Effects And Mechanism Of Silencing Arf6 With SiRNA On The Proliferation,Migration And Invasion Of The Human Prostate Cancer PC-3 Cell Line

BackgroundProstate cancer （PCa） is one of the most commonly diagnosed noncutaneous malignancy in men and secondary leading cause of cancer-associated mortality in males among western countries. Although the incidence of prostate cancer is relatively low in China, but the confirmed PCa is growing greatly in recent years, due to the diet habit and the changing life style. The morbidity of prostate cancer increased at an average annual rate of 12.07% between 1998-2008. Prostate cancer has become the highest incidence of cancer in the urinary system in China Since 2008.In China, Radical prostatectomy （RP） is generally used for treatment of early prostate cancer. The 5 years’survival rate of the patients is over 90%. The prostate cancer screening is not common and the majority prostate cancer patients have no symptoms at early stage, therefore,80-90% of patients were initially diagnosed with metastatic prostate cancer. At present, the mainly clinical treatment of prostate cancer metastasis is Androgen Deprivation Therapy （ADT）, which is less responsive to most patients. The median survival time is about 20 months. Metastatic prostate cancer is still the main obstacle to the treatment and the main cause of death in patients with prostate cancer. Metastasis of prostate cancer is an extremely complex biological process which requires polygenic participation and multiple stages. Cancer cells firstly invade and destroy the basement membrane and extracellular matrix, then intravasate into the blood vessels with the blood circulation migration to distant organ, and then tumor cell extravasation and adhesion to target organs, proliferate and form new tumors. Proliferation, migration and invasion are critical step of prostate cancer metastasis. Accordingly, better understanding of the molecules mechanism that regulates proliferative, migratory and invasive behaviors of prostate cancer cell is vital to provide novel therapeutic targets for prostate carcinoma.ADP-ribosylation factor （Arf） is the small GTP-binding protein belonging to the Ras superfamily. It was identified as a protein factor required for the ADP ribosylation of the adenylate cyclase activator Gs a by cholera toxin. Arf6, molecular weight about 20kD, located in the cell membrane and endosomal membrane, is different in its sequence and characteristics from other Arfs. Arf6 plays an important roles in endosomal recycling, exocytosis, endosytosis of the cell membrane, phospholipid metabolism, cytokinesis, intercellular adhesion formation and actin cytoskeleton reconstruction etc.Recent studies have found that Arf6 is highly expressed in many tumor tissues and cells, such as breast cancer, melanoma, and glioma, which are closely related to tumor cells migration, invasion and metastasis. Although the mechanism of Arf6 is to regulate all kinds of tumor cell motility, the invasion and migration remains to be studied. The decades studies have shown that Arf6 affect the tumor cell movement, invasion and migration ability mainly by following aspects. Firstly, the Arf6 activation could increase the E-cadherin internalization in tumor cells, and promote the destruction of cell-cell adhesion, which contribute to tumor cell epithelial-mesenchymal transition. Arf6 also regulates actin cytoskeleton remodeling of tumor cell, influences the formation of membrane protrusions involved in migration and invasion, including membrane ruffles, lamellipodia, filopodia, podosome and invadopodia. In addition, Arf6 regulates tumor cell invasion through the release of tumor cell-derived microvesicle containing proteolytic enzymes. Proteolytic enzymes adhesion to the extracellular matrix （ECM） is facilitated by microvesicle-associated integrin receptors, which enhance the distruction of extracellular matrix and increase the invasion and migration of tumor cells.Phosphoinositide 3-kinases （PI3K）/AKT signaling pathway plays a vital role in a wide variety of cellular processes such as cell survival, growth, translation, glucose metabolism, proliferation, migration and metastasis. Hyperactivity of the PI3K promotes cell proliferation, migration and invasion via phosphorylation of a downstream target of the serine/threonine protein kinase B （AKT）. A study found that Arf6 regulate the glioblastoma cell proliferation correlated with the PI3K/AKT signaling pathway.Extracellular signal-regulated kinase （ERR）, a