Zhi-liang Wang¹, Wei Zhou², Zheng-ai Xiong³, Li-mei Wu³,  Yu-tong Wu³, Yuan-yuan Hua³, Cheng-xiang Li⁴

¹Department of gynecology and obstetrics, The Second Affiliated Hospital of Chongqing Medical University, ²Department of obstetrics, Chongqing Health Center for Women and Children, ³The Second Affiliated Hospital of Chongqing Medical University, ⁴Chongqing University, College of Electrical Engineering

Objective:To verify the feasibility of using irreversible electroporation (IRE) to mediate HPV18 E6 shRNA plasmid into cervical cancer cell HeLa in vitro and in vivo, and their co-effect on cell and tumor growth. Method: HPV18 E6 targeted shRNA and CTL shRNA plasmid were constructed as previous report. HeLa cells suspension mixed with 10μg HPV18 E6 shRNA plasmid were received IRE treatment with fixed parameters (800V, 100us, 1Hz, 8pulses); the expression of GFP was confirmed by fluorescence microscope 24 hours later. Then HeLa cells were subjected to different treatments: CTL, IRE, CTL shRNA, IRE+ CTL shRNA, E6 shRNA, IRE+ E6 shRNA. The level of E6 mRNA was detected by RT-PCR. The levels of E6, P53 and PCNA proteins were determined by Western blotting. Cell growth was determined by CCK-8 assay. A xenograft model was used to observe the co-effect of IRE and E6 shRNA plasmid: HE staining and frozen sections were used to confirm the ablation effect and the feasible of mediating plasmid into tumor tissue. Then 20 female nude mice xenograft models were randomly divided into 4 groups: CTL, IRE, E6 shRNA and IRE+ E6 shRNA. Tumor growth curve was drawn after treatment according to the data obtained every 5 days. 30 days later, tumors were harvested to perform Western blotting on E6, P53 and PCNA proteins. Result: Enzyme digestion and DNA sequencing verified the plasmids was correctly constructed. Green fluorescence was seen under an inverted fluorescence microscope24 hours later confirming the feasible of IRE mediating plasmid into HeLa cells.The levels of E6 mRNA and protein decreased significantly (P<0.05)in IRE+ E6 shRNA group. Cell proliferative curve revealed that IRE+ E6shRNA inhibited cell growth significantly (P<0.05). HE staining and frozen sections showed that IRE induced cell death and mediated plasmid into peripheral tumor tissue at the same time. Western blotting and tumor growth curve obtained the similar trend of in vitro, which IRE and E6 shRNA have co-effect of inhibiting tumor growth in vivo. Conclusion: IRE mediated E6 shRNA into HeLa cell both in vitro and in vivo, and they had a co-effect of inhibiting growth; this combined treatment strategy may have a potential value in cervical cancer treatment. .

Key Words: Irreversible electroporation, shRNA plasmid