| Background:Cervical cancer is the most common malignant cancer in women in the worldwide, and the incidence in the developing countries is remain high, behind breast cancer. The persistent infection of high-risk human papillomavirus is the main reason. It is a continuous process from CIN to invasive cervical cancer, which usually lasts ten years. In recent years, due to the widespread application of cervical cancer screening based on liquid-based cytologic test, the morbidity and mortality of cervical cancer declined obviously because of the early detection and treatment. Surgery, chemotherapy and radiotherapy are common modes of treatment for cervical cancer. Photodynamic therapy (PDT) is a recently developed anticancer modality, whose effects result from direct action of singlet oxygen and reactive oxygen species through visible light irradiation of a photosensitive dye accumulated in the cancerous tissue. The cationic porphyrin,5,10,15,20-tetra-(N-methyl-4-pyridyl) porphine (TMPyP4), is a novel type of synthetic water-soluble photosensitizer. It locates in nucleus and stabled the G-quadruplex ligand and the activity of telomerase. As the active part, hTERT synthetizes telomeric repeats by using RNA component as template, then maintain the length of telomere and the stability of chromosome. This research will make the Lenti-hTERT-shRNA transfect the normal SiHa cells and those accepted TMPyP4-PDT to investigate the effects of hTERT on TMPyP4-PDT and the possible mechanism aimed at providing a novel adjuvant therapy for cervical cancer patients.Objective:To investigate the effects of hTERT silencing on apoptosis and invasion of cervical cancer SiHa cells and to study the possible mechanism of action.Methods:Identify the optimal MOI value by immunofluorescence (IF) and pick out the ideal RNA interference sequence by RT-PCR and Western Blot. The cervical cancer SiHa cells were divided into four groups:control group, gene therapy group, TMPyP4-PDT group, and combined treatments group. Cell Counting Kit-8 (CCK-8) was performed to monitor cell proliferative activity. The apoptotic cells were identi-fied using the AnnexinV-PE/7-AAD double dye kit. The ability of cellular invasion was assessed by Trans well assay. The expression of Bcl-2、Bax、Caspase-3、 Caspase-8 were detected by real time PCR, and the Western blot was chosen to test the expression of Bcl-2、Bax、Caspase-3、Caspase-8 proteins.Results:When the MOI value was 20, the transduction efficiency was up to 80%. The expression of hTERT gene was lowest in sh -1693 groups. The cell survival rate in control group, gene therapy group, TMPyP4-PDT group, and combined treatments group was respectively (1.0±0.03)%、(0.75±0.02)%、(0.62±0.05)%、(0.19±0.03)%, The difference was significant (P<0.05). The apoptosis rate increased significantly (P<0.05) and the total number of invaded cells decreased obviously in combined treatments group when compared with other three groups. The expression of Bcl-2, Caspase-8 mRNA and protein decreased significantly (P<0.05), meanwhile, the expression of Bax, Caspase-3 mRNA and protein increased (P<0.05) in the groups who were dealt with two treatments than those accepted gene therapy or TMPyP4-PDT alone.Conclusions:The hTERT down-regulation can promote the apoptosis and restrain the invasion ability, proliferation of SiHa cells in photodynamic therapy by decreasing target genes Bcl-2, Caspase-8 expression and increasing the target genes Bax, Caspase-3 expression. |
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