| Objective:(1)to establish different potential of invasion and metastasis of human ESCC subline,to screen candidate genes correlated to invasion and metastasis of ESCC by screening of gene chip.(2)Partly candidate genes expression in different ESCC cells and tissue were validated,to investigate the expression and roles of HSP90?,Annexin A1 and 14-3-3? in Xinjiang Kazakh ESCC.(3)To study the effects and mechanism of HSP90AA1 gene scilencing on the biological behavior of ESCC in vivo and vitro.Methods:(1)Transwell chamber was used to screen Eca109(Eca109-T0),Eca109-T4 ESCC sublins with high invasion and metastasis were obtained.Ability of migration and proliferation of Eca109-T0,Eca109-T4,CE81T-4 and CE81T-0 were compared.total RNA of Eca109-T0,Eca109-T4 were extracted.Then they were labeled c DNA probe respectively with Cy5 and Cy3,hybridizating with Affymetrix Gene Chip? Human Genome U133 Plus 2.0 Arry,Screening candidate genes related with invasion and metastasis of ESCC.The candidate genes were performed by GO and KEGG analyses.(2)HSP90AA1,ANXA1,YWHAB,CXCR7,SDC2,TNFRSF10 D m RNA relative abandance and HSP90?,Annexin A1,14-3-3?,CXCR7 relative protein expression were validated in Eca109-T0,CE81T-0,Eca109-T4 and CE81T-4 by q RT-PCR and Western blot.HSP90?,Annexin A1 and 14-3-3? expression in 86 cases of Xinjiang Kazakh ESCC and adjacent tissues were detected by immunohistochemistry,correlationship of their expression with clinical pathological parameters of age,gender,general type,location,tumor size,depth of invasion,differentiation,lymph node metastasis were analysized.(3)RNAi technology was applied to knockdown HSP90AA1 of CE81T-4,effect of RNAi was validated by green fluorescence,q RT-PCR and Western blot.Cellular proliferation was detected by MTT,cell cycle and apoptosis was measured by flow cytometry.Transwell chamber and Wound healing assay was used to detect invision and migrate ability.MET,MMP2,ERK and P-ERK protein expression were detected byWestern blot,MET and MMP2 m RNA were detected by q RT-PCR.increasing volum and weight of xenogaft of BALB/C nude mouse were observed.Results:(1)Eca109-T4 ESCC sublins with high invasion and metastasis were screened by transwell chamber.Comparison of biological characteristics in vivo and vitro showed: Migration,proliferation,volum and weight of xenogaft in Eca109-T4,CE81T-4 were higher than that in Eca109-T0 and CE81T-0.Migration and invision of Eca109-T4 exceeded that of CE81T-4.Result of screening candidate gene from Eca109-T0 and Eca109-T4 showed that 326 candidate genes were detected,including 123 up-expression genes and 203down-expression genes in Eca109-T4.GO clustering analysis showed that their subcelluar composition include cytoplasm,nucleus and cytosol,Major molecular function include protein binding,metal ion binding and DNA binding,they participated in biological process of signal transduction,transcription,DNA-templated,regulation of apoptotic process,regulation of cell proliferation,cell adhesion,and so on.Regulation of actin cytoskeleton,MAPK pathway and signal transduction pathway of cancer were involved.