Screening Of CD63-LEL Protein Related To Invasion And Metastasis Factors Of Tongue Cancer And Its Expression In Tongue Carcinoma Cells

ObjectiveScreening the protein（Integrinβ1）interacting with CD63-LEL in the total membrane protein of tongue cancer cells.The interaction between CD63-LEL and Integrinβ1 in vivo and in vitro and co-localization in tongue cancer cells was further confirmed by His-pulldown technique,CO-IP technique and immunofluorescence technique.The expression of integrinβ1 in different tongue cancer cell lines which was analyzed by in vitro transfection technique,real-time quantitative PCR and immunofluorescence.The interaction between integrin β1and CD63-LEL in tongue cancer cells was explored.Methods1.Specific primers for CD63-LEL were designed according to the m RNA sequence of human CD63（NM₀01780.5）.The total RNA of TCA8113 cells was extracted and the CD63-LEL gene was amplified by RT-PCR.The recombinant plasmid p ET29a（+）-CD63-LEL was constructed by inserting the prokaryotic expression vector p ET29a（+）by directional cloning.2.The expression of p ET29a（+）-CD63-LEL protein was optimized by His fusion protein purification column.The expression of p ET29a（+）-CD63-LEL was induced by IPTG.3.The total membrane protein of TCA8113 cells was cultured in TCA8113 cells.The CD63-LEL protein was used as a bait protein to extract the protein that could interact with CD63-LEL in the total protein of TCA8113 cells.Immunoblotting was performed by different antibody.The interaction between Integrinβ1 and CD63-LEL was successfully screened effect.4.The fusion gene of CD63-LEL and Integrinβ1 was designed according to the sequence of human CD63 m RNA（NM₀01780.5）and human integrin β1（gi21856332）.RT-PCR was used to amplify the target gene and Infusion method was constructed Recombinant plasmid pc DNA3.1（+）-3flag-CD63-LEL,p EFHA-Integrinβ1.5.The recombinant plasmid pc DNA3.1（+）-3flag-CD63-LEL and p EFHA-Integrinβ1 were transfected into TCA8113 cells by RT-PCR.The expression of CD63-LEL and Integrinβ1 were detected by two-way CO-IP method.Interaction of CD63-LEL and Integrinβ1 in tongue squamous cell carcinomaby fluorescence confocal microscopy.6.The expression of integrinβ1 in tongue squamous cell carcinoma was detected by real-time quantitative PCR and Western Blot.The expression of TCA8113 cells was detected by RT-PCR.The expression of TCA8113 cells and Integrinβ1 positioning were detected by confocal microscopy.Results1.The recombinant plasmid p ET29a（+）-CD63-LEL was successfully constructed.The results showed that p ET29a（+）-CD63-LEL protein could be expressed at 37 ℃ and IPTG at 0.1¹.0 mmol/L.IPTG concentration of 1.0mmol/L was the highest,SDS-PAGE electrophoresis showed that the purified protein was 13.3KD,and the protein concentration was 0.8 mg/ml by BCA protein quantitative kit.2.The total membrane protein was successfully obtained in TCA8113 cells.SDS-PAGE showed that the total membrane protein was between 15-170 KD and the total membrane protein concentration was 200 μg/ml.3.The purified CD63-LEL protein was used as bait protein to detect His-pull-down after hybridization with total membrane protein.The results of Western Blot showed that CD63-LEL protein and the total membrane protein in the interaction of Integrinβ1.4.The recombinant plasmid 3flag-CD63-LEL and HA-Integrinβ1 were successfully constructed.The eukaryotic expression vector pc DNA3.1（+）-3flag-CD63-LEL and p EFHA-Integrinβ1 were successfully constructed.The results of CO-IP showed that there were banded at 15 KD and 85 KD,which were consistent with the size of CD63-LEL and Integrinβ1.The results showed that CD63-LEL protein and Integrinβ1 were inoculated with intracellular interactions.5.The recombinant plasmids 3flag-CD63-LEL and HA-Integrinβ1 were co-transfected into TCA8113 cells.Immunofluorescence technique was used to detect co-localization in the cells.The results showed that CD63-LEL protein and Integrinβ1 were expressed in the cell membrane and cytoplasm.It was confirmed that Integrinβ1 and CD63-LEL protein were expressed in the cell membrane and cytoplasm of tongue squamous cell carcinoma.6.The expression of integrrinβ1 was detected by real-time PCR and Western Blot respectively.The results of Real-Time PCR showed that the expression of Integrinβ1 in CD63 high expression cell line was twice as high as that of CD63 low expression cell line（p<0.05）,but there was no significant difference between TCA8113 strain and CD63 low expression of TCA8113 strain,p>0.05.The results of immunoblotting showed that the expression of Integrinβ1 in TCA8113 group was higher than that in TCA8113 group andTCA8113 group.The results of comprehensive analysis showed that the expression of CD63 in TCA8113 cells was positively correlated with the expression of Integrinβ1.7.The results of confocal microscopy showed that the localization of CD63 and Integrinβ1 in TCA8113 cell line and CD63 high and low expression were different.The reduction of CD63 could decrease the expression of Integrinβ1.The expression of Integrinβ1 in CD63 cells was higher than that in normal cells significantly increased.Normal cells and CD63 overexpressed cell lines Integrinβ1 located in the cell membrane and cytoplasm,low expression is mainly located in the cytoplasm.Conclusions1.CD63-LEL protein interacts with Integrinβ1 in tongue cancer cell line TCA81132.The expression of CD63 in tongue squamous cell carcinoma cell lines was different,and the expression and alignment of Integrinβ1 were different.3.The expression of CD63 in TCA8113 cells was positively correlated with the expression of Integrinβ1.