two groups,P<0.05. RKIP-AD group inhibited the wound closure rate of RBE, only53.1%while74.13%of GFP-AD group, there's statistical difference betweenthe two groups, P<0.05.(8) The result of western blot showed that theexpression of RKIP in the RKIP-AD group was increased compared with theGFP-AD group (2.77±0.95vs0.85±0.44) P<0.05. Compared withNC-RNAi-GFP-AD group, the expression of RKIP in RKIP-RNAi-AD groupwas decreased (0.26±0.17vs0.88±0.37) P<0.05. Compared with the controlgroup, neither GFP-AD group nor NC-RNAi-GFP-AD group had difference.(9) Western blot revealed that there was no difference about the expression ofP-Raf and P-ERK among the groups.(10) Regarding to the expression ofGRK2, western blot revealed that RKIP-AD group was lower than GFP-ADgroup (0.41±0.44vs1.35±0.62) P<0.05. And compared with theNC-RNAi-GFP-AD group, the expression of GRK2of RKIP-RNAi-ADgroup was increased (1.43±0.53vs0.67±0.42) P<0.05. Whereas comparedwith the control group, there was no difference between the GFP-AD and theNC-RNAi-GFP-AD group.Conclusion: There is no effect of RKIP on RBE cell proliferation andapoptosis, but RKIP inhibits the cell invasion and migration. there is no effectof RKIP on MAPK pathway. RKIP has been demonstrated to regulateG-protein-coupled receptor signaling pathway.