Effects Of Smac Gene Overexpression Of MiaPaCa-2 Cells On Chemotherapeutic Sensitivity

Objective: Pancreatic cancer is a common tumor of the digestive system with a high degree of malignancy and prognosis is poor. Early diagnosis of pancreatic cancer is difficult, the effect of the treatment is bad with a low rate of surgery, and there isn't a breakthrough progression in the chemotherapy and radiotherapy. Molecular targeted therapy is a highlights in recent research,but there are not satisfactory results in the treatment of pancreatic cancer. Smac is in the mitochondria, which can regulate the cell apoptosis. It mainly antagonist of the inhibition from IAPs to caspases. Smac can promot cell apoptosis through mitochondria apoptosis pathway and death receptor apoptosis pathway. Previous studies found the high expression of Smac gene was closely related to the occrrrence, development, the stage and metastasis in many tumors. This experiment constructed pEGFP-C1-Smac recombinant plasmid to explor the effection of over expression of Smac gene on cell apoptosis, and whether it can increase the chemotherapy sensitivity.Method:1 Cell cultureMiaPaCa-2 cells and K562 cells were cultured in general conditions. MiaPaCa-2 cells were in DMEM medium, and K562 cells were in RPMI- 1640 medium.2 By RT-PCR to obtain cDNA from K562 cells.3 The construction of pEGFP-C1-Smac plasmidReclaim feagments from the step 2 and pEGFP-C1 of PCR product from agarose gel, digest the PCR product with HindⅢand BamHⅠ, and purify it. Ligase the digested product in 16℃for 3h. Transform the ligated product into E.coli in DH5αand select monoclone. The selected monoclone were identified by PCR and sequencing. 4 Exteaction of plasmid from transformated of DH5αovernight cultured with the plasmid exteaction kit.5 Transfect pEGFP-C1-Smac and pEGFP-C1 into MiaPaCa-2 cells,and under the fluorescence microscopy to observe fluoresecnt protein expression.6 After transfected with the Smac gene for 24h, the apoptosis rates of the untransfected group, the empty vector group and the transfected group were detected by flow cytometry assay. Then the apoptosis rates, cell cycle, and the expression of Survivin and Bcl-2 were detected after treated with gemcitabine (10μg/ml) and cisplatin (8μg/ml) for 24h.7 The growth inhibition rates of different groups treated with 10μg/ml gemcitabine and 8μg/ml cisplatin at 8, 16, 24, 36, 48 hour were investigated by MTT assay.Results:1 The pEGFP-C1 vector and pEGFP-C1-Smac plasmid identified by PCR. The construction was successful.2 The pEGFP-C1 vector and pEGFP-C1-Smac plasmid was transfected into Miapaca-2 cells, under fluorescene microscopy to analysis fluorescent protein expression. The transfected cells were observed the green fluorescene.3 The apoptosis rates in transfected cells(7.01±3.88)% were higher than the control cells(2.17±0.87)%, and it was also higher than the control vector groups(4.40±1.49)%, P<0.05. But there was no significant statistical difference between the untransfected group and the empty vector group. Treated with gemcitabine the apoptosis rates in transfected cells (7.20±3.26)% were higher than the cells treated with gemcitabine(6.65±1.76)%, P<0.05. But there was no significant statistical difference in the transfected MiaPaCa-2 cells treated with cisplatin, which apoptosis rates(6.07±1.53)% were higher than the cells treated with cisplatin(5.80±6.68)%.4 In the groups treated with cisplatin, the OD570 at 8, 16, 24, 36, 48 hour were 0.30±0.061, 0.37±0.065, 0.37±0.015, 0.25±0.021, 0.17±0.019. The growth inhibition rates were -6.28%, 26.11%, 47.11%, 66.65%, 85.70%, in which the growth inhibition rates at 36 hour was higher than 24 hour, and that at 48 hour was higher than 24 hour, P<0.05. But there was no significant statistical difference in the groups treated with gemcitabine, which OD570 at 8, 16, 24, 36, 48 hour were 0.25±0.010, 0.30±0.053, 0.32±0.066, 