The combinational effect of E6/E7 siRNA and anti-miR-182 on apoptosis induction in HPV16-positive cervical cells
The combinational effect of E6/E7 siRNA and anti-miR-182 on apoptosis induction in HPV16-positive cervical cells
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DOI：
10.1080/21691401.2018.1468770
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发表时间：
2018-01-01
影响因子：
5.8
通讯作者：
Kamali, Mehdi
中科院分区：
文献类型：
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作者：
Javadi, Hamidreza;Lotfi, Abbas Sahebghadam;Kamali, Mehdi
在本研究中，我们假设通过特定的siRNA序列减少E6和E7癌蛋白的量并恢复p53和RB蛋白，沿着通过应用anti-miR-182恢复FOXO 1蛋白，将增加癌细胞的凋亡并降低增殖率。使用HPV 16阳性CaSki宫颈癌细胞系。转染靶向E6和E7癌蛋白的siRNA和anti-miR-182后48小时，通过实时PCR、蛋白质印迹分析和免疫细胞荧光染色评估其细胞靶点p53、p21和FOXO 1的表达。在所有处理中，分别使用Annexin-V-FITC凋亡检测试剂盒和MTT法评估凋亡率和存活率。在设计的siRNA中，E6-1和E7-2证明在降低E6和E7表达方面最有效，48小时后凋亡率分别增加至12.4%和16%。此外，使用anti-miR-182在转染宫颈癌细胞后48小时将凋亡率增加至12.7%。E6-1或E7-2 SiRNA与抗miR-182的联合使用导致细胞凋亡分别增加至19.3%和26%，高于单独应用任一不含抗miR-182的情况下获得的结果。与单独使用顺铂相比，同时使用siRNA E6-1和siRNA E7-2与顺铂增加了对顺铂的敏感性并降低了癌细胞的活力。与单独使用顺铂相比，同时使用顺铂和抗miR-182对活力或凋亡率没有显著影响。
In the present research, we assumed that reducing the amounts of E6 and E7 oncoproteins by a specific siRNA sequence and recovering p53 and RB proteins, along with the recovery of the FOXO1 protein by applying anti-miR-182, would increase apoptosis and reduce proliferation rate in cancer cells. The HPV16-positive CaSki cervical cancer cell line was used. 48 hours after transfection of siRNA for targeting E6 and E7 oncoproteins and anti-miR-182, expression of its cellular targets p53, p21 and FOXO1 was assessed by real-time PCR, western blot analysis and immunocytofluorescence staining. In all treatments, apoptosis rate and viability were evaluated using Annexin-V-FITC apoptosis detection kits and MTT assays, respectively. Among the designed siRNAs, E6-1 and E7-2 proved the most effective in reducing E6 and E7 expressions by increasing the apoptotic rates to 12.4% and 16%, respectively, after 48 hours. Also, using anti-miR-182 increased apoptotic rate to 12.7% 48 hours after transfection of cervical cancer cells. The combinational use of either E6-1 or E7-2 siRNAs with anti-miR-182 resulted in a rise in apoptosis to 19.3% and 26%, respectively, higher than those obtained from the individual application of either without anti-miR-182. The simultaneous use of siRNA E6-1 and siRNA E7-2 with cisplatin increased sensitivity to cisplatin and reduced the viability of the cancer cells as compared to the use of cisplatin alone. The simultaneous use of cisplatin and anti-miR-182 had no considerable effect on viability or apoptosis rate compared to cisplatin alone.