| Objective: To explore the feasibility of applying the technique RestrictionFragment Differential Display—Polymerase Chained Reaction(RFDD-PCR) toscreening candidate genes which are related to salivary adenoid cysticcarcinoma metastasis; to investigate those candidate genes, e.g. matrixmetalloproteinases(MMPs), expression pattern in salivary adenoid cysticcarcinoma cell lines(ACC-M, high metastasis potential and ACC-2, lowmetastasis potential) as well as in transplanted tumor of salivary adenoid cysticcarcinoma, study the possible mechanism of these genes in the process ofsalivary adenoid cystic carcinoma metastasis.Methods: Total RNA of 2 salivary adenoid cystic carcinoma cell linesACC-M and ACC-2 was extracted respectively, gene expression profile of eachcell line has been constructed using RFDD-PCR technique at equal paceaccording to operating manual. Then all fragments of the two gene expressionprofiles had been separated and displayed by electrophoresis on 6% ureapolyacrylamide gel(electrophoresis power: 60W). The electrophoresis imageswere scanned by imaging system——Typhoon 9200, and analyzed using softwareImageQuant TL, Image Tool and Fragment Analysis, thus the differentialexpression fragments were detected. Bioinformatics analysis was applied foridentifying salivary adenoid cystic carcinoma metastasis related genes/ESTs.Then semi-quantitative RT-PCR was used to observed expression pattern of part of these putative salivary adenoid cystic carcinoma metastasis candidate genes.Expression pattern of one significant altered gene family——MMPs and itsinhibitor——tissue inhibitor of metalloproteinases(TIMPs), includingMMP-2,MMP-7,MMP-9,TIMP-1 and TIMP-2 were immunodetected in two rattumor models transplanted ACC-M,ACC-2 cell line respectively besides ofsemi-quantitative RT-PCR.Results: Electrophoresis of RFDD-PCR products efficiently separated5420 significant bands altogether, including 2619 in ACC-M and 2801 inACC-2 cell line. Sizes of these band were among the range of 50～1000bp.There were 1734 bands (32%) altered according to software GeneTools. 433bands showed higher intensity in ACC-M than in ACC-2, while 562 bandsindicated a down-regulated tendency in ACC-M. 416 fragments were detectedonly in ACC-M and 323 bands only in ACC-2.Bioinformatics analysis were used to identify gene information of these5420 bands. The expressed genes in the two cell lines were concerned withmetabolism enzymes, growth factors and their receptors, cell factors, signaltransduction molecules, ion channel proteins, cyclins, transmembraneglycoproteins and proliferation, apoptosis related genes, etc. Among them,MMPS gene family showed a significant difference. Expression of MMP-2,MMP-7, MMP-9, MMP-15, MMP-20, MMP-28 was observed down-regulated inACC-M, while MMP-8, MMP-11, MMP-12, MMP-19 showed no obviousdifference between 2 profiles. As to E-Cadherin family, except that CDH11,CDH12 showed up-regulated in ACC-M, and CDH2, CDH4, CDH5, CDH10,CDH19 were observed up-regulated in ACC-2, there was no difference on theexpression of CDH1, CDH9, CDH13, CDH20. CDH3, CDH6, CDH8 weredetected in ACC-2 only. Furthermore, integrin ITGB4, ITGB7, KAI1showedlower expression amount in ACC-M, while ITGB2 no difference. ITGB3,ITGB 5, ITGB 8 also were detected in ACC-2.The results of Semi-quantitative PCR also showed a higher expression amount of MMP-2, MMP-7, MMP-9, and MMP-15 in ACC-M among 12 MMPsgenes in expression profile we constructed. While MMP-14, MMP-24 had astronger band in ACC-2. No difference showed on the expression of MMP-8,MMP-11, MMP-12 and MMP-19. MMP-20, MMP-28 could not be detected atall.In the ACC-M transplanted tumor model, the expression of MMP-2,MMP-7, MMP-9, TIMP-1 were higher than that in ACC-2 model, while therewas no different as to TIMP-2.Conclusions: Total fragments displayed of all 64 "expression windows"of RFDD-PCR, all the candidate genes selected by bioinformatics analysis andidentified through other methods, shows that RFDD-PCR is a promisingtechnique with high sensibility, coverage and reliability. Our results prove itsfeasibility in high-through screening of disease related genes and detecting newclues.5420 significant fragments were detected from 2 cell lines, and 1734differentially expressed fragments (32%) were showed. The function of thesealtered genes are concerned with transmembrane proteins, cyclins, metabolicenzymes and proliferation or differentiation related genes, etc. Thus, theseeking of tumor molecular targets would be a great challenge cause all thedifferences we have detected between 2 cell lines only result from differentmetastasis potential.Our results unclose some salivary adenoid cystic carcinoma metastasiscandidate genes or gene family which had not been reported before or not beenrecognized clearly before, these are new clues for the study of salivary adenoidcystic carcinoma metastasis mechanism and new therapeutic method. We'vefound that the high metastasis potential of ACC-M may have correlation withMMP-2, MMP-7, MMP-9 and MMP-15. Different types and amount of cellstroma around different tumor cells may result in similarity of MMP-8, MMP-11,MMP-12, MMP-19 of 2 cell lines. This indicates certain metastasis potential of certain tumor cells may tightly related to certain family members of MMPs.However, the relationship between MMP-19, MMP-20, MMP-8 and the tumormetastasis is still to be proved. What is more, we have also noticed that ACC-Mcell line showed similar expression phenotype both in vivo and in vitro.TIMP-1 may play a role as growth factor as we observed in ourtransplanted tumor model, while TIMP-2 did not show this kind of function.Therefore, considering previous researchers' observation, we inferred thatmembers of TIMPs may have multiple function in the process of tumor genesisand metastasis, so the relationship between MMPs and this MMPsinhibitor——TIMPs is still open.
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