The gene FAT1 produces a
high molecular weight (~500 kD) membrane
protein which is a member of cadherin superfamily. Squamous cells are the flat
cells that line the cavity of oral and lips. Additionally, most oral cancers
occur in squamous cells. The squamous cells that cancer occurred are referred
as Squamous Cell Carcinoma. The aim of the study conducted at 2011 by Yukiko Nishikawa,
Miyazaki, Nakashiro, Yamagata, Isokane, Goda, Tanaka, Oka and Hamakawa; was to
examine the mechanisms that FAT1 takes role in the biological behavior of
OSCCs. Cytoplasmic domain of the gene FAT1 binds with ?-catenin. The domain has
a key role in cell polarity, morphology and migration. Hence, a deletion
occurrence may embroil the characteristics of OSCCs including cell migration
and adhesion which eventually cause prognosis to be insufficient. In addition,
FAT1 gene is also evaluated as a tumor suppressor gene. Ever since human FAT1
was duplicated as the homolog of a tumor suppressor gene in Drosophila, there
have been no research done which examined the tumor suppressor function or the
clinical associations of FAT1 yet.

Firstly, Western blot analysis was performed to
confirm anti-FAT1 antibody monoclonality. With the aim of synthesizing whole
molecule, extracting intact FAT1 from tissue was hard due to the fact molecular
weight of it being too big. So analysis on the synthesized fragments of FAT1
molecule is conducted. The crude and purified proteins were separated via
electrophoresis and then transbloted to PVDF (Polyvinylidene difluoride)
membranes. Then the PVDF membranes got blocked overnight at 4°C to prevent
non-specific bindings. After that probing is done by using monoclonal rat
anti-FAT1 antibodies then followed by usage of horseradish
peroxidase-conjugated secondary goat antibodies against rat IgG. The complexes
got visualized by using enhanced chemiluminescence. Secondly; the direct immunofluorescence of FAT1 staining is
done by using zenon® Alexa Fluor 488 Monoclonal Antibody labeling kit®. Double
immunofluorescence against ?-catenin is done in a simultaneous way by using
Alexa 647-conjugated Donkey anti-mouse and murine anti-human ?-catenin IgG
antibodies. The nuclei of the cell were stained with by Hoechst 33342®. The
Immunofluorescence got viewed and captured with a confocal
ultra-spectral-microscope system A1®. Thirdly, design and transfection of synthetic siRNAs are executed by
designing and producing three pairs of FAT1 specific synthetic siRNAs. B-Algo
and siPrecise were used while choosing the target sequence in order to have
high rate of target gene silencing alongside low rate of off-target effects. Transfection
is done by using 10nM siRNA with lipofectamine RNAiMax (Invitrogen) in the
assays. Cell proliferation analysis was
also executed by starting as the cells being planted and incubated for 4 days
in 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-time
RT-PCR was carried out using the comparative CT method (??CT method) to
quantify the mRNA levels with One Step SYBR PrimeScript RT-PCR kit II in a
lightCycler 1.5 System. As internal control Hydroxymethylbilane synthase (HMBS)
mRNA was used. The reaction mixture contained: forward and reverse primers, 2X
One-step SYBR RT-PCR buffer 4, PrimeScript® One-step enzyme mix 2, RNase Free water
dH2o and total RNA. The conditions of reaction were as for RT step holding at 42?C
for 5 minutes and for denaturation hold at 95?C for 10 seconds, followed by 40
PCR cycles of 95?C for 5 seconds and 60?C for 20 seconds. As last of RT-PCR, a
melting-curve analysis is performed by adjusting the heat to 95?C without
staying 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 finally
cooling to 40?C. In vitro wound-healing
assay got also performed where a 35-mm collagen-coated glass bottom dish
was covered with OSCC cells with a high density. Then for the next 3 days, the
dishes were cultured with or without siRNA against FAT1. By using the tip of a
plastic pipette, the monolayers of the cells scarred. After eight hours the
cells were fixed and stained. Lastly, the wound widths were evaluated using
NIS-element software program. As last in vitro cell invasion assay, CytoSelect™ Cell Migration and Invasion assay
kit was used to execute cell invasion assay. High density of OSCC cells with or
without siRNA transfection got plated on the inner chamber that contained matrix
coated 8µm pores with serum-free DMEM. After that, the cells got incubated for
24 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 alongside
FlexStation III fluorescent microplate reader (Nishikawa et al. 2011).

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Nishikawa et al. analyzed
the expression levels of mRNAs of FAT1, which exist in human OSCC cell lines with
RT-PCR. The levels showed diverse results for all cultured cell lines except
SAS and SCC66. Additionally, immunofluorescence was conducted with the same
aim. However, alike result got yield. Results got from double
immunofluorescence done with 3 FAT1-positive and 1 FAT1-negative cell lines
showed that ?-catenin is localized in the cell membrane of FAT1-expressing
OSCCs, however in the FAT1-negative cell since the gene is absent, ?-catenin’s
got 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 mentioned
before; the gene FAT1 can be considered to be as tumor suppressor.
Additionally, the gene can regulate cell proliferation in an indirect way due
to the fact expression of gene having a reverse correlation with proliferation
in vascular muscle cells. Thus, it was predicted that suppression of the gene
would also raise the proliferation of oral squamous cell carcinomas (OSCCs).  However, in this study when the mRNAs of FAT1
genes were silenced, circumscribed effects were observed on the proliferation
of OSCC cell lines. Therefore, it can be concluded that FAT cadherin has a more
minor role for cell proliferation of OSCC when it is compared with other tumor
suppressor genes. Occurrence of deletion in the product of the gene FAT1 might
have a major role in the metamorphosis of various characteristics of carcinoma
cells including; decrement of cell to cell adhesion, morphology and cell
polarity. One of the characteristic roles of gene FAT1 products is to maintain
morphology and polarity by collaborating with the stress fibers ?-catenin and
Vasp/Ena which binds with the cytoplasmic domain of the gene. Furthermore, the
gene contains a nuclear localization signal sequence and by translocating its
cytoplasmic domain into the nucleus, cell polarity and migration cell polarity
and migration can occur as result. This study also revealed that, when the gene
FAT1 was silenced a significant reduction of cell to cell adhesion occurred
alongside alteration in cell morphology due to the fact dispersed settlement
of ?-catenin. These results propose that deletions occurring in FAT1 gene
contributes to cancerous dispersed morphology. Consequently, restricted cell to
cell adhesion may bring about tumors which can effectively scatter and
metastasize. At the same time; in wound healing and cell migration assays, silencing
of the gene resulted as repressed mobility of OSCCs.

Overall, the discoveries
obtained from the study propose that, inhibition of FAT1 gene in OSCCs may outcome
as repression of tumor migration. Thus the gene can be evaluated as a possible
target for new therapeutic ways to have better prognosis of OSCCs (Nishikawa et
al. 2011).

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