The Effect Of ARID1A Gene Silenced On The Sensitivity Of Ovarian Clear Cell Carcinoma ES2 Cells To Cisplatin And Correlative Study

Objective:Design and chemically synthesize ARID1A-specific siRNA by siRNA technique and then transfect the ovarian clear cell carcinoma ES2 cells to select the most effective ARID1A-specific siRNA. To investigate the effect of ARID1A gene silenced on the sensitivity of ES2 cells to Cisplatin in vitro and the possible mechanism. To study the antitumor effect of protein kinase B inhibitor Perifosine combined with Cisplatin on ES2 cells in vitro and to provide certain theoretical basis for molecular targeted therapy of ovarian clear cell carcinoma in the future.Methods:The chemically synthetic siRNA was transfected to ovarian clear cell carcinoma ES2 cells in vitro by RNAiMax transfection reagent. Expressions of ARID1A in mRNA and protein level were evaluated by RT-PCR and western blot to select the most effective ARID1A-siRNA (that is siRNA-3). The most effective siRNA-3 was used to transfect ES2 cells. Different concentrations of Cisplatin were added 24 h after transfection. After 48 h of cultivation, the IC50 was measured by CCK-8 assay. The survival rates were detected in different groups after adding Cisplatin at the concentration of 50 μM for 48 h. The apoptosis rates of ES2 cells in different groups were measured by flow cytometry (FCM). Expressions of protein kinase B (AKT) in ES2 cells in different groups were measured by western blot.ES2 cells were treated with different doses of Perifosine for 48 h. CCK-8 assay was used to evaluate the minimal effective Perifosine dose. Then the ES2 cells were treated with Perifosine at minimal effective dose combined with different doses of Cisplatin. Biirgi formula was used to assess the synergy effect between Perifosine and Cisplatin. Apoptosis was studied by DAPI staining and flow cytometer analysis. Cellular mitosis was detected by Giemsa staining assay. The expressions of Caspase-3(cleaved) and PARP(cleaved) protein were measured by western blot.Results:CCK-8 method showed that the value of IC50 was significantly increased in siRNA-3 group compared with that in siRNA-NC group and blank control group (P<0.05). The cell survival rate was significantly increased in siRNA-3 group after treatment with 50 μM Cisplatin for 48 h compared with that in siRNA-NC group and blank control group (P<0.05). FCM analysis showed that the apoptosis rate of siRNA-3 with Cisplatin group was significantly lower than that of Cisplatin group (P<0.01). The apoptosis rate of siRNA-3 group was significantly lower than that of control group (P<0.05). Western blot demonstrated that expression of AKT protein was upregulated in siRNA-3 group compared with control group (P<0.01). In addition, expression of AKT protein in Cisplatin group was significantly decreased compared with control group (P<0.01). What’s more, expression of AKT protein in siRNA-3 with Cisplatin group was significantly increased compared with Cisplatin group (P<0.05).Perifosine at 4 μM was the minimal effective dose on ES2 cells. Combined treatment of Perifosine at 4μM and Cisplatin at 40 μM showed the best effect in cell growth inhibition. DAPI staining analysis showed that a large number of karyorrhexis was observed in the combination group. FCM analysis discovered that combination of Perifosine and Cisplatin synergistically enhanced apoptosis of ES2 cells, inhibited cellular mitosis, compared with Perifosine or Cisplatin alone. Western blot showed that treatment of Perifosine combined with Cisplatin induced increased level of Caspase-3(cleaved) protein and decreased level of PARP(cleaved) protein (P<0.01).Conclusions:ARID1A siRNA efficiently decreases expression of ARID 1 A, which reduces Cisplatin chemosensitivity and cell apoptosis in ovarian clear cell carcinoma ES2 cells, and the mechanism may be related to activating the signal pathway of protein kinase B(AKT). Combined treatment of AKT inhibitor Perifosine and Cisplatin could inhibit cell proliferation and induce apoptosis in ovarian clear cell carcinoma ES2 cells, and the mechanism of apoptosis may be related to up-regulating the expression of Caspase-3 (cleaved) protein and down-regulating the expression of PARP(cleaved) protein.