| Objective To research the specificity and efficiency of HPV16 E6 gene silence in CaSki cells of cervical cancer in vitro and in vivo by RNA interference. Methods To design the specific HPV16 E6 siRNA and transfect it into CaSki cells by liposome. To test the apoptotic rates of CaSki cells, the changes of HPVI6 E6 mRNA by RT-PCR and the expression of HPV16 E6 protein by Western blot and Flow cytometry at different times after transfection. To set up nude mice model of cervical cancer, HPV16 E6 siRNA was given through i.p or into tumor directly. To measure the changes of tumor volume, the expression of the HPV16 E6 protein by IHC, the apoptosis of tumor cells by TUNEL. Results The apoptotic rates of CaSki cell were 7.7%, 11.8%, 37.4% and 12.6% respectively at 24h, 48h, 5d and 9d after transfection. The HPV16 E6 mRNA was reduced by 77%, 83%, 59% and 41% at 24h, 48h, 5d and 9d after transfection, but the mRNA of β-actin as internal control and the siRNA negative control of HPV16 E6 did not change. The inhibition rates of HPV16 E6 protein were 79.7%, 80.4%, 71.3% and 57.4% at 24h, 48h, 5d and 9d after transfection measures by FCM, which were confirmed by the results of Western blot. However, the protein levels of Lamin A/C as internal control and the negative control of HPV16 E6 did not change after transfection. In vivo tests, the growth rates of tumor were markedly inhibited when given HPV16 E6 siRNA by injection intoperitoneal cavity or in1o tumor directly. The expressions of HPV16 E6 protein were inhibited and the apoptosis of tumor cells increased markedly after HPV16 E6 siRNA were given. The results were better when HPV16 E6 siRNA were given twice than once. Conclusion There really was RNA interference in CaSki cell of cervical cancer, the interference to HPV16 E6 are specific and high efficient no matter in vtro and in vivo tests. However, the time-efficiency may be improved by reconstructing siRNA or selecting the best vector for transfection. |
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