The gene FAT1 produces ahigh molecular weight (~500 kD) membraneprotein which is a member of cadherin superfamily. Squamous cells are the flatcells that line the cavity of oral and lips.
Additionally, most oral cancersoccur in squamous cells. The squamous cells that cancer occurred are referredas Squamous Cell Carcinoma. The aim of the study conducted at 2011 by Yukiko Nishikawa,Miyazaki, Nakashiro, Yamagata, Isokane, Goda, Tanaka, Oka and Hamakawa; was toexamine the mechanisms that FAT1 takes role in the biological behavior ofOSCCs. Cytoplasmic domain of the gene FAT1 binds with ?-catenin. The domain hasa key role in cell polarity, morphology and migration.
Hence, a deletionoccurrence may embroil the characteristics of OSCCs including cell migrationand adhesion which eventually cause prognosis to be insufficient. In addition,FAT1 gene is also evaluated as a tumor suppressor gene. Ever since human FAT1was duplicated as the homolog of a tumor suppressor gene in Drosophila, therehave been no research done which examined the tumor suppressor function or theclinical associations of FAT1 yet.Firstly, Western blot analysis was performed toconfirm anti-FAT1 antibody monoclonality. With the aim of synthesizing wholemolecule, extracting intact FAT1 from tissue was hard due to the fact molecularweight of it being too big. So analysis on the synthesized fragments of FAT1molecule is conducted. The crude and purified proteins were separated viaelectrophoresis and then transbloted to PVDF (Polyvinylidene difluoride)membranes. Then the PVDF membranes got blocked overnight at 4°C to preventnon-specific bindings.
After that probing is done by using monoclonal ratanti-FAT1 antibodies then followed by usage of horseradishperoxidase-conjugated secondary goat antibodies against rat IgG. The complexesgot visualized by using enhanced chemiluminescence. Secondly; the direct immunofluorescence of FAT1 staining isdone by using zenon® Alexa Fluor 488 Monoclonal Antibody labeling kit®.
Doubleimmunofluorescence against ?-catenin is done in a simultaneous way by usingAlexa 647-conjugated Donkey anti-mouse and murine anti-human ?-catenin IgGantibodies. The nuclei of the cell were stained with by Hoechst 33342®. TheImmunofluorescence got viewed and captured with a confocalultra-spectral-microscope system A1®.
Thirdly, design and transfection of synthetic siRNAs are executed bydesigning and producing three pairs of FAT1 specific synthetic siRNAs. B-Algoand siPrecise were used while choosing the target sequence in order to havehigh rate of target gene silencing alongside low rate of off-target effects. Transfectionis done by using 10nM siRNA with lipofectamine RNAiMax (Invitrogen) in theassays. Cell proliferation analysis wasalso executed by starting as the cells being planted and incubated for 4 daysin 60-mm dishes which contain complete medium, siRNA and lipofectamine RNAiMax.Then to collect, the cells were treated with 0.05% trypsin, 0.53mM EDTA (wako)and counted by using a z1 Coulter counter.
Real-timeRT-PCR was carried out using the comparative CT method (??CT method) toquantify the mRNA levels with One Step SYBR PrimeScript RT-PCR kit II in alightCycler 1.5 System. As internal control Hydroxymethylbilane synthase (HMBS)mRNA was used.
The reaction mixture contained: forward and reverse primers, 2XOne-step SYBR RT-PCR buffer 4, PrimeScript® One-step enzyme mix 2, RNase Free waterdH2o and total RNA. The conditions of reaction were as for RT step holding at 42?Cfor 5 minutes and for denaturation hold at 95?C for 10 seconds, followed by 40PCR cycles of 95?C for 5 seconds and 60?C for 20 seconds. As last of RT-PCR, amelting-curve analysis is performed by adjusting the heat to 95?C withoutstaying at this temperature rapidly changing to 65?C and holding for 15 seconds,then increasing the temperature gradually to 95?C at 0.1?C per second, and finallycooling to 40?C. In vitro wound-healingassay got also performed where a 35-mm collagen-coated glass bottom dishwas covered with OSCC cells with a high density.
Then for the next 3 days, thedishes were cultured with or without siRNA against FAT1. By using the tip of aplastic pipette, the monolayers of the cells scarred. After eight hours thecells were fixed and stained. Lastly, the wound widths were evaluated usingNIS-element software program. As last in vitro cell invasion assay, CytoSelect™ Cell Migration and Invasion assaykit was used to execute cell invasion assay. High density of OSCC cells with orwithout siRNA transfection got plated on the inner chamber that contained matrixcoated 8µm pores with serum-free DMEM. After that, the cells got incubated for24 hours. The cells which migrated into the outer chamber, that contains 10%FBS in DMEM, got lysed and quantified by using Cyquant® GR fluorescent dye alongsideFlexStation III fluorescent microplate reader (Nishikawa et al.
2011).Nishikawa et al. analyzedthe expression levels of mRNAs of FAT1, which exist in human OSCC cell lines withRT-PCR. The levels showed diverse results for all cultured cell lines exceptSAS and SCC66. Additionally, immunofluorescence was conducted with the sameaim.
However, alike result got yield. Results got from doubleimmunofluorescence done with 3 FAT1-positive and 1 FAT1-negative cell linesshowed that ?-catenin is localized in the cell membrane of FAT1-expressingOSCCs, however in the FAT1-negative cell since the gene is absent, ?-catenin’sgot present only in cytoplasm and nuclei due to cell to cell adhesion being lack,hence have a undifferentiated shape of cells. In the light of points mentionedbefore; the gene FAT1 can be considered to be as tumor suppressor.Additionally, the gene can regulate cell proliferation in an indirect way dueto the fact expression of gene having a reverse correlation with proliferationin vascular muscle cells. Thus, it was predicted that suppression of the genewould also raise the proliferation of oral squamous cell carcinomas (OSCCs). However, in this study when the mRNAs of FAT1genes were silenced, circumscribed effects were observed on the proliferationof OSCC cell lines. Therefore, it can be concluded that FAT cadherin has a moreminor role for cell proliferation of OSCC when it is compared with other tumorsuppressor genes.
Occurrence of deletion in the product of the gene FAT1 mighthave a major role in the metamorphosis of various characteristics of carcinomacells including; decrement of cell to cell adhesion, morphology and cellpolarity. One of the characteristic roles of gene FAT1 products is to maintainmorphology and polarity by collaborating with the stress fibers ?-catenin andVasp/Ena which binds with the cytoplasmic domain of the gene. Furthermore, thegene contains a nuclear localization signal sequence and by translocating itscytoplasmic domain into the nucleus, cell polarity and migration cell polarityand migration can occur as result. This study also revealed that, when the geneFAT1 was silenced a significant reduction of cell to cell adhesion occurredalongside alteration in cell morphology due to the fact dispersed settlementof ?-catenin. These results propose that deletions occurring in FAT1 genecontributes to cancerous dispersed morphology. Consequently, restricted cell tocell adhesion may bring about tumors which can effectively scatter andmetastasize. At the same time; in wound healing and cell migration assays, silencingof the gene resulted as repressed mobility of OSCCs.Overall, the discoveriesobtained from the study propose that, inhibition of FAT1 gene in OSCCs may outcomeas repression of tumor migration.
Thus the gene can be evaluated as a possibletarget for new therapeutic ways to have better prognosis of OSCCs (Nishikawa etal. 2011).