member of the Mitogen-activated protein kinase （MAPK） family, is the primary signaling molecule regulating gene expression, cell differentiation, mitosis, survival, and apoptosis. Elevated expression of phosphorylated ERK （p-ERK） have been associated with increased tumor cell proliferation, invasion and metastasis. Arf6 can promote the proliferation of glioma cells by activating the ERK signaling pathway. The inhibition of Arf6 by siRNA inhibited the cell proliferation by decreasing EGF-induced ERK activation. In addition, previous study indicated that in HepG2 cells the p-ERK exerted the activity through Ras-related C3 botulinum toxin substrate 1 （Rac1）, a cytoskeleton reorganization to reform membrane protrusions and ruffles.Although previous studies showed Arf6 maybe associated with malignancy in many human tumors, the relationship between Arf6 and prostate cancer has not been reported. Whether there is a direct correlation between Arf6 activation and prostate cancer invasion, and the roles of Arf6 and its molecular mechanism in the migration and invasion of prostate cancer cell or not is still ambiguous. Here, we used human prostate cancer PC-3 cell to explore the effects of Arf6 depletion on prostate cancer cell proliferation, migration and invasion.ObjectiveIn this study, we used small interference RNA to inhibit the expression of Arf6, then observed the effects of Arf6 on proliferation, migration and invasion of androgen independent prostate cancer PC-3 cell line in vitro and preliminary discuss its molecular mechanism.Methods1、Design and identification of Arf6 siRNAâ‘ Three siRNA duplexes targeting different encoding regions of human Arf6 （GenBank:NM₀0 1663.3） were designed and synthesized. The sequences of siRNA for Arf6 were as follows:siRNA-1 （792-810）,5’-GGGACGCCAUAAUCCUCAU dTdT-3’（sense）,5’-AUGAGGAUUAUGGCGUCCCdTdT-3’（antisense）;siRNA-2（53 4-552）,5’-CAACAAUCCUGUACAAGUUdTdT-3’（sense）,5’-AACUUGUACAGG AUUGUUGdTdT-3’（antisense）, and siRNA-3 （950-968）,5’-CUCACAUGGUUAA CCUCUAdTdT-3’（sense）,5’-UAGAGGUUAACCAUGUGAGdTdT-3’（antisense）.â‘¡Cells, when approximately 60-70% confluent, were transfected with Arf6 siRNA in Lipofectamine（?）2000, according to the manufacturer’s instruction. One sequence which had better interfering effect on the expression of Arf6 was evaluated and selected through Real Time-PCR and Western Blot. Finally, the same method was used to determine the optimal concentration and the siRNA sequence.2、MTT assayPC-3 cells were divided into the Arf6 siRNA-3,the negative control siRNA （NC） and the parental cell group （Con）.The effect of siRNA on cell proliferation was examined for 48,72 and 96 h after transfection with siRNA-3 or negative control siRNA by the MTT assay following the manufacturer’s instructions.Cell growth curves were portrayed by using OD490 nm value of each group, and calculated cell inhibitory rate of siRNA-3 group. The cell survival rate（%） was calculated using the formula:Cell survival rate（%）=[OD （treatment）-OD （blank）/OD （con）-OD （blank）]× 100%. Inhibition rate （%）=1-cell survival rate （%）.3-. Wound healing assayCells grew to reach full confluence and then wounded by creating with a 10 μl pipette tip in a sterile environment. The wounded monolayers were then kept in 1% serum medium for 18 h and then photographed under an inverted phase-contrast microscope for each replicate at Oh,6h,12h and 18h respectively with a lOx objective lens. The migration distance was calculated using the formula:migration distance=（width0 h-Widthxh）/2. Wound covered rate=[（wound areaoh-wound areaxh） /wound areaoh]x 100%.4、Transwell migration and invasion assayUsing the Matrigel-uncoated and -coated transwell invasion assay, we investigated the migratory and invasive ability of single cell transfected with siRNA-3 for 48 h. After 24 h or 48 h incubation, migration and invasion were stopped by scraping the residual cells on the top chamber with a cotton swab. Cells on the lower membrane surface were fixed in 4% paraformaldehyde for 15 mins and stained with 1% crystal violet for 10 mins. Membrance was photographed at five randomly picked fields using microscope Olympus DP71 with a 10x objective lens.5、Western blot was used to examine the proteins related to the Arf6 signal pathwaysTo determine whether siRNA-3-induced silencing of Arf6 affects PC-3 cell proliferation, migration and invasion through the PI3K/AKT or ERK signal pathway, western blot was used to detect the protein expression levels of AKT, p-AKT, ERK 1/2,p-ERK1/2 and Racl in three