Effecs Of Silencing HPV16 E6 Gene By RNA Interference On Autophagy In Human Cervical Cancer SiHa Cell

Objective:Investigating whether the HPV16 E6 siRNA can specifically inhibit the expression of HPV16 E6 gene in human cervical carcinoma SiHa cell and observing the effect of silencing HPV16 E6 gene expression on autophagy in SiHa cell from both the autophagosome morphology and the autophagosomal marker aspects in order to study the effect of HPV infection on autophagy and offer a new idea for the development of cervical cancer prevention and treatment.Methods:(1)Recombinant eukaryotic expression vector sh138-HPV16 E6 siRNA was constructed and identified.Then transfected it into human cervical cancer SiHa cells taking a liposome approach.Moreover,the transfection efficiency was observed under fluorescence microscope and the expression of HPV16 E6 mRNA before and after transfection in each group of cells was measured by Real-Time fluorescence quantitative PCR.(2)The formation of autophagosomes in each group of cells after silencing HPV16 E6 gene was observed through by transmission electron microscope and the expression of Beclin 1 protein in SiHa cell among differengt groups was detected by immunocytochemistry.Results:(1)The agarose gel electrophoresis result of recombinant plasmid PCR products and the DNA sequence analysis have proved that the recombinant eukaryotic expression vector sh138-HPV16 E6 siRNA was constructed successfully.After liposome-mediated transfection of recombinant plasmid into SiHa cells a small amount of green fluorescence could be observed under fluorescence microscope inaround 12 hours, which could be a proof of successful transfection. By selecting 1020 X field of view around 48 hours after transfection, when the fluorescence was brightest, and counting the number of cells with green fluorescence and all cells, it could be calculated that the transfection efficiency was about( 250 ±) %. The result of real-time PCR 48 hours after transfection showed that the expression of HPV16 E6 mRNA in the SiHa-sh138-HPV16 E6 siRNA group of cells decreased distinctly,which was statistically significant compared to other groups(P<0.001).(2)The number of autophagosomes in SiHa cells showed that there were less autophagosomes in the group of SiHa-sh138-HPV16 E6 siRNA(P<0.05) and more of them in starvation group(P<0.05),compared to SiHa group and SiHa-sh138-NC group.(3)The result of applying immunohistochemistry to detect the Beclin 1 protein expression in SiHa cells showed that the Beclin 1 protein expression in the group of SiHa-sh138-HPV16 E6 was lower than SiHa group and SiHa-sh138-NC group(P<0.05), whereas it was clearly higher in starvation group(P<0.05).Conclusion:(1)The Recombinant plasmid sh138-HPV16 E6 siRNA expressed by silencing HPV16 E6 gene was successfully constructed and the small interfering RNA could effectively silence the expression of HPV16 E6 gene in SiHa cells.(2)The silencing of HPV16 E6 gene could reduce the formation of autophagosomes and down-regulate the expression of Beclin 1 protein distinctly in SiHa cells.