Correlation Analysis Of XAGE-1b Gene With Metastasis Of Adenoid Cystic Carcinoma And Study On Its Expression In Malignant Tumors Of Head And Neck

ObjectiveMetastasis is associated with a series of changes such as invasion to the peripheral tissues, migration along vessels and lymphoid nudes, proliferation in the target organ. The mechanism is very complex and not well understood. Adenoid cystic carcinoma (ACC) is a common form of malignant secretary glands neoplasm in head and neck that is known to metastasize to the lung quite often. The metastasis rate is estimated higher than 40%, the mechanism of the metastasis process is intimately associated with mutations in gene and protein expression level. It is necessary to find out tumor metastasis related genes which might be the possible targets for cancer therapy. The following study was based on the heterogeneity of high metastasis adenoid cystic carcinoma clone Acc-M and low metastasis adenoid cystic carcinoma clone Acc-2, which the metastasis of Acc-M is 96% whereas that of Acc-2 is 18%. Since Acc-M was evolved from Acc-2, the differences in metastasis rate must be conferred by genetic differences between the two, making them a good template for the research of tumor metastasis related genes.In this study, the differences of gene expression between high metastasis Acc-M and low metastasis Acc-2 cell lines were screened by suppression subtractive hybridization (SSH) techniques; the function of XAGE-1b, a suspected metastasis related gene identified by the former experiment, was investigated by RNA interference (RNAi) techniques; the expression of XAGE-1b mRNA was detected in the samples of partly oral-maxillofacial malignant tumor patients to further investigate the role of XAGE-1b in the metastasis process of oral-maxillofacial tumors.Methods1．Cell linesHuman high metastasis adenoid cystic carcinoma clone Acc-M and low metastasis adenoid cystic carcinoma clone Acc-2 cell lines were provided by oral-maxillofacial tumor Department of Shanghai No.9 People's Hospital.2．mRNA isolation, purification and suppression subtractive hybridizationAcc-2 an Acc-M cell lines were incubated in RPMI 1640 supplemented with streptomycin, penicillin and 10% FBS in a 5% CO₂ atmosphere and harvested by trypsinization after 3d culture. Total RNA was extracted by Trizol solution and mRNA was isolated by Oligotex^TM according to the manufacturer's instructions. All SSH procedures were carried out according to CLONTECH manual PT-1117．3．Differentially expressed gene clones and homologous comparisonThe differentially displayed DNA fragments were cloned into pGEN-Tvector according to the manufacturer's instructions. Positive clones were sequenced and homologous analyzed by the NCBI's Blast N program.4．RNA dot blotting and RT-PCR analysisSemi-quantitative analysis of the differentially expressed gene fragments were performed by RNA dot blots and RT-PCR.5．Configuration of XAGE-1b siRNA expression vectorpcDNA3．1siRNA expression vectors were synthesized by MolecularImmunology Lab of School of Life Sciences, Fudan University. U6 promotor was synthesized according to GenBank sequence (Accession number X06980) and inserted into pcDNA3．1B(-) vector to substitute original CMV promoter, thus siRNA expressed vector pcDNA3．1-U6 was constructed.6．TransfectionXAGE-1b siRNA expression plasmids were transfected into Acc-2 and Acc-M cells by lipofectamine 2000 according to the manufacturer's instructions. The transfected cells were named Acc2-X and AccM-X respectively. Transfection with pEGFP plasmid and empty vector were used as control. Cells were collected 24-48h after transfection.7．Inhibitive efficiency analysis of XAGE-1b expression by RT-PCRTotal RNA of transfected cells and control cells were extracted respectively.0．6g RNA was used to synthesize cDNA, which was taken as template to detect the alteration of XAGE-1b expression.8．Migration ability of tumor cells detected by Boyden Room9．Metastasis ability of tumor cells in vivoTumor cells suspension was injected into the caudal veins of nude mice and another injection was repeated 1 week later. The nude mice were killed 8 week later and lungs were taken out. Metastasis tumors were carefully stripped and weighted. Data were analyzed by SPSS 11．5 Software.10．