(2)m RNA abandance of HSP90AA1,YWHAB,CXCR7 and SDC2 in Eca109-T4,CE81T-4 were higher than that in Eca109-T0,CE81T-0(P < 0.05).TNFRSF10 Dm RNA in CE81T-4 was lower than that in Eca109-T4(P<0.05).ANXA1 m RNA of Eca109-T4 and CE81T-4 were lower than that in Eca109-T0,CE81T-0(P<0.05).protein expression of HSP90?,14-3-3?,CXCR7 in Eca109-T4 and CE81T-4 were higher than that in Eca109-T0,CE81T-0(P < 0.05).Annexin A1 protein expression in Eca109-T0 and CE81T-0 exceed that in Eca109-T4 and CE81T-4(P<0.05).The above results are consistent with the gene chip screening.HSP90AA1 m RNA and HSP90?protein in CE81T-4 were more than that in Eca109-T4(P < 0.05).The positive expression of HSP90? and 14-3-3? protein in Xinjiang Kazakh ESCC were significantly higher than in the adjacent tissues of ESCC(87.2%18.6%,80.23%41.86%,P=0.000,0.0002),The expression of Annexin A1 protein in ESCC was lower than in the adjacent tissues(32.6%?59.3%,P=0.0002).HSP90? expression was related with tumor invasion degree,differentiation and lymph node metastasis(P=0.024,0.021,0.011),was not related with age,gender,type,location and size of the tumor(P>0.05).Annexin A1 expression was associated with differentiation and lymph node metastasis(P=0.037,0.014),was not associated with age,gender,invasion degree,type,location and size of the tumor.14-3-3? expression was related to invasion and differentiation degree(P=0.008,0.035),had nothing to do with age,gender,lymph node metastasis,type,location and size of the tumor(P > 0.05).(3)Effect of RNAi interference with CE81T-4 wereconfirmed by visible green fluorescence,q RT-PCR and Western blot detection,which showed decrease in HSP90AA1/HSP90? of HSP90AA1-RNAi group compared with NC-RNAi and NC groups after scilencing HSP90AA1(P < 0.05).there were no difference for MTT detection in 0 and 24 h,and cell proliferation of HSP90AA1-RNAi group was suppressed in 48 h,72h and 96h(P<0.05).HSP90AA1-RNAi leaded to cell cycle arrest G0/G1 phase,down-expression of HSP90AA1 induced cellular apoptosis,the apoptosis rate in HSP90AA1-RNAi,NC-RNAi and NC group was respectively(27.30±4.33)%,(5.63±1.12)%,(10.20±2.04)(P<0.05).After scratching,the CE81T-4migration distance of 24 h,48h and 72 h of HSP90AA1-RNAi group were decreased(P<0.05).Transwell invasion experiment results showed that invasive cell numbers in HSP90AA1-RNAi group decreased(P<0.05).Western blot showed that MET,MMP2 and P-ERK protein expression were reduced in HSP90AA1-RNAi group(P<0.05).there was no difference for ERK protein expression in three groups(P > 0.05).MET and MMP2 m RNA were decreased after HSP90AA1-RNAi(P<0.05).increasing volum and weight of xenogaft of BALB/C nude mouse in HSP90AA1-RNAi group were inhibited(P < 0.05).Conclusion:(1)The establishment of different invasion and metastasis-associated ESCC sublines provides a researching model.Screening and establishing of candidate genes correlated to invasion and metastasis of ESCC provides plentiful informations for further research in the future.(2)HSP90AA1,ANXA1,YWHAB,CXCR7 and SDC2 were related with invasion and metastasis of ESCC.HSP90? and 14-3-3? were high expression in Xinjiang Kazakh ESCC,positively correlated with depth of invasion and negatively correlated with differentiation,HSP90?were positively correlated with lymph node metastasis.Annexin A1 was low expression in Xinjiang Kazakh ESCC,higher expression in well-differentiation and no lymph node metastasis of ESCC compared with those low-differentiation and lymph node metastasis.Level of HSP90?,Annexin A1 and 14-3-3? reflect status of differentiation,depth of invasion and lymph node metastasis of ESCC in some degree,therefore they maybe expected to the biomakers for early diagnosis,prodiction for metastasis,invasion and differentiation of ESCC.(3)RNAi could effectively inhibit the HSP90AA1 expression in CE81T-4.down-expression of HSP90AA1 induced CE81T-4 cell cycle arrest in G0/G1 phase,promote apoptosis,inhibit the proliferation,migration and invasion,which maybe performed by down-regulation of p-ERK,MET and MMP2 mediated mechanism. |
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