fetal bovine serum was purchased from hyclone ( logan , utah ) , lipofectamine 2000 from invitrogen ( carlsbad , ca ) , tripure isolation reagent from roche molecular biochemicals ( mannheim , germany ) , gapdh ( glyceraldehyde phosphate dehydrogenase ) antibody from abmart ( shanghai , china ) , and phospho - stat3 ( tyr705 ) ( d3a7 ) xp rabbit monoclonal antibody . phospho - p44/42 mitogen - activated protein kinase ( mapk ) ( erk1/2 ) ( thr202/tyr204 ) ( 197g2 ) rabbit monoclonal antibody were all purchased from cell signaling technology ( beverly , ma ) . primer synthesis was performed by sangon biological engineering technology & service co ( shanghai , china ) . the cell cycle detection kit , cell lysis solution , and cell counting kit 8 ( cck-8 ) were from beyotime ( shanghai , china ) . dna marker , t4 dna ligase , and taq enzyme were all from takara ( dalian , china ) . rabbit immunoglobulin g antibody was purchased from abgent ( san diego , ca ) , dna rapid purification kit was from omega bio - tek ( norcross , ga ) . the primers were designed based on the coding regions of the arhi gene as follows : sense , 5-ccggaattcatgggtaacgccagctt-3 ; antisense , 5-cgcggatcctcacatgattatgcacttgt-3 ( the underlined gaattc and ggatcc sequences are the ecori and bamhi recognition sites , respectively ) . polymerase chain reaction ( pcr ) was performed using pcdna3.0-arhi as the template ( kindly donated by dr d. b. badgwell , at the university of texas m. d. anderson cancer center , houston , tx ) . the pcr products were separated by electrophoresis on a 1.5% agarose gel , followed by ethidium bromide staining . the fragments of interest were recovered from the agarose gel , purified , and ligated by t4 dna ligase to express the pires2-egfp - arhi plasmid . the positive recombinant products were selected on lb agar plates using 100 g / ml ampicillin and confirmed by pcr and dna sequencing . skov3 cells were purchased from shanghai institute of cell biology , chinese academy of sciences ( shanghai , china ) and were maintained in rpmi 1640 media supplemented with 10% fetal bovine serum , penicillin 100 u / ml , and streptomycin 100 u / ml at 37c in a humidified 5% co2 atmosphere . the cells were seeded in 6-well plates at a density of 34  10 cells per well in 2 ml of antibiotic - free medium and incubated for 1 day before transfection at 90% to 95% confluence . transfection was performed by first diluting 2 l plasmid and 5 l lipofectamine 2000 per well in serum - free medium to a final volume of 250 l , gently mixing , and incubating at room temperature for 5 min . this was followed by further gentle mixing and incubation at room temperature for 20 minutes . the cells were washed with serum - free medium before the diluted plasmid / lipofectamine complex was added to the 6-well plates for 4 to 6 hours , after which the complex was replaced with normal cell culture medium . the cells were visualized 24 hours after transfection under a nikon eclipse fluorescence microscope ( nikon , japan ) . the skov3 cells were divided into the following 4 groups : cells transfected with pires2-egfp - arhi plasmid ( treated group ) , cells transfected with pires2-egfp plasmid ( positive control group ) , cells without transfection ( negative control group ) , and a blank control without cells . cells were passaged at the logarithmic growth phase , trypsinized , and adjusted to 5  10 cells / ml . the cells were then seeded in 96-well plates in 100 l 10% fetal bovine serum and cultured for 24 , 48 , 72 , 96 , and 120 hours at 37c in a humidified 5% co2 atmosphere . after incubation for 4 hours under the same condition , 10 l cck-8 reagent was added to each well . the optical density value of each well was measured using a microculture plate reader at a wavelength of 450 nm . the growth inhibitory rate ( ir ) was calculated using the following formula : the skov3 cells were divided into the following 3 groups : pires2-egfp - arh  transfected group ( treated ) , pires2-egfp  transfected group ( positive control ) , and cells without transfection ( negative control ) . the cells were harvested after 48 and 72 hours , and cell apoptosis was detected using cell cycle and apoptosis analysis kits ( haimen , china ) , following the manufacturer s instructions . aliquots of cell lysates containing 50 g of proteins were separated by 10% sodium dodecyl sulfate the membranes were blocked with tbst ( tris - buffered saline and tween 20 ) buffer ( 10 mm tris - hcl , ph 8.0 , 0.15 m nacl , 0.05% tween 20 ) containing 5% skimmed milk ; incubated with rabbit polyclonal antibodies to phospho - stat3 , p44/42 mapk ( erk1/2 [ extracellular signal regulated protein kinase 1/2 ] ) , or phospho - p44/42 mapk ( erk1/2 ) at 4c overnight ; and followed by the addition of horseradish peroxidase  linked anti rabbit immunoglobulin g. the bands were visualized by electrochemiluminescence , and the intensity of each western blot band was analyzed using quantity one software ( biorad ) . comparisons of growth irs and apoptotic rates were performed by  test , and protein relative expression levels were compared by student t test . fetal bovine serum was purchased from hyclone ( logan , utah ) , lipofectamine 2000 from invitrogen ( carlsbad , ca ) , tripure isolation reagent from roche molecular biochemicals ( mannheim , germany ) , gapdh ( glyceraldehyde phosphate dehydrogenase ) antibody from abmart ( shanghai , china ) , and phospho - stat3 ( tyr705 ) ( d3a7 ) xp rabbit monoclonal antibody . phospho - p44/42 mitogen - activated protein kinase ( mapk ) ( erk1/2 ) ( thr202/tyr204 ) ( 197g2 ) rabbit monoclonal antibody were all purchased from cell signaling technology ( beverly , ma ) . primer synthesis was performed by sangon biological engineering technology & service co ( shanghai , china ) . the cell cycle detection kit , cell lysis solution , and cell counting kit 8 ( cck-8 ) were from beyotime ( shanghai , china ) . dna marker , t4 dna ligase , and taq enzyme were all from takara ( dalian , china ) . rabbit immunoglobulin g antibody was purchased from abgent ( san diego , ca ) , dna rapid purification kit was from omega bio - tek ( norcross , ga ) . the primers were designed based on the coding regions of the arhi gene as follows : sense , 5-ccggaattcatgggtaacgccagctt-3 ; antisense , 5-cgcggatcctcacatgattatgcacttgt-3 ( the underlined gaattc and ggatcc sequences are the ecori and bamhi recognition sites , respectively ) . polymerase chain reaction ( pcr ) was performed using pcdna3.0-arhi as the template ( kindly donated by dr d. b. badgwell , at the university of texas m. d. anderson cancer center , houston , tx ) . the pcr products were separated by electrophoresis on a 1.5% agarose gel , followed by ethidium bromide staining . the fragments of interest were recovered from the agarose gel , purified , and ligated by t4 dna ligase to express the pires2-egfp - arhi plasmid . the positive recombinant products were selected on lb agar plates using 100 g / ml ampicillin and confirmed by pcr and dna sequencing . skov3 cells were purchased from shanghai institute of cell biology , chinese academy of sciences ( shanghai , china ) and were maintained in rpmi 1640 media supplemented with 10% fetal bovine serum , penicillin 100 u / ml , and streptomycin 100 u / ml at 37c in a humidified 5% co2 atmosphere . the cells were seeded in 6-well plates at a density of 34  10 cells per well in 2 ml of antibiotic - free medium and incubated for 1 day before transfection at 90% to 95% confluence . transfection was performed by first diluting 2 l plasmid and 5 l lipofectamine 2000 per well in serum - free medium to a final volume of 250 l , gently mixing , and incubating at room temperature for 5 min . this was followed by further gentle mixing and incubation at room temperature for 20 minutes . the cells were washed with serum - free medium before the diluted plasmid / lipofectamine complex was added to the 6-well plates for 4 to 6 hours , after which the complex was replaced with normal cell culture medium . the cells were visualized 24 hours after transfection under a nikon eclipse fluorescence microscope ( nikon , japan ) . the skov3 cells were divided into the following 4 groups : cells transfected with pires2-egfp - arhi plasmid ( treated group ) , cells transfected with pires2-egfp plasmid ( positive control group ) , cells without transfection ( negative control group ) , and a blank control without cells . cells were passaged at the logarithmic growth phase , trypsinized , and adjusted to 5  10 cells / ml . the cells were then seeded in 96-well plates in 100 l 10% fetal bovine serum and cultured for 24 , 48 , 72 , 96 , and 120 hours at 37c in a humidified 5% co2 atmosphere . each experimental group included 6 wells , and all experiments were repeated in triplicate . after incubation for 4 hours under the same condition , 10 l cck-8 reagent was added to each well . the optical density value of each well was measured using a microculture plate reader at a wavelength of 450 nm . the skov3 cells were divided into the following 3 groups : pires2-egfp - arh  transfected group ( treated ) , pires2-egfp  transfected group ( positive control ) , and cells without transfection ( negative control ) . the cells were harvested after 48 and 72 hours , and cell apoptosis was detected using cell cycle and apoptosis analysis kits ( haimen , china ) , following the manufacturer s instructions . aliquots