Preliminary Study Of The Impact Of CD63 On Invasion And Metastasis Of TCA8113 Cells

ObsjectTo explore the relationship between the expression level of CD63 protein and the histological differentiation of tongue squamous cell carcinoma using the immunohistochemistry technology.In order to preliminary detect the impact of different CD63 expression level on invasion and metastasis of tongue squamous cell carcinoma cell line,we changed the expression of CD63 in TCA8113 squamous cell carcinoma cells by RNA interference and gene transfection technology,and screened TCA8113 cell lines with low and high level expression of CD63Methods1? 32 cases of tongue cancer tissue samples with different pathological degree were selected to analyze the relationship between the differentiation degree of tongue cancer tissue and CD63 expression levels using the immunohistochemical technology.2?According to the sequence of CD63 m RNA?NM₀01780.5?,we designed3 pieces of sh RNA clone,inserted them into the eukaryotic expression vector GV102,and then transfected the plasmids into 293 T cells.We gained the most efficient plasmids CD63-RNAi-4041 using real-time PCR and western blot technology.The plasmids were transfected it into TCA8113 cells and a CD63 gene silenced cell line named CD63-L was obtained after screening by G418.3 ? A pair of primers of CD63 gene were desighed according to the sequence of CD63 m RNA?NM₀01780.5?.Total RNA was extracted fromTCA8113 cells and the CD63 gene was amplified by RT-PCR method and cloned into PEGFP-N3 vector.The recombinant plasmids PEGFP-N3-CD63 were transfected into TCA8113 cells after screened by restriction enzyme digestion and DNA sequencing.Then we obtained a TCA8113 cell line named CD63-H with high level expression of CD63?uploaded by 32%?after G418 screening.Western blot and confocal microscope were used to detect the expression and location of CD63 protein.4?We detected the difference of the expression level of MMP-2 and MMP-9genes using western blot technology.Wound healing assay was used to detect the difference of the wound healing speed between the 3 TCA8113 cell lines.Transwell assay was used to analyze the effect of different CD63 expression level on the invasion ability of the 3 cell lines.Results1?CD63 protein was deep brown-yellow colored like granular in normal tongue tissue and mainly expressed in the cell membrane and cytoplasm.The color was weaker and weaker than normal tongue tissue as the decreasing of differentiation degree of tongue squamous cell carcinoma.Variance analysis of the difference between the groups,P<0.05,indicated a positive correlation between CD63 expression level and histopathological differentiation of the tongue cancer.Tested by t test,and P<0.05,indicating reduced differentiation of tongue cancer,CD63 expression was reduced,and increased likelihood of lymph node metastasis in tongue cancer,that is to say a negative correlation between expression level of CD63 and lymph node metastasis in tongue cancer.2 ? sh RNA plasmids can be efficiently transfected into 293 T cells,real-time PCR and western blot results showed that the CD63-RNAi-4041 can obviously silencing the expression of CD63,and the interference efficiency in m RNA and protein levels were respectively 88% and 74%,P<0.05.And aCD63 gene silenced TCA8113 cell line named CD63-L was successfully obtained after screening by G418.3?PEGFP-N3-CD63 plasmids were successfully obtained after restriction enzyme digestion and DNA sequencing.And a CD63-H cell line with high level expression of CD63 was constructed by G418 screening.The CD63 protein was located in the outer membrane of TCA8113 cells observed with confocal microscope.4?Western blot results showed that,the expression of MMP-2 and MMP-9were uploaded 60% and 61% respectively in CD63-L cells but was reduced29% and 41% respectively in CD63-H cells than CON cells.CD63-L cells were able to repair the wound 34% more rapidly as compared with CON cells,but the CD63-H cells were 59% slower than CON cells.The transwell assay showed that cells invaded though matrigel-coated filters of CD63-L were 70% more than that of CON cells,however,CD63-H cells invaded were 71% less than CON cells.Variance analysis of the difference between the groups,P<0.05,indicated a negative correlation between CD63 expression level and the invasion ability of TCA8113 cells.Conclusions1?There was a positive correlation between cell surface expression level of CD63 and histological differentiation of tongue squamous cell carcinoma and a negative correlation between CD63 expression level and lymph node metastasis in tongue squamous cell carcinoma.2?A CD63 silenced TCA8113 cell line,CD63-L,was successfully obtained.3?A CD63 over-expressed TCA8113 cell line,CD63-H,was successfully obtained.4?The MMP-2 and MMP-9 expression level,the wound healing speed and the invasion ability were all negatively correlated with the CD63 expression level.This sdudy preliminary proved a negative correlation between CD63 expressionand invasion and metastasis potential of tongue cancer cell.