1. Maternal and Children Health Care of Shanxi Province
Silencing Folate Receptor alpha Induced Proliferation Decrease and Apoptosis Increase of Cervical Cancer Cells through Mediating the ERK Signaling Pathway FRα介导ERK信号通路对宫颈癌细胞增殖和凋亡的作用
Maternal and Children Health Care of Shanxi Province
Children’s Hospital of Shanxi Province
山西省妇幼保健院 山西省儿童医院
Dr. Li-xia Bai 白丽霞

2. Contents
Introduction
Objective
Methods
Results
Conclusion

3. Introduction
Cervical cancer is the fourth most common malignancy in women in the world and it remains a leading cause of cancer-related death of women.
HPV is considered as the primary risk factor in the development of cervical cancer. However, only HPV infection may not induce cervical cancer, many cofactors of cervical cancer have been established for female with persistent infection.

4. However, the relationship of FRαand cervical cancer is unclear.
Introduction
Folate receptor alpha (FRα), called as folate binding protein (FBP), is a membrane-bound protein linked to cell surfaces via a glycosylphosphatydilinositol (GPI) anchor.
FRα has a high affinity to mediate
folate uptake by endocytosis.
FRαis highly expressed in human
cancer tissues and cells, but lower in normal tissues.
FRαis highly expressed in human cervical cancer Hela cells.
However, the relationship of FRαand cervical cancer is unclear.

5. Introduction
Raf
MEK
ERK signaling pathway has been reported in many human carcinomas, but the association between ERK signaling pathway and cervical cancer is inconclusive.
ERK1 and ERK2 proteins are the important factors in ERK signaling pathway and associate with many cancers.
ERk1/2 is highly expressed in human cervical cancer.
ERK
We assumed that cervical cancer might associated with ERK signaling pathway.

6. Introduction
It is reported that both FR-targeted liposomes loaded with carboplatin or empty FR-targeted liposomes exhibited the significantly increase ERK phosphorylation (Chaudhury A et al, 2012).
We assumed that FRαassociated
with ERK protein.

7. Introduction Src protein is one of the members of Src family kinase.
Src  protein could be activated by cell membrane receptors, such as growth factor receptor and integrin receptor and G protein coupled receptor.
Folate
FRα
We assumed that Src protein could be activated when FRαmediates folate uptake by endocytosis.
Src family
kinase

8. Introduction P
Src
Phosphorylated Src protein could further activate the ERK signaling pathway.
When phosphorylated ERK1/2 enter the nucleus, many transcription factors could be phosphorylated, such as c-Fos, c-Jun, c-myc, cyclin-D1 and Bcl-2.
ERK signaling pathway P P
ERK1
ERK2 P P
c-Jun
cyclin-D1 P P P
c-Fos
c-myc
Bcl-2
Nucleus

9. Introduction
The c-Fos and c-Jun are the major nuclear transcription proteins and they usually play a key role in cell proliferation and apoptosis.
It is reported that the c-Fos and c-Jun are high expressed in cervical cancer tissues, while lower in normal tissues.
We assumed that c-Fos and c-Jun might have a
role in FRαmediating ERK pathway.

10. ？ ？ Cervical cancer Folate FRα Src FRα Involved in cervical cancer
progression
through
mediating
the
ERK
signaling
pathway
Src
Cervical
cancer
Raf ？
MEK
ERK1
ERK
ERK2
Bcl-2
c-Fos
cyclin-D1
c-myc
c-Jun

11. Objective
The aim of the present study was to evaluate the hypothesis that FRαinvolved in cervical cancer progression through mediating the ERK signaling pathway in vitro.

