The Mechanism Of Raf Kinase Inhibitor Protein Inhibiting The Invasion Of The Esophageal Carcinoma Cells

Esophageal cancer is a malignant tumor of the esophageal epithelialtissues, which occurs more frequently in males than females and which istraditionally more prevalent in subjects older than40years. However, inrecent years there has been an increased tendency for the disease to appear atyounger age. The development of esophageal cancer is a complex processinvolving many pathogenic factors, multiple stages, and accumulation ofmultiple gene mutations and interactions. These factors cause dysregulation ofoncogenes, tumor suppressor genes and signaling protein molecules at themolecular level.Raf kinase inhibitor protein (RKIP) is a small, cytosolicphosphatidylethanolamine-binding proteins originally purified from bovinebrain. This protein family is highly conserved and is rarely homologous toother types of protein. RKIP is widely distributed in many tissues in variousmammals such as mouse, monkey, and human. It acts as a signal regulator,which not only inhibits Raf-mediated MAPK and ERK activities, but alsoinhibits NF-κB signaling pathway and regulates the activity of Gprotein-coupled receptors. Reduced RKIP expression has been shown to affectcell growth, angiogenesis, apoptosis and gene integrity. Increased RKIPexpression was shown to suppress invasion and reduce metastasis to basilarmembranes in mouse models of prostate cancer. It has also been shown toinhibit growth and invasion of ovarian cancer. Reduced RKIP expression isclosely associated with progression and prognosis of hepatocellular carcinoma,colorectal cancer, gastric cancer and gastrointestinal stromal tumor. However,the underlying mechanisms of RKIP in esophageal cancer cells remain unclear.The present study was, therefore, designed to investigate the mechanisms ofRKIP in esophageal cancer progression. Part1: The expression of RKIP in the esophageal carcinoma tissuesObjective: To investigate the expression of RKIP in the esophagealcarcinoma tissuesMethod: With hematoxylin-eosin (HE) staining, we observed thepathology changes of40patients by optical microscope. Then we detected theexpression of RKIP in the carcinoma tissues, peritumoral tissues and normaltissues with Western blot and immunohistochemical. At last, we collected theclinical and pathological data of patients that after operation and investigatedthe relationship between the expression of RKIP and pathological parameterthrough statistical analysis.Results:(1) The results of HE staining showed that the pathological types of40patients were squamous cell carcinoma.(2) The results of immunohistochemical staining showed that theexpression of RKIP in esophageal carcinoma tissues was much lower than inthe peritumoral tissues and normal tissues. The mean density among the threegroups was respectively0.0030.001,0.0280.003and0.0390.004, P<0.05.The expression of RKIP in the peritumoral tissues and normal tissues had noobvious difference, P>0.05. The frequency of RKIP positive expression was70%,55%and20%in the normal esophageal epithelia, tumor-adjacent tissuesand esophageal squamous cell carcinoma, respectively. These results indicatethat the RKIP expression was significantly lower in the esophageal cancertissues than in normal esophageal epithelia (P <0.05, Chi-square test).(3) Western blot showed that the expression of RKIP in the esophagealcarcinoma tissues was0.320.05, the peritumoral tissues was0.560.80, andthe normal tissues was0.700.11, which proved the expression of RKIP in theesophageal carcinoma tissues was reduced significantly.(4) The analysis of pathological parameter showed that the reduced levelof the RKIP expression was related to the distant metastasis and lymphmetastasis, but had no direct relation with gender, age, differentiation andpathological classification. Conclusion: The expression of RKIP in esophageal carcinoma tissues isreduced，which is related to the distant metastasis or lymph metastasis.Part2: The effect of RKIP on the invasion of the esophageal carcinomacells.Objective: To observe the effects of RKIP on the proliferation of TE cell,apoptosis, cell adhesion and invasion in esophageal carcinoma.Methods: By the over-expression of RKIP and the reconstruction ofRNA interference adenovirus of targeting RKIP, proliferation was evaluatedby3-(4,5-dimethyl)2,5-diphenyltetrazolium bromide (MTT) assay andBrdU assay. Cell apoptosis was detected by flow cytometry, TUNEL assay andtransmission electron microscopy (TEM). Cell adhesion was detected bymatrigel-treated24well plate. Cell invasion was examined through cellinvasion assay in vitro.Results:(1) The result of MTT showed that there was no significant difference inany of the four groups compared with the control at different time.