| PURPOSE:To investigate the clinicopathological significance of expression of the critical molecules in MICA-NKG2D passway in oral squamous cell carcinoma(OSCC) and construct the stable MICA-transfected Tca8113-Tb cell line, and explore the effect on NK and CTL-mediated cytotoxicity by genetic overexpression of MICA in vivo and in vitro.METHODS:1.The expression of MICA protein and mRNA in OSCC tissues were detected by immunohistochemistry and real time PCR,and the lever of sMICA in sera and the expression of NKG2D on PBMCs of OSCC patients were assessed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Data was analyzed with SPSS16.0 software package.2.cDNA of MICA gene from pCMV-SPORT6-MICA was amplified by PCR, and subcloned into eukaryotic expression vector pEGFP-N1.The recombinant plasmid was sequenced and transfected into Tca8113-Tb cell line by lipofectamineTM 2000.After screen culture by G418,stable tranfected Tca8113-Tb cell line was established using definite dilution method. The expression of GFP protein was viewed directly with fluorescence microscopy and the overexpression of MICA was identified by RT-PCR, real time PCR and immunocytochemistry.3.The Tca8113-Tb cell by genetic overexpression of MICA were detected to identify the biological features including cell growth curve,cell cycle distribution, plate clone forming rate and tumorigenicity in nude mice. The expression of NKG2D receptor and the cytotoxicity to target tumor cells of NK92 and CTL cells, which co-cultured with transfected Tca8113-Tb cells,non-transfected or blank vector-transfected controls, were measured by flow cytometry and lactate dehydrogenase (LDH) release assay.RESULTS:1.MICA was located in cytoplasm and plasm membrane. The expression of MICA in para-cancerous tissue was higher than cancerous tissue(P<0.05),but no statistical difference was found between para-cancerous tissue with normal mucosa and normal mucosa with cancerous tissue, and also among different clinicopathological parameters in OSCC (P>0.05). The level of MICA mRNA in OSCC was higher than para-cancerous tissue and was coorelated with regional lymph node status and disease stage(P<0.05). There was no correlation between the mRNA and protein expression.2. The positive rate of assay for sMICA in sera of OSCC patients was 98.7%(77/78), with the 95% confidence interval 74.30-93.95pg/ml and the median 82.17pg/ml. In the healthy controls, the positive rate of assay was 94.7%(18/19), with the 95% confidence interval 29.48～50.30pg/ml and the median 37.54pg/ml.The sMICA in sera of OSCC patients were higher than that of the controls (P<0.01).And the sMICA in sera of OSCC patients was significantly different among different clinicopathological parameters such as tumor size,disease stage and regional lymph node status(P<0.05). But no difference was found statistically among different gender, age, and tumor differentiation (P>0.05).3. The expression rate of NKG2D on PBMCs in OSCC patients (12.49±5.26)% was lower than that of the controls (22.93±8.14)% (P＜0.05). And the NKG2D expression on PBMCs in OSCC patients was significantly different among different clinicopathological parameters such as tumor size and disease stage(p＜0.05). But no difference was found statistically among different gender,age,tumor location,tumor differentiation and regional lymph node status (P>0.05).4. The recombinant plasmid was confirmed by PCR,restrictin enzyme digestion and sequencing. The stable MICA-transfected Tca8113-Tb cell line was established and the MICA was overexpressed successfully.5. There is no difference on biological features between before and after MICA gene transfection to OSCC cells. Flow cytometry and LDH release assay showed that MICA-overexpressed OSCC cells enhanced the cytotoxicity to target tumor cells and up-regulated the expression of NKG2D on NK92 and CTL.CONCLUSION:The critical molecules in MICA-NKG2D passway may be closely related with the occurrence and development of OSCC.The MICA-overexpressed OSCC cells can up-regulated the expression of NKG2D on NK and CTL,and enhance the cytotoxicity to target tumor cells,and the study suggests that MICA-NKG2D passway can be considered as a promising immunotherapy target of oral squamous cell carcinoma.
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