Study Of Killing Effect Of TMPyP4-PDT Combined With Nucleolin Silence On Human Cervical Cancer SiHa Cells In Vitro

Background:Cervical cancer is one of the most common malignant carcinoma in women and does harm to women. Photodynamic therapy (PDT) is a promising and effective approach which has gradually gained much attention. It killed tumor cells selectively by cells’specific absorption of a given photosensitizer (PS) with a proper light. PDT has been approved to cure several tumors. Meso-5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)(TMPyP4) is a kind of porphyrin derivatives and it is able to selectively gather in the tumor tissue, but quickly metabolized in normal tissues. So much attention is drawn in cancer treatment over a long period of time especially in photodynamic therapy aspect. Recent years, scientists kept trying for combination therapy of PDT in order to enhance its antitumor effects. Recent studies demonstrated that nucleolin are overexpressed in many tumer cells including cervical cancers. In this study, we attempted to study for the first time the antitumor effect of combination of PDT and gene silence. According to interference principle of RNAi, we designed siRNA targing human nucleolin and transfected into cervical cancer SiHa cell to investigate its effect on TMPyP4-PDT, after which proliferation, apoptosis and related molecules were assessed, furthermore offered the theoretical foundation of PDT combined with nuleolin silence in treating cervical cancer.PART1. Effect of nucleolin-specific siRNA transfection on nucleolin expression in cervical cancer SiHa cells and Screening for the most effective siRNAObjective:Design and chemically synthesize nucleolin-specific siRNA by siRNA technique and then transfect the cervical cancer SiHa cells to select the most effective nucleolin-specific siRNA for the followup experiments.Methods:The cervical cancer SiHa cells were used as subject and four groups were rolled in the experiment, including the siRNA-1, siRNA-2, siRNA-3groups, which targeted human nucleolin gene, and three control groups, including the NC-siRNA group, blank control group and normal control group, which served as negatice control. The chemically synthetic siRNA was transfected to SiHa cells in bitro by RNAi Max transfection reagent. Expression of nucleolin in mRNA and protein level in nucleolin-siRNA groups and negative control groups was evaluated by RT-PCR and Western blot to select the most effective nucleolin-siRNA.Results:Nucleolin-specific siRNA effectively down-regulated nucleolin, compared with negative control siRNA (P<0.05). The protein expression was also down-regulated in nucleolin-specific siRNA group (P<0.05). siRNA-2was the most effective nucleolin-specific siRNA.Conclusions:Nucleolin-specific siRNA effectively down-regulated nucleolin in both mRNA and protein level and Nucleolin-specific siRNA-2can be used in the follow-up experiment. PART2. Killing Effect of TMPyP4-PDT Combined with Nucleolin Silence on Human Cervical Cancer SiHa Cells in vitroObjective:To study the antitumor effect of new photodynamic therapy (PDT) applying TMPyP4combined with nucleolin silence on cervical cancer SiHa cells in vitro, and to explore an available combination treatment project for cervical cancer.Methods:1. SiHa Cells were divided into:blank control group, RNAi group, PDT group and PDT-RNAi group.2. The proliferation activities of SiHa cells were assessed by Cell Counting Kit-8(CCK8) assay. 3. The apoptotic rates were measured by flow cytometry (FCM) with Annexin V/PI staining.4. Expressions of Bax and Bcl-2were detected by Western blot.Results:1. Compared with blank control group, the inhibitory rates of growth of SiHa cells in experiment groups (RNAi group, PDT group, PDT-RNAi group) were increased significantly (P<0.01);the inhibitory rates of growth of SiHa cells inRNAi-PDT group were higher than those in PDT group and RNAi group (P<0.05). The Q value was1.27.2. FCM analysis showed that compared with blank control group, apoptotic rates of SiHa cells in experiment groups were increased, and the latter showed a higher late apoptotic rate (P<0.05).3. Western blot showed that the expression of Bax was increased (P<0.05), while the expression of Bcl-2was decreased in three experiment groups (P<0.05), compared with the control group.Conclusions:1. TMPyP4-PDT showed a strong photodynamic effect, which can inhibit the proliferation and induce cell apoptosis of SiHa cells in vitro.2. Nucleolin silence showed a good synergy effects to TMPyP4-PDT through up-regulating the expressions of Bax and down-regulating the expressions of Bcl-2.3. TMPyP4-PDT combined with nucleolin may be a new potential therapy for cervical cancer and even other cancers.