groups.6、Statistical analysisValues reported in the results are mean values±standard deviation. Data were analyzed by Image J and statistical analyses were carried out using the SPSS software version 19.0. Data were analyzed by analysis of variance （ANOVA） （followed by post hoc analysis） or via Student’s t test to check the statistical difference among groups with P value less than 0.05 being considered significant.Results1±. Specific siRNA-3 suppressed the Arf6 RNA and protein expression in prostate cancer PC-3 cellsâ‘ There was no remarkable difference of Arf6 RNA and protein between the cells transfected with negative control siRNA and parental PC-3 cells, which revealed that negative control siRNA did not affect the expression of Arf6 RNA and protein. The cells transfected with siRNA-1, siRNA-2 or siRNA-3 were decreased the Arf6 RNA expression （34.82 ± 4.79）%, （56.85 ± 1.52）% and （91.88 ± 3.13）%, respectively, and the protein expression （25.73 ± 1.25）%, （67.11±1.08）% and （86.37 ± 0.57）%. Results indicated that siRNA-3 exerted the most efficient in suppressing the Arf6 RNA and protein expression in PC-3 cells.â‘¡ Moreover, PC-3 cells transfected with different concentrations （10 nmol/L, 20 nmol/L and 50 nmol/L）of siRNA-3 for 48 h, observed a decrease of Arf6 RNA and protein expression in a dose-dependent manner. In addition, the Arf6 RNA and protein expression was suppressed after 48 h and suppression was stable up to 96 h after transfection by 50 nmol/L of siRNA-3.2、 siRNA-3-induced silencing of Arf6 inhibited PC-3 cellular proliferationThe rate of cell proliferation was examined for 48,72 and 96 h following transfection with siRNA-3 （50 nmol/L） or negative control siRNA. Cell growth curves were portrayed by using OD490 nm value of each group, and calculated cell inhibitory rate of siRNA-3 group. The inhibition rate of cell proliferation were （10.68±0.04）%, （16.51 ± 0.04）% and （26.35 ± 0.03）% in the siRNA-3 group at 48, 72 and 96 h, respectively. Results revealed that the inhibitory effect of siRNA-3 on PC-3 cell proliferation initiated at 48 h, and the proliferation of PC-3 cells was significantly inhibited at 96 h in the siRNA-3 group. These results indicated that silencing of Arf6 could inhibit PC-3 cell proliferation in a time-dependent manner.3、 siRNA-3-induced silencing of Arf6 decreased migration and invasion of PC-3 cells￤ound healing assay showed that silencing of Arf6 by siRN A-3 significantly reduced the migration distance of PC-3 cells at the end of 18 h compared with the parental cells and the cells transfected with negative control siRNA （P<0.001）. The cells transfected with siRNA-3 covered only about 45% to 50% of surface covered by parental PC-3 cells at the end of 18 h. There was no remarkable difference between the cells transfected with negative control siRNA and parental PC-3 cells （P >0.05）.â‘¡ The Matrigel-uncoated Transwell migration assay show that PC-3 cells transfected with siRNA-3 migrated across 8 micro pores were decreased 61.61% when compared to parental cells and the NC group （P<0.05）. The Matrigel-coated transwell membrane is the most commonly used model for the invasion of tumor cells traverses this basement membrane. Accordingly, transwell invasion assay was employed to examine the effect of siRNA-3 silencing of Arf6 on PC-3 cells invasion capacity. Results showed that siRNA-3-induced silencing of Arf6 reduced cell invasion by 89.44%（P<0.05） in the PC-3 cells compared with parental cells and the NC group. No remarkable difference was detected between the cells transfected with negative control siRNA and parental cells （P>0.05）. These results revealed that siRNA-3-induced silencing of Arf6 significantly reduced the migratory and invasive capacity of PC-3 cells in vitro.4、siRNA-3-induced silencing of Arf6 inhibited ERK and Rac1 activationThese results indicated that the expression level of AKT, p-AKT, and ERK1/2 were remained unchanged, but p-ERK1/2 and Rac1 expression was remarkable reduced in the PC-3 cells after transfection with siRNA-3 （50 nmol/L） for 48 h, while there were no statistical changes in parental cells and negative control siRNA transfected cells.Conclusions1、The specific siRNA can significantly down-regulate the expression of Arf6 both in the level of RNA and protein.2、Down-regulation of Arf6 expression can remarkably inhibit proliferation, migration and invasion of PC-3 cell in vitro.3、The proposed mechanism underlying the role of Arf6 in PC-3 cells was correlated with down-regulation of p-ERK1/2 and Rac1.