of cell lysates containing 50 g of proteins were separated by 10% sodium dodecyl sulfate  polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes . the membranes were blocked with tbst ( tris - buffered saline and tween 20 ) buffer ( 10 mm tris - hcl , ph 8.0 , 0.15 m nacl , 0.05% tween 20 ) containing 5% skimmed milk ; incubated with rabbit polyclonal antibodies to phospho - stat3 , p44/42 mapk ( erk1/2 [ extracellular signal regulated protein kinase 1/2 ] ) , or phospho - p44/42 mapk ( erk1/2 ) at 4c overnight ; and followed by the addition of horseradish peroxidase  linked anti rabbit immunoglobulin g. the bands were visualized by electrochemiluminescence , and the intensity of each western blot band was analyzed using quantity one software ( biorad ) . spss version 13.0 software was used for statistical analyses . comparisons of growth irs and apoptotic rates were performed by  test , and protein relative expression levels were compared by student t test . 1a - c ) showed a high level of green fluorescence protein ( gfp ) in pires2-egfp - arhi - skov3 cells ( fig . the high level of gfp in the treated group compared with the control groups confirms successful transfection . green fluorescence protein expression 48 hours after transfection : ( a ) pires2-egfp - arhi - skov3treated group ; ( b ) pires2-egfp - skov3 plasmid control group ; ( c ) untransfected skov3 cells negative control group . the high level of gfp in the treated group compared with the control groups confirms successful transfection . after the cells had been cultured for 24 , 48 , 72 , 96 , and 120 hours , the growth irs were 64.69% , 70.17% , 67.01% , 66.87% , and 67.70% , respectively , in the test group , and were 27.20% , 31.10% , 29.80% , 30.73% , and 31.28% , respectively , in the plasmid control group ( fig . this showed that the growth ir was significantly increased in the test group compared with the plasmid control group ( p < 0.01 ) . the effect of arhi on cell proliferation in skov3 , measured by cck-8 assays , shows that arhi significantly inhibits growth of skov3 cells . the proportions of s - phase cells and apoptosis rates in skov3 cells were determined by flow cytometry . after 48 hours of culture , the mean proportions of s - phase skov3 cells in the test , plasmid control , and negative control groups were 64.18% , 38.43% , and 15.15% , respectively , and the mean apoptosis rates were 47.97% , 26.53% , and 9.33% , respectively . after 72 hours of culture , the mean proportions of s - phase skov3 cells in the test , plasmid control , and negative control groups were 43.29% , 10.37% , and 10.89% , respectively , and the apoptosis rates were 51.34% , 24.70% , and 4.39% , respectively ( table 1 ) . data show that the proportions of s - phase cells , and thereby s - phase arrest , were clearly higher in the pires2-egfp - arhi - skov3 cell group compared with the control group at both 48 and 72 hours ; the result at 72 hours was much more significant in the test group . in addition , the apoptosis rate was significantly increased in the test group compared with the control group ( p < 0.01 ) . influence of arhi on the cell cycle phase distribution and apoptosis rate the western blot images for p - stat3 ( phosphorylated signal transduction and activators of transcription 3 ) and p - erk protein expression for the control and treated groups : skov3 , pires2-egfp - skov3 , and pires2-egfp - arhi - skov3 , are shown in figure 3 . compared with gapdh reference protein , the relative expression levels of p - stat3 were 1.1473  0.0002 , 1.0913  0.0021 , and 0.7424  0.0006 , respectively , and the relative expression levels of p - erk were 1.2260  0.0011 , 1.1289  0.0018 , and 0.5866  0.0013 , respectively . in comparison to the 2 control groups , the expression levels of p - stat3 and p - erk proteins were significantly lower in the pires2-egfp - arhi - skov3 group ( p < 0.05 ) . changes in the expressions levels of p - erk1/2 and p - stat3 in the different skov3 groups : lane 1 , protein expression in the untransfected skov3 control group ; lane 2 , protein expression 48 hours after pires2-egfp was transfected into skov3 cells ; lane 3 , protein expression 48 hours after pires2-egfp - arhi was transfected into skov3 cells . the results show that arhi significantly reduces p - erk1/2 and p - stat3 expression levels . 