Journal of Jilin University Medicine Edition ›› 2015, Vol. 41 ›› Issue (06): 1107-1112.doi: 10.13481/j.1671-587x.20150602

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Influence of irreversible electroporation mediated HPV16 E6 shRNA interference plasmid in proliferation of cervical cancer SiHa cells

WANG Zhiliang1, YU Tenghua2, QIN Qin1, WU Yutong1, ZHANG Wenqian1, HUA Yuanyuan1, XIONG Zhengai1, ZHOU Wei3   

  1. 1. Department of Gynecology and Obstetrics, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China;
    2. Department of Endocrine and Breast Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China;
    3. Department of Obstetrics, Chongqing Health Center for Women and Children, Chongqing 400013, China
  • Received:2015-04-03 Online:2015-11-28 Published:2016-01-11

Abstract:

Objective To explore the feasibility of using irreversible electroporation(IRE) mediating HPV16 E6 shRNA into cervical cancer cell line SiHa,and to clarify the influence of their co-effect on the proliferation of SiHa cells and its mechanism. Methods A HPV16 E6 gene specific interference sequence was inserted in pGenesil-1 to build a interference vector.10 pulses of IRE with 800 V,100 μs, and 1 Hz were applied to the suspension of SiHa cells and vectors.According to the treatment factors,control group,IRE group,pGenesil-N group,pGenesil-N+ IRE group,pGenesil-E6 group and pGenesil-E6+IRE group were set up.The expression of green fluorescent protein (GFP) and transfection efficiency were confirmed by inverted fluorescence microscope 24 h after the vector was transfected by IRE,and the expression efficancy of GFP was calculated.The expression levels of E6 mRNA and protein were detected by RT-PCR and Western blotting method which was also applied to detect the expressions of P53 and PCNA.The proliferative activity of SiHa cells was determined by CCK-8 assay. Results Enzyme digestion and DNA sequencing verified that the vectors were correctly constructed. GFP was seen under inverted fluorescence microscope 24 h after IRE transfection.Compared with IRE group,the expression levels of E6 mRNA and protein were decreased detected by RT-PCR and Western blotting method after the vectors were treated with IRE,the P53 protein expression level was increased(P<0.05),and the PCNA expression level was decreased(P<0.05). The CCK-8 assay results showed the proliferative activity of SiHa cells in pGenesil-E6+IRE group was decreased more obviously than that in pGenesil E6 group (P<0.05). Conclusion IRE can play the role of gene transfection of mediating HPV16 E6 shRNA into SiHa cells,and their co-effect can significantly inhibit the proliferation of SiHa cells.

Key words: electroporation, interference plasmid, uterine cervical neoplasms, cell proliferation

CLC Number: 

  • R737.33