OBJECTIVETo construct the eukaryotic expression vector, encoding major histocompatibility complex class I-related chain A gene (MICA), for the further research of transfecting Tca8113-Tb cell line(a metastatic cell line of brain metastasis from human tongue cancer Tca8113 cells in nude mouse), and to establish a stable MICA overexpression oral squamous cell line.

METHODScDNA of MICA gene from pCMV-SPORT6-MICA was amplified by PCR, and subcloned into eukaryotic expression vector pEGFP-N1 marked with green fluorescent protein (GFP). The recombinant plasmid was sequenced and transfected into Tca8113-Tb cell line by lipofectamine 2000. After screen culture by G418, stable tranfected Tca8113-Tb cell line was established using definite dilution method. The expressions of GFP protein was viewed directly with fluorescence microscopy and the overexpression of MICA was identified by RT-PCR, real time PCR and immunocytochemistry.

RESULTSThe MICA gene was amplified by PCR and then cloned into the vector, whose sequence was identical to that in the GenBank. The transfected cells showed the expression of GFP. And the overexpression of MICA gene in transfected cells was detected by RT-PCR, real time PCR and immunocytochemistry.

CONCLUSIONThe recombinant eukaryotic expression vector pEGFP-N1-MICA has been constructed successfully and stably expressed in Tca8113-Tb cell line, providing a foundation for further studies on the function of MICA in vitro.

Animals ; Cell Line ; Genetic Vectors ; Green Fluorescent Proteins ; Humans ; Mice ; Mice, Nude ; Tongue Neoplasms ; Transfection

OBJECTIVETo investigate the effect on natural killer (NK) and cytotoxic T lymphocyte (CTL)-mediated cytotoxicity by genetic overexpression of MHC class I chain-related protein A (MICA) in oral squamous cell carcinoma (OSCC).

METHODSThe OSCC cells 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 natural killer group 2, member D (NKG2D) receptor and the cytotoxicity to target tumor cells of NK92 and CTL cells, which co-cultured with the transfected OSCC cells or the non-transfected or blank vector-transfected controls, were measured by flow cytometry and lactate dehydrogenase (LDH) release assay.

RESULTSThere was no difference in biological features 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 (P<0.05).

CONCLUSIONMICA may be considered as a promising immunotherapy target of OSCC.

Animals ; Carcinoma, Squamous Cell ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Genetic Therapy ; Histocompatibility Antigens Class I ; Humans ; Killer Cells, Natural ; Mice ; Mice, Nude ; Mouth Neoplasms ; NK Cell Lectin-Like Receptor Subfamily K ; Staphylococcal Protein A ; T-Lymphocytes, Cytotoxic ; Transfection

OBJECTIVE: To examine the expression of soluble major histocompatibility complex class I-related chain A (sMICA) in the serum in patients with oral squamous cell carcinoma (OSCC) and to explore its clinicopathological significance. METHODS: Seventy-eight OSCC patients were selected as an experiment group, and 19 healthy persons as a control group. The sMICA in the serum in the experiment group and the control group was detected by enzyme-linked immunosorbent assay. RESULTS: The detection rate of sMICA in the serum in the experiment group was 98.7% (77/78), with the 95% confidence interval 74.30-93.95 pg/mL and the median 82.17 pg/mL, The detection rate in the control group was 94.7% (18/19), with the 95% confidence interval 29.48-50.30 pg/mL and the median 37.54 pg/mL. The sMICA in the serum in the experiment group was higher than that in the control group (P<0.01). There was statistic difference in the serum sMICA in the experiment group among the different clinicopathological parameters such as tumor size, disease stage and regional lymph node status (P<0.05), but no difference was found in gender, age, and tumor differentiation (P>0.05). CONCLUSION The sMICA in the serum in the OSCC patients increases, and is related with the tumor size, disease stage and regional lymph node status. Determination of sMICA in the serum may provide useful information to evaluate the immune state of OSCC patients.
Aged ; Carcinoma, Squamous Cell ; blood ; immunology ; pathology ; Case-Control Studies ; Female ; Histocompatibility Antigens Class I ; blood ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Mouth Neoplasms ; blood ; immunology ; pathology ; Solubility