(2) The result of BrdU assay showed that there was no differencebetween any of the four groups compared with the control group.(3) After48h of transfection, the combined marked Annexin-V/PEshowed that the apoptotic rates of TE cells had no significant differenceamong the five groups.(4) The result of TUNEL assay showed that there was the same tendency,and no apoptotic cells were observed.(5) TEM revealed that there was no apoptotic cell in RKIP-AD group andcontrol group.(6) The cell adhesion assay revealed that the number of adhesive cells inRKIP-AD group was decreased compared with GFP-AD group (64.60±13.67vs96.20±19.73, P<0.05). At the same time we observed the number ofadhesive cells in RKIP-RNAi-AD group was increased compared withNC-RNAi-GFP-AD group (174.80±22.67vs107.60±20.37, P<0.05).Compared with control group there was no difference between GFP-AD and NC-RNAi-GFP-AD group (107.60±20.37,96.20±19.73vs98.60±19.31,P>0.05).(7) The cell invasion assay revealed that compared with control groupthere was no difference between GFP-AD and NC-RNAi-GFP-AD group(60.67±22.02,52.67±16.62vs54.33±16.21). We also observed that theinvasive cells in the RKIP-RNAi group were increased compared with theNC-RNAi-GFP-AD group (115.00±18.30vs52.67±16.62, P<0.05), andinversely the invasive cells in RKIP-AD group were decreased compared withthe GFP-AD group (17.00±6.48vs60.67±22.02, P<0.05).Conclusions: There are no effects of RKIP on TE cell proliferation andapoptosis, but the cell invasion and adhesion could be inhibited by RKIP.Part3: The molecular mechanism of RKIP inhibiting the invasion of theesophageal carcinoma cells.Objective: To investigate the relevant mechanism about the invasion ofRKIP on TE cells.Methods: The RKIP, p-RKIP, Raf, p-Raf, ERK, p-ERK, GRK2andGAPDH protein expressions were investigated by Western blot. Theexpressions of MMP-1, MMP-2, MMP-3, MMP-13, MMP-14, Bcl-xL, Bcl-2,caspase-8, caspase-9, LIN28, TIMP-1, Elk, RKIP and GAPDH mRNA wereobserved by real-time Q-PCR respectively.Results:(1) The result of western blot showed that the expression of RKIP in theRKIP-AD group was increased compared with the GFP-AD group (2.31±0.36vs0.43±0.09, P<0.05). Compared with NC-RNAi-GFP-AD group, theexpression of RKIP in RKIP-RNAi-AD group was decreased (0.05±0.18vs0.38±0.08, P<0.05). Compared with the control group, neither GFP-AD groupnor NC-RNAi-GFP-AD group had difference.(2) Western blot revealed that there was no difference about theexpression of p-Raf and p-ERK1,2among the groups.(3) Regarding to the expression of GRK2, the western blot revealed thatRKIP-AD group was lower than GFP-AD group (0.52±0.10vs1.01±0.11, P<0.05). Moreover, compared with the NC-RNAi-GFP-AD group, theexpression of GRK2of RKIP-RNAi-AD group was increased (1.37±0.15vs0.84±0.10, P<0.05). Whereas compared with the control group, there was nodifference between the GFP-AD and the NC-RNAi-GFP-AD group.(4) Using the relative quantification of2-Ctmethod, we compared theexpression of MMP-1, MMP-2, MMP-3, MMP-13, MMP-14, Bcl-xL, Bcl-2,caspase-8, caspase-9, TIMP-1, Elk, RKIP, LIN28and GAPDH mRNAexpression in each group, real time Q-PCR suggested that①Compared withthe GFP-AD group, the expression of RKIP mRNA in RKIP-AD group wasincreased (166.46±0.09vs1.00±0.00, P<0.05). Compared with theNC-RNAi-GFP-AD group, the expression of RKIP mRNA inRKIP-RNAi-AD group was decreased (0.17±0.09vs1.00±0.00, P<0.05).②Compared with the GFP-AD group, the expression of MMP-14mRNA ofRKIP-AD group was decreased (0.26±0.20vs1.00±0.00, P<0.05). Comparedwith the NC-RNAi-GFP-AD group, the expression of MMP-14mRNA ofRKIP-RNAi-AD group was increased (6.89±0.84vs1.00±0.00, P<0.05).③Compared with the GFP-AD group, the expression of LIN28mRNA ofRKIP-AD group was decreased (0.28±0.06vs1.00±0.00, P<0.05). Comparedwith the NC-RNAi-GFP-AD group, the expression of LIN28mRNA ofRKIP-RNAi-AD group was increased (1.86±0.12vs1.00±0.00, P<0.05).While there were no difference of other factors among all the groups.Conclusions: There is no effect of RKIP on the MAPK pathway. RKIPregulates G-protein-coupled receptor signaling pathway. RKIP inhibits theinvasion of TE cells by downregulating the expression of MMP-14and LIN28mRNA.