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

Contents Introduction Objective Methods Results Conclusion

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.

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.

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.

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.

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

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

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.

？ ？ 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

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.

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

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.

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

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

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

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

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

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

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

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

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

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

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

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

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.

Thank you! This study was supported by National Natural Science Foundation of China (Grant No. No.81273157)