The Initial Investigation Of Wnt Signal Pathway's Potential Role On Human Nephroblastoma Cell Line SK-NEP-1

Backgrounds Nephroblastoma is the most common primary malignant tumor of kidney in children.It accounts for about 8 percent of all pediatric malignant tumors and it is confirmed as a kind of embryonal tumor.Wnt signal pathway is a conservative pathway in organic evolutions.It participates in the embryonal development of many organs and is accommodated strictively.In recent years,wnt signal pathway has been found to play a key role in tumorigenesis.The extensive activation of wnt signal pathway is common in many tumors and the relationship between wnt signal pathway and tumor is becoming more and more attractive. Therefore,wnt signal pathway,the common pathway of embryonal development and tumorigenesis,maybe become the clue to reveal the mechanism of nephroblastoma.If any new anticancer target would be found in wnt signal pathway,it could be significant to improve the therapeutic effect and survival rate of nephroblastoma.Up to now,such kind of research has not been reported in the world.Purpose We cultured nephroblastoma cell line SK-NEP-1 in vitro and detected the expression of wnt related genes and proteins to confirm the activation of wnt signal pathway.After that,we investigated the effect of sFRP-1 on the proliferation of SK-NEP-1 and the role of siRNAs againstβ-catenin on the proliferation and apoptosis of SK-NEP-1 respectively.The expression level of downstream genes and proteins concerned with wnt signal pathway in each group was detected by real time PCR to explor the relationship ofβ-catenin and the proliferation of nephroblastoma.We also observed the effect of 17β-estradiol and bisphenol A on the proliferation and apoptosis of SK-NEP-1 and detected the expression change of downstream genes and proteins concerned with wnt signal pathway.We would try to explore the role of wnt signal pathway on nephroblastoma cell line SK-NEP-1 and its possible mechanism.It would conduce to find new targets for targeted therapy of Wilms'tumor and provide the scientific evidence.Materials and Methods1.Nephroblastoma cell line SK-NEP-1 culture and detection of wnt signal pathway's activation:SK-NEP-1 cell line was cultured in McCoys 5A medium with 15%fetal bovine serum and 1%mycillin under routine condition.Growth curve of SK-NEP-1 was obtained by viable cell counting at each time-point with hemocytometer and trypan blue exclusion.The expression of Vimentin,β-catenin, WT1 and CK in cell line were tested by Envision immunohistochemistry.The RT-PCR was applied to detect the mRNA level of WNT4,sFRP1,CTNNB1,CCND1,MYC genes in SK-NEP-1 and 293 cells respectively.The effect of sFRP-1 on Wnt signal pathway and the proliferation of SK-NEP-1 cells was tested by CCK-8 kit.Cells were divided into control group,low-dose group of sFRP-1(100～500ng/mL),high-dose group of sFRP-1(10μg/mL),high-dose group of Anti sFRP-1 (10μg/mL).The sFRP-1 at different concentrations were added to complete growth medium and the absorbance value(AV) was tested at 0,24,48,72hours after interference respectively.2.Transient transfection with siRNAs againstβ-catenin into SK-NEP-1 cell: Transfecection of siRNA againstβ-catenin was implemented by HiPerFect Transfection Reagant according to the manufacturer's instructions.Cells were typically seeded in six-well plates(400,000 cells per well) before transfection. Transfection complexes were prepared in serum-flee medium(McCoys 5A). Immediately before transfection,complete growth medium was removed from all wells,and wells were rinsed with PBS.Cells were typically exposed to transfecion complexes for 6 hours before replacement of complete growth medium depending on the particular experiment.The concentration of each siRNAs within HiPerFect is indicated in each experiment.Cells were divided into four groups:no transfection group(UT),negative control group(NS),siRNA1 group(S1),siRNA2 group (S2).The effect of siRNAs againstβ-catenin on the proliferation and apoptosis of SK-NEP-1 were tested by CCK-8 kit and flow cytometry at each time-point respectively.The change of wnt signal pathway were detected by real time PCR and immunofluorescence.3.The effect of 17β-estradiol and bisphenol A on wnt signal pathway and the proliferation of SK-NEP-1 cells:Cells were divided into three groups and different concentrations of 17β-estradiol and bisphenol A were added into each group respectively.The AV value at 48 hours was tested to discriminate the effective