Seminario Biologia Molecular

2. Introduction
Cancer: An abnormal growth of
cells which tend to proliferate in an
uncontrolled way and, in some
cases, to metastasize
One of those is
Is the fourth most
commonly diagnosed
cancer and the third
leading cause of cancer
death among females.
Characteristics:
• forms in tissues of the cervix
(the organ connecting the
uterus and vagina).
• It is usually a slow-growing
cancer
• May not have symptoms
• One of the causes is the
human papillomavirus (HPV)
infection.

3. ■ A member of the FHL protein
family.
■ Is an adaptor molecule.
■ Can interact with many different
proteins to play diverse roles in
regulating cell survival,
proliferation, differentiation,
adhesion, and motility as well as
gene expression and signal
transduction.
■ the role of FHL2 in CC remains
unclear
■ Numerous studies have reported that
FHL2 might act as a tumour suppressor
in a cell-type-dependent manner in
human cancers because FHL2 can
function as a transcriptional co-
activator for several transcription
factors and can also serve as a
transcriptional co-repressor.
■ Previous evidence has demonstrated
that FHL2 is overexpressed in breast
cancer, gastrointestinal cancer, prostate
cancer and glioblastoma, though low
expression levels have been observed in
human rhabdomyosarcoma, liver
cancer and neuroblastoma.

4. Objective
This study aimed to investigate the
prognostic significance of
FHL2 expression in CC and its effects on
cell proliferation, apoptosis
and tumorigenicity via in vivo and in vitro
analyses. Furthermore, we
preliminarily explored the role of FHL2 and
the possible molecular
signaling pathway underlying its
involvement in CC development.

5. MATERIALS AND METHODS
PATIENTSANDTISSUE
SPECIMENS
A group of
samples stored
at - 80° C.
A group of
samples was
fixed with
formalin and
embedded in
paraffinNumber of patients: 52
Age: 39-71 Diagnostics: CC

6. The CC human
cell lines were
maintained in
Dulbecco's
modified
Eagle.
It contained
Remained
in incubator
10% fetal
bovine serum
37°with a
humidified 5%
CO2
atmosphere.
CELL CULTURE

7. M
E
T
H
O
D
S
Immunohistochemical
(IHC) staining
Small interfering RNA
(siRNA) and transient
transfection
Artificially introduce DNA or RNA
into cells to generate changes in
the characteristics of the cell.
Identification of a tissue through a specific
interaction between an antigen and an antibody.
48 hours after
Transfection, were
Measured:
Expression of
proteins
Cell viability.
Apoptosis
In this case was to identify tumor cell.

8. A
N
A
L
Y
S
I
S
Western
blot
Apoptosis
A series of molecular processes that lead to cell death. In
cancer cells, it may be locked.
CELLS Was analysed
using a FACScan
flow cytometer
Equal amounts of total protein
were separated by sodium
dodecyl sulphate-
polyacrylamide gel
electrophoresis (SDS-PAGE),
then the proteins are
incubated with antibodies that
react with the interest protein
In this case was to identify
specific proteins from a
complex mixture of
proteins extracted from
cells.

9. Results
The expression
of FHL2 in the
adjacent normal
cervical tissue is
less than cancer
tissues.
FHL2 was
upregulated in
patients' samples
at protein level by
Western blotting.
Use B-actin like
load control.
1 2

10. 3
FHL2 protein levels was assessed in CC
cell lines (CaSki, SiHa, HeLa and C33a).
The results showed that FHL2 protein
levels were higher in CaSki and HeLa
cells than in SiHa and C33a cells.
To select the best FHL2 siRNA, the inhibition efficiencies
of three different siRNAs (siRNA-485, siRNA-623 and
siRNA-955) were determined in CaSki and HeLa cells.
Compared with the control scrambled siRNA, all siRNAs
showed significant inhibition efficiency in both cell
lines.
The analysis showed that CaSki and HeLa cells
transfected with siRNA-955 had significantly
higher levels of apoptosis than did NC-treated
cells
4
5

11. 6
7
FHL2 knockdown in CC cells caused reduced
levels of p-AKT and p-mTOR.
AKT/mTOR pathway plays an important role in
regulating apoptosis and proliferation.
FHL2 knockdown attenuates CC cell proliferation.To further confirm the role of FHL2 in
cell proliferation, FHL2 was transiently overexpressed in CaSki and HeLa cells, and cell
viability was evaluated. FHL2 was successfully overexpressed (as FLAGFHL2) in both cell
lines.
6

12. 8
9
10
GFP detection via
fluorescence microscopy
showed high infection
efficiency in Hela cells by
transfected FHL2 shRNA.
FHL2 protein expression was
detected byWestern blotting.
Western blotting confirmed
that FHL2 protein levels were
decreased in xenograft
tumour tissues.

13. Previous studies have reported that FHL2 levels are higher in
numerous cancers than in normal tissues, and such overexpression
has been associated with poor prognosis.
(Zienert et al., 2015).
Regarding the role of FHL2 in CC, Jin et al. found it to be
significantly overexpressed in the squamous epithelium of CC
tissues and that FHL2 regulated CC cell proliferation by facilitating
MDM2- mediated degradation of immediate early response 3 (IER3)
(Jin et al.,2016).
Numerous studies have shown that FHL2 is a critical factor
affecting the biological be- haviours of cancer cells. In
osteosarcoma, colon cancer and ovarian granulosa cell tumours,
FHL2 knockdown suppresses tumour cell growth, reduces cell
viability and inhibits cell migration (Zhang et al., 2010; Brun et al.,
2013; Hua et al., 2016).
Discussion

14. Conclusion
• FHL2 is overexpressed in the tissues of patients
with CC.
• FHL2 can have either a tumour-promoting or
tumour-suppressing role in tumorigenesis and
development, and this dual role is dependent on
the type of tumour cell.
• FHL2 might be a potencial target for cancer
therapy. However, more research is needed to
confirm the specific role of FHL2 in different
tumours.