| Objective: Hepatocyte growth factor can promote cell regeneration cell division andmovement, unmoral expression of HGF and its receptor c-met play an important role in thedevelopment of ovarian cancer. Apoptosis inhibiting protein XIAP may also involved inthe development of ovarian cancer, but the function of them in the regulation of ovariancancer has not yet been reported. The immunohistochemical was used to detection theexpression of HGF and c-Met in ovarian cancer, than using c-Met the specific inhibitorsof PHA665752affect ovarian cancer SKOV-3, observe the changes in cell proliferation,apoptosis and the effects of XIAP. Provide the theoretical basis for clinical treatment ofovarian cancer.Methods:(1) The expression of HGF and its receptor in ovarian serouscystadenocarcinoma organization border, ovarian serous cystic adenoma, ovarian tumorsand normal ovarian tissue were by immunohistochemical (EnVision method) and imageanalysis software Image-ProPlus.(2) Choiced human papillary serous cystadenocarcinomaovarian cell line SKOV-3, cultured it at37℃and5%CO2, O2and20%to95%humidity,extract growth index cells and well growed cell for experiments. At various concentrations(0ng/ml, and20ng/ml, and40ng/ml, and80ng/ml) of HGF and (0.25umol/l, and1umol/l,2-umol/l) PHA665752cells, at a different time (24h,48h,72h) and collect cells, usingMTT method, flow cytometry for Annexin V-FITC/PI assay for detection of groups ofproliferation and apoptosis in ovarian cancer cells. By using different concentrations (0ng/ml,20ng/ml,40ng/ml,80ng/ml) of HGF and (0.5umol/l,1umol/l,2umol/l)PHA665752to cell, and than collected cells in different time (24h,48h,72h), By usingMTT method, flow cytometry Annexin V-FITC/PI method to detect the cell proliferationand apoptosis in different group of ovarian cancer.(3) The expression of HGF，XIAP andc-Met in ovarian cells treated by PHA6657522umol/l, XIAP HGF40ng/ml for48hourswere by PT-PCR and Western Blot.(4) Data derived from said groups were comparedusing analysis of variance, using analysis of variance between groups, p <0.05wasstatistically significant, statistical use SPSS17.0software processing.Results:(1) The expression of HGF and c-met between ovarian benign tumor and controlgroup was not statistically significant (P>0.05), the positive rate was20%and20%; levels of ovarian malignant tumor expression of HGF and c-met in benign, increased significantly,a statistically significant difference (P<0.05), the positive rate was60%,70%. Differentconcentrations (0ng/ml, and20ng/ml, and40ng/ml, and80ng/ml) of HGF and (0, umol/l,and0.5umol/l, and1umol/l,2-umol/l) PHA665752effect on the survival rate of ovariancancer:24h, respectively (44.58%,53.77%, and63.97%) and (60.05%, and,52.05%,44.58%);48h, respectively (38.48%,84.33%, and81.85%) and (57.05%,46.72%,39.28%,37.66%);72h respectively (29.26%,30.18%, and32.28%) and (49.26%, and,32.41%,34.43%).(3)20ng/ml HGF with role time extended, cell proliferation no obviously change,differences no statistics meaning (p>0.05); and40ng/ml, and80ng/mlHGF with role timeextended, cell survival increased, the group comparison has statistics differences (p<0.05)When the24hours later as the concentration increases, survival rates are not significantlychanged, the difference was not statistically significant (p>0.05);48h,72h, extended itsrole, survival increases with concentration increases, a statistically significant difference(p<0.05). PHA665752effect of different concentration of24h cellular proliferation nosignificant changes, compare without differences in statistics (p>0.05); And with theincrease in concentration when48h,72h (1umol/l,2, umol/l), the cell survival rate reduced,comparison groups had statistically significant differences (p<0.05)， When theconcentration is0.5umol/lPHA665752with prolonged cellular proliferation were notsignificantly changed, compare without differences in statistics (p>0.05),1umol/l,2umol/lPHA665752with time extension of the cell survival rate decline comparison groupshad statistically significant differences (p<0.05).(3) with different concentrations (0ng/ml,and20ng/ml, and40ng/ml, and80ng/ml) of HGF and (0.5umol/l, and1umol/l,2-umol/l)the effect of PHA665752on apoptosis of ovarian cancer:24h, respectively (13.63%,13.27%, and13.2%) and (9.71%,10.33%,14.13%,15.61%);48h respectively (14.37%,13.75%, and10.9%) and (10.66%,15.49%,30.61%,19.26%);72h respectively (14.18%,12.26%, and13.76%), and (14.09%,17.45%, and20.28%). HGF apoptosis rate betweenexperimental24h the concentration of groups are not significantly different, comparewithout differences in statistics (p>0.05), and48h,72h with increased concentrations ofHGF (40ng/ml, and80ng/ml), reduced the apoptosis, statistically significant differences(p<0.05). Same as PHA665752concentrations, as extensions of the time, increased rate ofapoptosis, thedifferences were statistically significant (p<0.05);0.25umol/lPHA66575224h no significant change in the rate of apoptosis, the difference was not statisticallysignificant (p>0.05),48h,72h, along with increased concentrations of1umol/l,2umol/l, increase in apoptosis rate and the differences were statistically significant (p<0.05).(4)control group expression of HGF mRNA content of0.88±0.11, expression of c-metmRNA content of0.85±0.14, XIAP mRNA expression of concentration level of0.92±0.08, the experimental group when you have ovarian cancer cell line SKOV348h HGF40ng/ml, expression of HGF mRNA content of1.32±0.25, expression of c-met mRNAcontent was1.69±0.16, XIAP mRNA expression levels of1.92±0.07,When you haveovarian cancer cell line SKOV348h PHA6657521umol/l,0.71±0.16expression of HGFmRNA content, expression of c-met mRNA content of0.49±0.05, XIAP mRNAexpression level was0.42±0.02. PHA665752protein in the treatment group relative lowexpression than blank group and the differences were statistically significant (p<0.05),HGF protein in the treatment group than the relative expression level high in the gap group,a statistically significant difference (p>0.05).(5) control group XIAP protein content of0.70±XIAP protein content of0.99±0.05,HGF group0.03,PHA665752group expressionof XIAP protein0.34±0.03. HGF group XIAP protein content higher than blank group, astatistically significant difference (p<0.05)、PHA665752group XIAP protein content lowerthan the blank group, a statistically significant difference (p<0.05). Expression of c-metprotein control group0.03,HGF Group c-met protein content of0.69±1.00±0.35±0.050.03,PHA665752treatment group of c-met protein expression. HGF treatment group ofc-met protein content higher than blank group, a statistically significant difference(p<0.05), PHA665752treatment group of c-met protein expression than blank group, astatistically significant difference (p<0.05).Conclusions:(1) The expression of HGF and c-Met were high in malignant ovariancancer.(2) PHA665752can inhibits the expression of c–Met and make the activity ofHGF/c-Met signaling pathways down, thus make the expression of c–Met HGFmRNA,c–MetmRNA and XIAP mRNA reduced, their protein expression were also reduced,eventually promote proliferation of ovarian cancer SKOV3cell, increase cell apoptosis.(3)HGF can make the expression of HGFmRNA, c-MetmRNA, XIAPmRNA increased, theexpression of protein were also increased, and promote the growth of ovarian cancer cellSKOV3. To sum up, the expressionof HGF， c–Met and XIAP were regulated by theHGF/c-Met pathways in ovarian cancer SKOV3cells. |
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