An Experimental Study On The Screening Of Stem Cell-Related Genes In Tongue Squamous Cell Carcinoma Tca8113Cells By Suppression Subtractive Hybridization

In recent years, people believe that the cancer stem cell (CSC)hypothesis could be applied to cancers. According to the hypothesis, atumor can be viewed as an aberrant organ initiated by a smallsubpopulation of cells called cancer stem cells (CSCs), which exhibitself-renewing capacities and are responsible for tumor maintenance andmetastasis. This hypothesis provides novel insight into the understandingof tumorigenesis. To validate this hypothesis, the isolation andidentification of CSCs became a “hot topic” in current cancer biology.Emerging evidence has supported the validity of this hypothesis in anumber of malignant diseases. The existence of cancer stem cells has alsobeen verified in HNSCC and the expression of aldehyde dehydrogenase(ALDH) is crucial. Cells with high ADLH activity (ALDHbr) displayCSC-associated properties such as radioresistance and the ability toproduce tumors with very low cell numbers, which is in contrast to cells with low ALDH activity (ALDHlow). However, the gene expression profilein the two cell subpopulations remains unknown, hindering a deepunderstanding of the underlying mechanisms of CSC in HNSCC.In this study, we isolated ALDHbrand ALDHlowcells from theoral squamous cell carcinoma cell line Tca8113, and then performedsuppression subtractive hybtidization (SSH) to identify differentiallyexpressed genes in ALDHbrand ALDHlowcells.Methods1. Sorting and identification of ALDHbrsubpopulation in Tca8113squamous cell carcinoma cellswe isolated ALDHbrand ALDHlowcells from the oral squamous cellcarcinoma cell line Tca8113using fluorescenceactivated cell sortingtechnology, and the cancer stem cell characteristics of ALDHbrcells wereidentified by proliferation assay, differentiation assay and tumorsphereformation assay.2. The construction of sbutracted library between ALDHbrandALDHlowcells in Tca8113squamous cell carcinoma cellscDNA from ALDHbrcells was used as the “Tester” and cDNA fromALDHlowcells as the “Driver” in forward subtracted library. conversely，in reverse subtracted library, the cDNA form ALDHlowcells was used as the“Tester”and cDNA from ALDHbrcells was used as the “Driver”. colonyPCR was performed to confirm the presence and the size of the inserts before sequencing.3. The preliminary screening of sbutracted library and bioinformaticanalysis of differentially expressed genesThe positive clones were selected randomly and sequenced at theBeijing Genomics Institute. ESTs were compared with non-redundant publicdatabases using the BALSTN and BLASTX algorithms of the NCBI.The putativephysiological functions of these ESTs were classified according to GeneOntology. Pathway analysis was performed using the Gene Set AnalysisToolkit V2online system.Resaults1. The enzymatic activity of ALDH in Tca8113tongue squamouscell carcinoma cell line is heterogeneous, and this cell line barbored1.3%of ALDHbrcells, which exhibited higher proliferation capacity than theirALDHlowcounterparts. Sorted ALDHbrcells were able to differentiate andgenerate ALDHlowcells in vitor. Moreover,in serum-free medium, ALDHbr,but not ALDHlowcells, survived and formed tumorspheres.2. White colonies were randomly selected from forward and reverselibrary. These clones were subjected to colony PCR using nested primers.All of the recombinants detected revealed amplicons ranging from200bpto800bp, indicating that the substracted librarys were onstructedsuccessfully.3. All240clones were picked and sequenced. Sequences were not obtained for14clones and those were omitted from our study. Comparisonof the unique sequences obtained from each library against the GenBankdatabases identified104unique clones,62of which corresponded to knowngenes. Signal pathway analysis revealed genes in10pathways changedsignificantly in the ALDHbrcell subpopulation.ConclusionA small subset of Tca8113cells with high ALDH enzymatic activitydisplay characteristics of cancer stem cells, suggesting that ALDH activitymay be a cancer stem cell marker for tongue squamous cell carcinoma.Using suppression subtractive hybridization, we identified some genesrelated to tongue squamous carcinoma stem cells, further study of thesegenes will provide crucial insights into their specific roles in thebiomolecular regulatory mechanisms of CSCs and help explain thedevelopment, recidivation and metabasis of oral squamous carcinoma.