concentration of 17β-estradiol and bisphenol A on the proliferation of SK-NEP-1. Their effect on apoptosis of SK-NEP-1 was tested by flow cytometry.The change of wnt signal pathway was detected by real time PCR and immunofluorescence.Results1.Nephroblastoma cell line SK-NEP-1 culture and detection of wnt signal pathway's activation:SK-NEP-1 cell line was suspension cell and its double time was about 24 hours.The expression of Vimentin,β-catenin,WT1 and CK were all detected by Envision immunohistochemistry.The RT-PCR confirmed the activation of Wnt signal pathway and that the mRNA level of WNT4 in SK-NEP-1 was very low whereas sFRP-1 and CTNNB1 were extremely high,which were similar to 293 cell line.The result of sFRP-1 on wnt signal pathway and proliferation of SK-NEP-1 cells showed that sFRP-1 at low concentrations of 100 to 500 ng/mL and Anti sFRP-1 at high-dose concentration had no effect on proliferation.Whereas sFRP-1 at high concentrations of 10ug/mL could inhibit the growth of SK-NEP-1.The AV value of control group was 1.23 times more than sFRP-1 group at 24 hours time-point(p＜0.05) and at 48 hours time-point it was 1.25 times(p＜0.05).72hours later the AV value between groups had no difference((P＞0.05,Oneway,respectively).2.Transient transfection with siRNAs againstβ-catenin into SK-NEP-1 cell: Optimization of transfection indicated that the satisfactory transfection efficiency (＞70%) could be obtained with cell density at 4.0×10~5/mL～1.0×10~6/mL and 6ul Hiperfect Transfection Reagant per 100ul transfection complexes in 24 orifice plate system.Growth inhibition was obvious when the working concentration of siRNAs againstβ-catenin reached 100 nM.24 hours after transfection,the AV value of UT was 1.25 times of S1 and 1.23 times of S2 respectively(p＜0.01)and after 48 hours it was 1.48 times of S1 and 1.43 times of S2 respectively(p＜0.01).72hours later the AV value of UT was 1.27 times of S1(p＜0.05) and 1.1 times of S2(P＞0.05,Oneway, respectively).100 nM siRNA1 and siRNA2 againstβ-catenin at 24hours inhibited theβ-catenin mRNA by 67%and 73%(P＜0.05,Oneway,respectively).Real time PCR showed the down-regulated expression of CCND1 and MYC in S1 and S2 groups compared with UT group(p＜0.05).Immunofluorescence disclosed the shutdown ofβ-catenin translation in siRNA groups.48hours after transfection,IOD/Area analysis showed the decrease value of S1 and S2 groups compared with UT and NS group (p＜0.01).Apoptosis of SK-NEP-1 increased in siRNA1 and siRNA2 groups compared with UT group(p＜0.05).3.The effect of estradiol and bisphenol A on proliferation of SK-NEP-1 cells and wnt signal pathway:The expression of ER was negative in SK-NEP-1 detected by Envision immunohistochemistry.17β-estradiol at concentration of 5 nM,and bisphenol A at concentration of 5μM had effect on the proliferation of SK-NEP-1 cells(P＜0.05,Oneway,respectively).Real time PCR showed that the expression of CCND1 and MYC had no difference between groups(P＜0.05,Oneway,respectively). Immunofluorescence found a stable level ofβ-catenin in three groups.IOD/Area analysis showed no difference between goups(p＞0.05).There was also no difference of apoptosis between groups(p＞0.05).Conclusions1.There is activation of wnt signal pathway in SK-NEP-1 cell line.The expression level of sFRP-1 andβ-catenin is high and Wnt-4 is not obvious in SK-NEP-1 cell.2.During the differentiation from embryonic cell to epithelial cell in human metanephron,malmoduration of wnt signal pathway would be probably one of the mechanism leading to the tumorigenesis.That means the inhibition of Wnt-4 would lead to dysdifferentiation.The activation of sFRP-1 andβ-catenin would result in the continuous proliferation of SK-NEP-1 cell.3.The addition of low-dose sFRP-1 and high-dose Anti sFRP-1 has no effect on the proliferation and downstream gene regulation,but high concentration of sFRP-1 can inhibit the proliferation of SK-NEP-1 cell.This implicate that high-dose sFRP-1 would be the inhibitor of wnt signal pathway.4.Growth inhibition and down-regulated expression of CCND1 and MYC are the result of siRNAs againstβ-catenin,and it also promotes the apoptosis of SK-NEP-1 cell.The activation ofβ-catenin act as a key role in the development of nephroblastoma.5.No estradiol receptor(ER) is expressed in SK-NEP-1.17β-estradiol at concentrations of 5 nM and bisphenol A at concentration of 5 uM have effect on the proliferation of SK-NEP-1 cell.6.There is no change of the wnt signal pathway after the interference of estradiol and bisphenol A.