| Background:As the second gynecological malignancy worldwide, the incidence of cervical carcinoma was just after the breast cancer. BEZ235is the dual targeted inhibitors of PI3K and mammalian target of rapamycin (mTOR.) which is downstream factors of Phosphatidylinositol-3-kinase (PI3K), and it could inhibit a variety of tumor, but we found the report on cervical cancer is less. S-phase kinase-associated protein2(Skp2), which plays an important role in the cell cycle, was closely related to cell proliferation and apoptosis. To date, many studies have found that the poor prognosis was associated with the overexpression of Skp2protein in a variety of malignant tumor. It was reported that Skp2could promote the development of tumor by regulating PI3K signaling pathway. In a variety of malignant tumors, PI3K signaling pathways regulated Skp2protein either before or after the transcription and translation.Object:To study on the inhibiting effects of BEZ235on cervical cancer, and the clinical pathological significance of Skp2overexpression in the progression of cervical cancer.Materials and methods:Part1:Cervical cancer cell lines Hela, C33A, SiHa cells were cultured; MTT assays were used to detect the effects of BEZ235on cells growth and proliferation. The growth curves were drawn, and screening of drug concentration; BEZ2350.1μM,0.4μM was added to the cells, and the same amount volume of DMSO was added to the control group. After48hours, extracted the total RNA and protein, and RT-PCR was used to detect the Skp2mRNA expression with or without treatment of BEZ235. Western-blot was used to detect the change of Skp2, Ezrin, Sixl expression with or without treatment of BEZ235in Hela and C33A cells. The effect of BEZ235on migration ability of Hela cells in cervical cancer were detected by scrach test. Part2:The Skp2expression in25cases of normal cervical epithelium,84cases of cervical intraepithelial neoplasia (CIN) and163cases of Squamous Cell Carcinoma (SCC) were investigated by immunohistochemical (IHC), and the human papillomavirus (HPV) expression in different tissues in cervical cancer were detected by RT-PCR. Combined with HPV infection status, biological characteristics, the clinical significance of Skp2protein expression in cervical cancer and the survival status of patients with SCC were analyzed by statistical analysis.Results:Part1:MTT assays showed that the growth rates of Hela, C33A, SiHa cells were decreased after blocking PI3K signaling pathway, and the results of RT-PCR and Western blot indicated that the expression of both Skp2mRNA and Skp2, Ezrin and Sixl protein in cervical carcinoma Hela and C33A cells are decreased with the concentration of BEZ235(0.1μM,0.4μM) inceased; In the scratch test results, we found that the cells moving distance was shorter than the control group after the treatment of BEZ235on cervical cancer Hela cells for24h or48h. Part2:HPV mRNA expression was highly positive in11cases of SCC tissues by RT-PCR assay in all of14cases. Compared with the control, the positive rates of Skp2in CIN-1, CIN-2and CIN-3were higher in order (P<0.01); In cervical squamous cell carcinoma, positive rate rate of Skp2protein was obviously higher than the normal cervical epithelium (P<0.01), and closely related with the FIGO staging and high-risk HPV infection (P<0.05); The patients with positive expression of Skp2protein expression in cervical SCC had a lower tumor free survival rate and total survival rate than those with negative expression (P<0.01).Conclusions:1. PI3K inhibotor BEZ235inhibited the proliferation and migration of cervical cancer cells by drownregulating Skp2protein;2. Skp2might be a useful biomarker for predicting the poor prognosis of cervical SCC. |
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