| Objective: To detect the different expression of ovarian cancer celllines of HO-8910, HO-8910PM and ovarian cancer tissues.To study howHtrA1expression on ovarian cancer cell lines to effect those cell’metastasis and invasion, so as to explore the regulation mechanism duringovarian cancer cells’ metastasis and invasion.Methods:1.Ovarian cancer tissue microchip were detected byimmunohistochemistry to quantity the expression level of HtrA1,including30cases of ovarian cancer tissue with different pathological stages,10cases of matched metastatic tissues, and8cases of nomal tissue.2. UsingImmunocytochemistry to detect the expression and cellular location ofHtrA1in HO-8910and HO-8910PM cell lines，and Western blot to detectthe different protein expression levels of HtrA1.3.The recombinanteukaryotic expression vector pcDNA3.1-HtrA1was constructed andidentified by double digestion and sequencing.4.The HO-8910andHO-8910PM cell lines were recruited as the objects of this study, the cellswhich were transfected with pcDNA3.1-HtrA1and HtrA1-siRNA were as treated group, and that were transfected with pcDNA3.1and scrambledsiRNA were as control groups.The expression levels of HtrA1weredetected by RTFQ-PCR and Western blot.5.The cell proliferation levels ofdifferent groups after transient transfection were detected by CCK8Kit.6.The different apoptosis degree of each group after transient transfectionwere evaluated by DAPI staining.7.To observe the migration and invasionchanges by Transwell chambers and cell scratch expriments.Results:1. Immunohistochemistry analysis showed a statisticallysignificant difference in HtrA1expression between tumor tissue andnormal tissue, and the same as between the metastatic tumor and primarytumor tissues(（P<0.05）, but no significant differences between differentstages of ovarian cancer tissues（p>0.05）.2.Immunocytochemistry analysisshowed that HtrA1were all expressed in HO-8910and HO-8910PM celllines,and mainly localized in the cytoplasm. HtrA1protein expression levelin HO-8910was significantly higher than HO-8910PM by Western blotanalysis.3.The pcDNA3.1-HtrA1eukaryotic expression vector wassuccessfully constructed and identified to be true by double digestion andsequencing.4. RTFQ-PCR and Western blot analysis showed that theexpression of mRNA and protein levels were significantly differentbetween treated and control groups after transfection,so the exogenousexpression and interference effect were obvious.5. There were nosignificant differences in cell proliferation and apoptosis（P>0.05）.6. In the scratch assay experiment,the cells’ healing rate of exogenous expression ofHO-8910and HO-8910PM was31.4%and40.6%,which was significantlylower than40.8%and48%in the control groups. The siRNA groups’healing rate was49.8%and60.4%,which was significantly higher than42.2%and51.0%in control groups.7. In migration test of Transwellchamber, the average penetrating cell numbers of HO-8910andHO-8910PM with over-expression of HtrA1were41.2±3.7and53.6±4.2,which were significantly smaller than53.2±4.0and62.2±5.7in thecontrol groups（P<0.05）. The siRNA groups’ average cell numbers were61.2±5.4and80.4±6.3,which were significantly larger than51.0±3.2and64.8±7.0in control groups（P<0.05）.8. In invasion test of Transwellchamber, the average penetrating cell numbers of HO-8910andHO-8910PM with over-expression of HtrA1were25.2±4.0and47.6±5.9, which were significantly smaller than37.6±5.9and56.6±4.9in thecontrol groups（P<0.05）。The siRNA groups’ average cell numbers were47.0±5.0and65.2±4.8，which were significantly larger than37.4±4.4and57.4±5.3in control groups（P<0.05）。Conclusion:1.HtrA1were generally expressed with low level inovarian cancers compared to narmal tissues, indicating that the role ofHtrA1as a tumor suppressor gene.2.The expression level of HtrA1inHO-8910cell line was significantly higher than that of HO-8910PM,andthe expression level of metastatic tumor tissues were significantly lower than the primary site, suggesting that HtrA1was associated with tumormetastasis.3. The migration and invasion of ovarian cancer cells could benotably inhibited by exogenous expression of HtrA1,but to lower theexpression of HtrA1could enhance ovarian cancer cells’ ability tometastasis and invasion. |
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