Research On The Role Of New Gene F10 In The Regulation Of Proliferation And Cell Cycle Of Choriocarcinoma Cell Line JAR

Choriocarcinoma,a trophoblastic tumor secondary to normal or abnormal pregnancy,metastasis early and extensive as a malignant tumor.F10 gene(GenBank number:AB196290)was a new gene screened out from the difference of cDNA library between the mole and normal early gestation villi by the research team,which is related to the occurrence and invasive behavior of choriocarcinoma.It is not fully clearly understood about the influence of F10 gene on cell proliferation and cell cycle of choriocarcinomaWe previously obtained two groups of choriocarcinoma cells JAR which were stably expressed as F10 gene silenced and F10 stably over-expressed by screening and packaging after transfecting recombinant into choriocarcinoma cells through eukaryotic plasmid vector and RNAi lentivirus vector.We investigated the relationships between F10 gene and some cyclin/Cdks to preliminary explore the role of new gene F10 in the regulation of proliferation and cell cycle of choriocarcinoma cell line JAR.First,the research contents and methodsThree groups of JAR cells were classified as,F10 gene silenced,F10 stably over-expressed,and non-treated control.cell growth curves of the three groups were created to observe the influence of F10 gene on the growth of choriocarcinoma cells by cell counting kit-8(CCK8)and the cell circle was detected by Flow Cytometry(FCM).The expressions of cyclin and cyclin-dependent kinase(CDKs)were detected by employing Western-blot and immuno-fluorescence technique such as:cyclinA2,cyclinBl,cyclin C,cyclinDl,cyclinE2,CDK1,CDK2,CDK3,CDK4,CDK5,CDK6,CDK7,CDK8,CDK9,P16,P21,and other regulators aiso related to cell cycle:proliferating cell nuclear antigen(PCNA),focal adhesion kinase(FAK),a serine/threonine protein kinase 4(PAK4).And we localizated and detected the expression levels of the proteins above by immuno-fluorescence technique.The Second,the results1.Growth curve reflected that three groups of cells began to proliferate on the first day,as exponential growth.The F10 over-expressed group had the fastest growth rate,followed by the untreated group,as the F10 silent group had the slowest one.Therefore,The F10 gene silenced group slows cell growth of choriocarcinoma cells to inhibit cell proliferation and the over-expression of F10 gene can promote the proliferation.2.Flow cytometry showed that the F10 gene silenced group had an obvious apoptosis peak.The G2/M phase cells' proportion of F10 gene over-expressed group was 41.37%,which was 2.4 times compared with the proportion of F10 gene silenced group(17.06%)when the untreated group(20.77%)was 1.2 times compared to the F10 gene silenced group.Two groups was significant difference(p<0.05).It showed that the proportion of G2/M phase cells increased in turn between F10 gene silenced group,untreated group and F10 gene over-expressed group.Implying that the F10 gene silenced may lead to choriocarcinoma cell apoptosis while the F10 gene over-expressed inhibited apoptosis and promote cell proliferationThe third,The difference on protein levels of choriocarcinoma cellsPositive regulators of cell cycle:Western blot results showed that:compared with the untreated control group,the F10 gene silenced had a significantly lower protein expression levels of cyclinB1,cyclinD1,cyclinE,CDK6,CDK8,CDK9.(p<0.001).The F10 gene over-expressed group had a significantly higher protein expression level of cyclinA2,B1,C,D1,E and CDK6,7,8,9 protein levels were significantly higher(p<0.05).And the immuno-fluorescence technique results showed:compared with the untreated control group,the F10 gene silenced had a significantly lower protein expression level of cyclinD1,cyclinE,CDK1,3,4,5,6,8,9(p<0.05),and The F10 gene over-expressed group had a significantly higher protein expression level of cyclinBl,cyclinDl,cyclinE,CDK1,2,4,5,6,7,8,9(p<0.05).Negative regulator of cell cycle:Western blot test results were shown:Expression level of P16 protein of the F10 gene silenced group was significantly lower than the untreated control group(p<0.05),and the level of the F10 gene over-expressed group was significantly higher than the untreated control group(p<0.05).Expression level of P16 protein had the result in accord with that detected by the immuno-fluorescence technique.The results of Western blot and the immuno-fluorescence technique showed there were no significant differences on the protein level of P21 between the three groups.Serine/threonine protein kinase 4(PAK4):Western blot test results were shown:Compared with the untreated control group,the F10 gene silenced group had a significantly lower protein expression level of PAK4(p<0.05),and the level of the F10 gene over-expressed group was significantly higher than that of the untreated control group(p<0.05).Expression level of PAK4 protein had the results in accord with those detected by the immuno-fluorescence technique.Focal adhesion kinase(FAK):Western blot test results are shown:Compared with the untreated control group,the F10 gene silenced group and the F10 gene over-expressed group had significantly higher protein expression levels of FAK(p<0.05),and the F10 gene over-expressed group had a significantly lower protein expression level of FAK compared with the group F10 gene silenced group.The immuno-fluorescence technique test results are shown:The F10 gene silenced group had a significantly higher protein expression level of FAK compared with the untreated control group,while there is no significant difference between the F10 over-expression group and the control group.The location of cells cycle regulate factor in Choriocarcinoma cells:CyclinA2,cyclinD1,CDK7 fully expressed in tumor cell nuclei.CyclinE,CDK1,CDK2,CDK6,CDK8,CDK9,P21 protein mainly express in the nucleus while a small amount express in the cytoplasm.CyclinB1,cyclinC,CDK3,CDK4,CDK5,P16,PAK4,FAK mainly localized in the cytoplasm,while PCNA was expressed both in the nucleus and cytoplasm.Conclusions1.We found that compared with the untreated control group,the F10 gene silenced group appeared slower growth rate by drawing growth curve through CCK8 method,while the F10 gene over-expressed accelerated the growth rate of choriocarcinoma cells line JAR.Implying that F10 gene promoted choriocarcinoma cells growth and thus speculate F10 gene may participate in its proliferation process.2.It showed that the G2/M phase cells of the F10 gene silenced group,the F10 gene untreated group,F10 over-expression group G2/M phase cells increased successively detected by the flow cytometry experiments suggesting that F10 gene accelerated cell cycle and intervened cell proliferation of choriocarcinoma cells.3.It was proved that the F10 gene regulated the expression level of some proteins as cell cycle regulating factors to accelerate cell cycle progression,promote the proliferation of choriocarcinoma cells by Western blot and immunofluorescence cell technology.