12. Experiment program Hela、Siha、Caski、C33A screening
FRα siRNA interference
FRαPositive cells：Hela
Positive FRα
siRNA interference group
Negative FRα
siRNA interference grouop
No interference
group
Biological Characteristics
FRαmRNA and protein levels
ERK signaling
pathway factors
Cells proliferation
Cells cycle
Cells apoptosis
FRα mRNA and protein levels
ERK、C-fos、C-jun
mRNA and protein levels
The role of FRαfor cervical cancer Hela cells through mediating ERK signaling pathway

13. Methods FRα was silenced by siRNA interference in Hela cells.
Cell proliferation was measured by CCK8.
FRα protein level and cell cycle and apoptosis were measured by Flow cytometry (FCM).
The mRNA level of FRαwas detected by RT-PCR.
The protein levels of ERK1/2 and c-fos and c-jun were measured by Western Blot.
All experiments were independently repeated three times.
SPSS 16.0 software package was used for statistical analysis.

14. Results FRαpositive cells
time and concentration
FRαpositive siRNA sequence
Screen for the best FRαpositive siRNA sequence
The effects of down-regulation of FRαon biological functions
the proliferation
cell cycle
apoptosis
The effects of down-regulation of FRα on ERK signaling pathway
ERK1/2 and p-ERK1/2
c-Fos and p-c-Fos protein
c-Jun and p-c-Jun protein

15. 1. Screen for FRαpositive cervical cancer cells
The
strongest
Positive
cell
Hela
Siha
weak
positive
Caski
C33A
Figure 1: FRαexpression of cervical cancer cells

16. The transfection rate was above 90% at 48h after transfection
2. Screen for the best time and concentration of FRαsiRNA transfection for Hela cell
The transfection rate was above 90%
at 48h after transfection
（48h，10 x 10）
（48h，10 x 10）
Figure 2: The transfection effect pictures of Hela cells after 48h transfected with FRαsiRNA sequence

17. 40 µg/ml group showed the highest transfection rate
2. Screen for the best time and concentration of FRαsiRNA transfection for Hela cell
(95%)
40 µg/ml group showed the highest transfection rate
Figure 3：The transfection rates of different concentration of FRαsiRNA sequences interference groups

18. 3. Screen for the best FRαpositive siRNA sequence
FRα protein level
was decreased dominantly by siRNA sequence1 *
Figure 4：The protein expression levels of FRαin the different siRNA interference groups

19. 3. Screen for the best FRαpositive siRNA sequence*
The mRNA level of FRα was also decreased dominantly
by siRNA sequence1
Figure 5：The relative mRNA expression level of FRαin different groups

20. 4. Down-regulation of FRα inhibited the proliferation
the inhibition rate of siRNA sequence 1 group at different time points were 43.9%, 71.5%, 72.7% and 80.5%, respectively
Figure 6: The effect of down-regulation of FRα
on the proliferation of Hela cells

21. 5. The effects of down-regulation of FRα on cell cycle
G0/G1
G2/M S PI
Figure 7: The effect of down-regulation of FRα
on the cell cycle of Hela cells

22. 6. Down-regulation of FRα induced apoptosis
Figure 8: The effect of down-regulation of FRα
on the apoptosis of Hela cells

23. 7. Down-regulation of FRα decreased the p-ERK1/2 protein level
Figure 9：The effects of down-regulation of FRα
on the expression of ERK1/2 and p-ERK1/2 protein

24. 8. Down-regulation of FRα decreased the c-Fos and p-c-Fos protein levels
Figure 10：The effects of down-regulation of FRα
on the expression of c-Fos and p-c-Fos protein

25. 9. Down-regulation of FRα decreased the c-Jun and p-c-Jun protein levels
Figure 11：The effects of down-regulation of FRα
on the expression of c-Jun and p-c-Jun protein

26. Conclusion
Hela is the strongest FRαpositive cell，the next are Siha and Caski, and C33A is the weakest.
Down-regulating FRαinhibited the biological functions of cervical cancer Hela cells.
Down-regulating FRα might block cervical cancer progression through mediating ERK signaling pathway.
FRα might be responsible for developing a new therapeutic target for cervical cancer.

27. Thank you! This study was supported by
National Natural Science Foundation of China
(Grant No. No )