1a - c ) showed a high level of green fluorescence protein ( gfp ) in pires2-egfp - arhi - skov3 cells ( fig . the high level of gfp in the treated group compared with the control groups confirms successful transfection . green fluorescence protein expression 48 hours after transfection : ( a ) pires2-egfp - arhi - skov3treated group ; ( b ) pires2-egfp - skov3 plasmid control group ; ( c ) untransfected skov3 cells negative control group . the high level of gfp in the treated group compared with the control groups confirms successful transfection . after the cells had been cultured for 24 , 48 , 72 , 96 , and 120 hours , the growth irs were 64.69% , 70.17% , 67.01% , 66.87% , and 67.70% , respectively , in the test group , and were 27.20% , 31.10% , 29.80% , 30.73% , and 31.28% , respectively , in the plasmid control group ( fig . this showed that the growth ir was significantly increased in the test group compared with the plasmid control group ( p < 0.01 ) . the effect of arhi on cell proliferation in skov3 , measured by cck-8 assays , shows that arhi significantly inhibits growth of skov3 cells . the proportions of s - phase cells and apoptosis rates in skov3 cells were determined by flow cytometry . after 48 hours of culture , the mean proportions of s - phase skov3 cells in the test , plasmid control , and negative control groups were 64.18% , 38.43% , and 15.15% , respectively , and the mean apoptosis rates were 47.97% , 26.53% , and 9.33% , respectively . after 72 hours of culture , the mean proportions of s - phase skov3 cells in the test , plasmid control , and negative control groups were 43.29% , 10.37% , and 10.89% , respectively , and the apoptosis rates were 51.34% , 24.70% , and 4.39% , respectively ( table 1 ) . data show that the proportions of s - phase cells , and thereby s - phase arrest , were clearly higher in the pires2-egfp - arhi - skov3 cell group compared with the control group at both 48 and 72 hours ; the result at 72 hours was much more significant in the test group . in addition , the apoptosis rate was significantly increased in the test group compared with the control group ( p < 0.01 ) . the western blot images for p - stat3 ( phosphorylated signal transduction and activators of transcription 3 ) and p - erk protein expression for the control and treated groups : skov3 , pires2-egfp - skov3 , and pires2-egfp - arhi - skov3 , are shown in figure 3 . compared with gapdh reference protein , the relative expression levels of p - stat3 were 1.1473  0.0002 , 1.0913  0.0021 , and 0.7424  0.0006 , respectively , and the relative expression levels of p - erk were 1.2260  0.0011 , 1.1289  0.0018 , and 0.5866  0.0013 , respectively . in comparison to the 2 control groups , the expression levels of p - stat3 and p - erk proteins were significantly lower in the pires2-egfp - arhi - skov3 group ( p < 0.05 ) . changes in the expressions levels of p - erk1/2 and p - stat3 in the different skov3 groups : lane 1 , protein expression in the untransfected skov3 control group ; lane 2 , protein expression 48 hours after pires2-egfp was transfected into skov3 cells ; lane 3 , protein expression 48 hours after pires2-egfp - arhi was transfected into skov3 cells . the results show that arhi significantly reduces p - erk1/2 and p - stat3 expression levels . cell cycle progression is regulated by multiple control points at different phases of the cell cycle ; the 3 principal ones being g1/s , g2/m , and at metaphase / anaphase transmission during mitosis . the g1/s check point is the most critical for control of cell proliferation via intracellular and extracellular signals related to transportation and integration of molecules into the nucleus.14 by reconstructing the pires2-egfp - arhi plasmid and successfully transfecting it into hoc skov3 cells with low arhi expression levels , as shown by high levels of green fluorescence from the plasmid observed by fluorescent microscopy , in addition , by using a cck-8 assay to analyze the growth irs , we observed significantly higher growth inhibition in pires2-egfp - arhi - skov3 cells compared with the control cells ; furthermore , flow cytometry showed the proportion of s - phase cells and the apoptosis rates were significantly higher after promoting expression of the arhi gene . these results suggested that arhi inhibited proliferation , arrested the cells at s - phase of the cell cycle , and induced apoptosis in skov3 cells . we therefore proposed that the arhi gene is involved in cell cycle regulation in hoc skov3 cells , preventing proliferation and inducing apoptosis . multiple signaling pathways are involved in cell growth and apoptosis , including the ras / raf / mek / erk1/2 signaling pathway , which is central for transporting the complex signals received by the cell surface receptors to transcription factors in the nucleus . extracellular signal regulated protein kinase 1/2 is a key physiological substrate of mek ; mek1 kinase can cause double phosphorylation of threonine and tyrosine at erk1/2 residues to activate erk1/2 ; activation of erk1/2 can subsequently