0.30±0.027, 0.33±0.013, and the growth inhibition rates were 22.31%, 39.09%, 54.94%, 60.77%, 72.50%. In transfected cells the OD570 were 0.35±0.038, 0.41±0.021, 0.26±0.010, 0.48±0.047, 0.41±0.047. The growth inhibition rates at the five times were -24.62%, 16.25%, 62.87%, 63.58%, 65.55%, in which the growth inhibition rates at 48 hour was higher than 24 hour, P<0.05. In transfected cells treated with cisplatin the OD570 were 0.19±0.035, 0.18±0.020, 0.16±0.034, 0.19±0.020, 0.14±0.013. The growth inhibition rates at the five times were 44.81%, 63.86%, 78.02%, 74.66%, 88.27%. But there was no significant statistical difference in these groups. After the treatment with gemcitabine, in transfected cells the OD570 were 0.27±0.028, 0.30±0.016, 0.26±0.032, 0.22±0.033, 0.17±0.047. The growth inhibition rates at the five times were 5.91%, 39.21%, 63.31%, 70.42%, 85.71%, in which the growth inhibition rates at 24 hour was higher than 16 hour, and that at 48 hour was higher than 24 hour, P<0.05. In the blank vector groups treated with cisplatin and gemcitabine, the growth inhibition rates were increased, But there was no significant statistical difference.5 In the transfected groups the population of G0/G1 phase(50.45±0.78)% was higher than the untransfected groups (45.60±35.68)%, and the population of S phase(35.70±0.71)% was lower than the untransfected groups (38.05±5.61)%, but there was no significant statistical difference. Treated with cisplatin the population of G0/G1 phase(55.3±9.22)% of the transfected groups was higher than the groups with cisplatin (34.36±6.48)%, and the population of S phase(20.13±10.76)% was lower than the groups with cisplatin (29.30±11.75)%, but there was no significant statistical difference. Under the treatment with gemcitabine, the population of G0/G1 phase(61.93±0.68)% of the transfected groups was higher than the groups with gemcitabine (55.56±9.34)%, and the population of S phase(27.18±6.35)% was lower than the groups with gemcitabine (36.38±10.14)%, but there was no significant statistical difference.6 In the transfected groups the expression of Survivin(143.56±136.78) was lower than the untransfected groups(190.19±90.39). Treated with cisplatin the expression of Survivin in the transfected groups (455.98±98.42) was lower than the groups with cisplatin (471.93±25.02). Under the treatment with gemcitabine, the expression of Survivin in the transfected groups (471.21±98.42) was lower than the groups with gemcitabine ( 562.17±85.43). But there was no significant statistical difference.In the transfected groups the expression of Bcl-2(52.06±22.29) was significantly lower than the untransfected groups(83.55±88.04), P<0.05. Under the treatment with gemcitabine, the expression of Bcl-2 in the transfected groups (221.3±115.2) was significantly lower than the groups with gemcitabine (284.89±11.8), P<0.05. The expression of Bcl-2 in the transfected groups treated with cisplatin (220.23±132.3)was lower than the groups with cisplatin (264.86±63.59), but there was no significant statistical difference.Conclusions:1 Overexpression of Smac gene on human pancreatic cancer MiaPaCa-2 cells could increase the apoptosis rate significantly.2 Induced by gemcitabine,overexpression of Smac gene in MiaPaCa-2 cells could increase the apoptosis rate significantly. That could increase the chemotherapeutic sensitivity of gemcitabine.3 Overexpression of Smac gene in MiaPaCa-2 cells could inhibit cell proliferation, and the inhibition induced by gemcitabine and cisplatin on the proliferation of MiaPaCa-2 cells could be a time-dependant manner.4 Induced by chemotherapeutics, overexpression of Smac gene in MiaPaCa-2 cells could induce cell cycle arrest, in which the population of G0/G1 phase was increased, and the population of S phase was decreased.5 Treated with gemcitabine for 24 hour, the expression of Bcl-2 of the transfected group were down regulation significantly.