Construction of Recombinant Adenovirus Adv-AFPsiRNA and Study of Inhibition Effect of Adv-AFPsiRNA on Ovarian Carcinoma
|School||Tianjin Medical University|
|Keywords||Alpha-fetoprotein RNA interference Ovarian Cancer AFP silent Gene therapy|
[Objective]: In view of the recent years, many scholars and clinicians found through repeated studies and detailed clinical observation and detection of alpha-fetoprotein in the tumor development process is not only an epiphenomenon, but a close with the growth of tumor cells related endogenous protein. And in a variety of diseases (such as: primary hepatocellular carcinoma, acute, chronic hepatitis, of testicular ovarian Kawasaki fetal tumor, congenital gastrointestinal diseases, mixed malignant ovarian germ cell tumor, immature teratoma, embryonal carcinoma endodermal sinus tumor) serum can be increased AFP. In these diseases, although serum AFP levels have been found associated with the non-normal growth phenotypes AFP whether tumor directly lead to the growth of phenotypic changes, but still not sure. My room the previous research results have shown that the liver AFP protein levels in HepG2 cells and mRNA levels can be AFPsiRNA significantly and specifically silence. It also found that the AFP the silent lead to the growth inhibitory effects is not mainly due to the occurrence of apoptosis, but cell cycle G1 / S phase transition stagnation. Recently although ovarian germ cell tumor expression of endogenous AFP reported, but whether with liver cancer have a similar situation? Has yet to see the AFP RNAi technology for the treatment of ovarian cancer reported. Accordingly, the purpose of this study is: First, by Western blot and Semi-qRT-PCR detection, observation commonly used strain built ovarian cancer cell lines and ovarian cancer, living tissue and cells of the normal nest cancer organization specimens AFP expression for follow-up research to lay the theoretical foundation; based RNAi technology can silence specific genes and to construct the recombinant target given the AFP the gland the virus rAdv-AFPsiRNA observe its inhibitory effect on the growth of ovarian cancer cells in vitro, and provide a new mode of treatment for ovarian cancer . [Method]: In the present study, we first detected by Western Blot and Semi-qRT-PCR, observe AFP protein levels in the several built strains ovarian cancer cell lines and human ovarian cancer living tissue and normal ovarian tissue and mRNA levels expression of the situation. And use of biological information technology design an AFP the target of shRNA. After chemically synthesized shRNA cDNA duplexes, constructed the the target recombinant fixed AFP adenoviral vector. The building, with its first transfected HepG2 cells, its function is identified. The recombinant target given AFP adenovirus rAdv-LacZsiRNA are respectively transfected ovarian cancer cell line SKOV3, OVCAR3 and ES-2. By Western blot and semi-quantitative RT-PCR analysis of expression of the protein levels of AFP in cancer cells and mRNA levels to determine the the AFP silent of its AFPsiRNA of the three ovarian cancer efficiency and specificity; using the MTT assay and soft agar cloning The formation of the experimentally observed AFP adenovirus recombinant target given the growth inhibition of ovarian cancer cells. [Result 1:1. Vitro AFP ovarian cancer cell lines express the results of Western blot results showed that the: ① in vitro culture of ovarian cancer cell line SKOV3, OVCAR3 and ES-2 cell lysate AFP expression in varying degrees, and the negative In comparison, in normal skin fibroblasts BJ no expression; 2 Semi-RT-PCR results show that: both the mRNA levels of the different degrees of the ovarian cancer and ovarian cancer live tissue specimens cell lysates. Ovarian cancer live tissue and ovarian living expression of tissue cell lysates mRNA levels compared to an average increase of 2.3 times. 2. Experimental results (1) restructuring AFP adenovirus targeting ovarian cancer cell growth inhibition with recombinant target set AFP adenovirus rAdv-AFPsiRNA were infected with ovarian cancer SKOV3, the OVCAR3 and ES-2 cells after 48 hours, through, vestern blot detection AFP expression level was The reduced specificity protein expression inhibition rate of 64%, 28% and 69%. Correspondingly, Adv-LacZsiRNA AFP expression levels are not significantly affected. ② by semi-quantitative RT-PCR method to verify rAdv-AFPsiRNA AFP mRNA level interference. The results show that the internal control β-actin expression levels are relatively consistent with the negative control normal skin fibroblasts BJ compared to ovarian cancer SKOV3, OVCAR3 and ES-2 cell AFPmRNA level significantly lower inhibition rates were 60%, 39% and 67%. Correspondingly, Adv-LacZsiRNA For AFPmRNA expression levels had no significant effect. ③ formed by cloning experiments confirmed OVCAR3 ovarian cancer cells infected with recombinant target set AFP adenovirus and ES-2, the number of colony forming with the control (corresponding uninfected cells) respectively, compared to 39% and 67%. ④ in MTT: cell activity measurement with ovarian cancer cells and the control cell activity was no significant difference. [Conclusion: The results show that: 1 three ovarian cancer cell lines in vitro and ovarian cancer live tissue specimens cells have different levels of expression the AFP protein and AFPmRNA of level; 2.AFP SKOV3, OVCAR3 and ES-2 intracellular protein levels with mRNA levels reorganization targeting AFP adenovirus AFPsiRNA significantly and specifically silence. This result indicates that the target recombinant rAdv-AFPsiRNA given AFP adenovirus can effectively and specifically inhibit the proliferation of human ovarian carcinoma cells, and provide a new gene therapy for the treatment of ovarian cancer.