lead to phosphorylation of downstream transcription factors , such as p90rsk kinase , creb , and c - myc . changes in cell proliferation , apoptosis , differentiation , and other critical physiological activities are a consequence of phosphorylation of these transcription factors.15 meloche and pouyssgur16 proposed that erk1/2 , which is a key regulatory factor in mitosis , is also involved in the evolutionary control of the cell cycle . a certain study showed that arhi plays roles in the egf - egfr - ras - raf - mapk / erk1/2 pathway.17 extracellular signals activate jak in the jak - stat signaling pathway through associated receptors , leading to phosphorylation of specific tyrosine residues on the receptors ; these phosphorylate stats , which are isolated from the receptors , transferred into the nucleus after dimerization , and combined with specific dna response elements . this initiates transcription at the target genes and promoting synthesis of a variety of proteins and their subsequent biological roles.18 studies have shown that inhibiting the jak - stat signaling pathway can significantly reduce p - jak2 , p - jak3 , and p - stat3 expression levels ; down - regulate cyclin - d1 , which is linked to cell cycle regulation ; and up - regulate p16 , p21 , and p27 expression , thereby inhibiting tumor cell proliferation and growth.19,20 stat3 , an important member of the stat family , is found in a variety of tumor tissues and cell lines and is now recognized as an oncogene.21,22 stat3 sirna down - regulates cyclin d1 , survivin , and vascular endothelial growth factor expression in hoc cells ; inhibits stat3 and its related genes ; inhibits hoc cell growth ; and induces apoptosis in vitro . as such , stat3 has recently become a target for hoc therapies.23 when the yeast 2-hybrid system was used to search for arhi - interactive proteins by screening the human mammary epithelial cell cdna gene pool , arhi was found to act with signal transduction proteins and the stat3 transcription activator , but not stat1 or stat5a . furthermore , when arhi and stat3 were coexpressed in skov3 cells , they did not form a complex in the cytoplasm . this suggests that by regulating stat3 phosphorylation , arhi can significantly reduce stat3 binding to dna , and thereby reduce the activity of the stat3-dependent promoter.24 our data showed that the expression levels of p - erk1/2 and p - stat3 proteins in the ras / raf / mek / erk1/2 and jak - stat3 signaling pathways were significantly reduced 48 hours after arhi transfection . in our study previously , we also measured the expression levels of stat3 and erk1/2 and did not find statistically significant . so we proposed that the mechanism by which arhi influences both the ras / raf / mek / erk1/2 and jak - stat3 signaling pathways to inhibit hoc skov3 cell proliferation is through regulating arhi gene expression by altering the phosphorylation levels of erk1/2 and stat3 protein kinases , thereby blocking the signaling pathway for tumor cell proliferation sufficiently to prevent cell proliferation and induce apoptosis . a study by lu et al25 revealed that the arhi gene maintains hoc cells in transplanted tumors in a resting state ; conversely , when the arhi level was decreased , the transplanted tumor grew rapidly , and when arhi expression subsequently recovered to normal levels , the hoc cells died through autophagy . the authors concluded that the arhi gene is a key factor in initiating autophagy in hoc cells ; a further study by mathias et al26 proposed that this may lead to cell death . an in - depth investigation is required to determine the molecular mechanism for autophagy initiation in the arhi gene , with the aim of developing an application based on arhi for the clinical treatment of hoc .
objectiveaplasia ras homolog member i ( arhi ) is associated with human ovarian cancer ( hoc ) growth and proliferation ; however , the mechanisms are unclear . 
 the purpose of this study was to investigate arhi effects in hoc skov3 cells.methodswe transfected skov3 cells with pires2-egfp - arhi and measured growth inhibition rates , cell cycle distribution , apoptosis rates , and expression of p - stat3 ( phosphorylated signal transduction and activators of transcription 3 ) and p - erk ( phosphorylated extracellular signal regulated protein kinase).resultsour data showed significant inhibition of growth , significantly increased s - phase arrest and apoptosis rates , and reduction of p - stat3 and p - erk1/2 expression levels.conclusionswe propose the mechanism may involve arhi - induced phosphorylation of erk1/2 and stat3 protein kinases , thereby blocking proliferation signaling pathways , to induce hoc skov3 apoptosis .