Total RNA extraction from oral-maxillofacial malignant tumor specimensTotal RNA was isolated from frozen tumor specimens that were surgically obtained from oral-maxillofacial malignant tumor patients according to Invitrogen's manual and analyzed by 1% agarose gel electrophorisis and ultraviolet spectrophotometer.11．Quantitative detection of XAGE-1b mRNA expression by Real-time Quantitative PCREach sample was tested two times. The copy numbers XAGE-1b and GAPDH gene were calculated by plotting Ct value on standard calibration curves. Relative amounts of XAGE-1b mRNA expression are defined as the ratio of XAGE-1b copy numbers to GAPDH copy numbers. Results1．Differentially expressed gene clones and homologous comparisonIn this study, Acc-M was designed as tester and Acc-2 as driver to carry SSH test. 6 highly differentially expressed genes in Acc-M were identified compared to Acc-2, all of which are known genes.2．RNA dot blotting and RT-PCR analysis2．1 RNA dot blotting:Semi-quantitative analysis of the differentially expressed gene fragments were performed by RNA dot blots and G3PDH was used as internal control. The result was quite coincidence with SSH. Among the differentially expressed gene fragments, 6 genes were highly expressed in Acc-M cells. The expression amounts of these genes in Acc-M cells were more than 2 times higher than in Acc-2 cells.2．2 RT-PCR analysis:RT-PCR analysis showed there was significant amplified band of m-12(XAGE-1b gene for cancer-associated protein) in high metastasis Acc-M. G3PDH was used as internal control. The results showed that m-12 gene was highly expressed in Acc-M, which the expression level was more than 3 times higher than in Acc-2 according to Image Scanning Quantitative Analysis.3．Alteration of XAGE-1b gene expressionXAGE-1b siRNA expression plasmids were successfully transfected into Acc-2 and Acc-M cells. XAGE-1b gene expression was significantly reduced in positive transfected AccM-X cells compared with negative transfected cells.4．Migration ability of tumor cells detected by Boyden RoomBoyden room was used to mimic extra cellular matrix to test migrationability of tumor cells. Migration ability of AccM-X cells was significantly reduced compared with empty vector or negative plasmid transfected cells (p<0．01) .5．Metastasis ability of tumor cells in vivoNo nodule or swelling lymphoid nodule was found in nude mice besides lungs in the three groups. There was almost no metastasis change or very slightly change observed by naked eyes in nude mice transfected with XAGE-1b siRNA compared with control groups.6．XAGE-1b mRNA expression in normal tissues and other oral-maxillofacial malignant tumorsXAGE-1b mRNA expression can be detected in normal tissues and clinically obtained oral-maxillofacial malignant tumor specimens. Relative amounts of XAGE-1b mRNA expression in ACC, tongue cancer without metastasis, tongue cancer with metastasis, carcinoma of the floor of the mouth and cancer of larynx were 0．0056±0．0070, 0．00034±0．00046, 0．018±0．021, 0．0010±0．0010, 0．00051±0．00091 respectively, which showed tongue cancer with metastasis >ACC > carcinoma of the floor of the mouth > cancer of larynx > tongue cancer without metastasis ranked from high to low. The results showed XAGE-1b gene related with metastasis. Average relative amounts of XAGE-1b mRNA expression in 6 normal tissues and 26 oral-maxillofacial malignant tumor specimens were 0．00272±0．005 and 0．00478±0．011 respectively. There was significant difference between the two groups (p<0．01) .Conclusions1．6 differentially expressed genes were identified by SSH technology between Acc-M and Acc-2 cells. These highly expressed genes in Acc-M might play important roles in metastasis process of adenoid cystic carcinoma.2．When XAGE-1b gene is down regulated by RNAi technique, the migration ability in vitro and metastasis ability in vivo of Acc-M cells is significantly reduced, which suggests XAGE-1b gene play an important role in metastasis process of adenoid cystic carcinoma.3．Expression of XAGE-1b in oral-maxillofacial malignant tumor specimens is higher than in normal tissues. Expression amounts of XAGE-1b are related to lymphoid nodule metastasis of squamous carcinoma in head and neck, which suggests XAGE-1b might be a monitoring target of metastasis.