A Preliminary Study On The Role Of SIRT1 In The Development Of Cervical

Objective : The class III histone deacetylase belongs to Sirtuins, namely the homologous protein of silent information regulator 2. It was well known that SIRT1 is closely related to the occurrence of tumor and might be a novel target for tumor treatment. The purpose of this study was to detect the expression of SIRT1 in cervical cancer tissue and its relationship with the clinical charaters, to investigate the effect of RNA interference targeting SIRT1 gene on the proliferation, migration, invasion,apoptosis and Notch signaling pathway in human cervical cancer Si Ha cells, to explore the mechanisms of SIRTl in the development of cervical cancer and the its regulation effect on the Notch-Hes signaling pathway.Methods:A total of 221 paraffin specimens were collected from the department of pathology in the affiliated hospital of Qingdao university between Sep 2012 to Mar 2014,including CINI grade, CINII-CINIII grade, cervical squamous carcinoma and normal cervical tissue. Immunohistochemical staining was used to detect the SIRT1 protein expression in the clinical specimences, and analyze the correlation of the expression and clinical indexes. Chemical synthesis of small interfering RNA(si RNA) targeting SIRT1 gene were transfected into cervical cancer Si Ha cells by Lipofectmine RNAi-MAX liposomes, 72 h after transfection, the total RNA and protein were extracted, the expression level of SIRT1 m RNA was detected by RT-PCR, and the expression of SIRTl,Notch1, Hes1 and Cyclin D1 protein were detected by Western blot. The cell proliferation was assessed by CCK-8 method, the migration and invasion of Si Ha cells were measured by Transwell method, the the apoptosis was evaluated by Annexin V staining and Hoechst33258 fluorochrome staining respectively, and Notch signaling pathway protein Notch1,Hes1, cyclin D1 were detected by Western blot.Results:Immunohistochemical staining indicated that the expression of SIRT1 in normal cervical tissue and lesser degree of cervical intraepithelial neoplasia(CINI grade)were not observed, while 16 cases were positive in CINII-III grade(16/67) cervical tissue,29 are positive in the cervical cancer tissues(29/54). RT-PCR results showed that the m RNA expression of si RNA-SIRT1 group was significantly down-regulated(P<0.05)compared to the control groups. Western blot showed the expression of SIRT1 protein in the si RNA-SIRT1 group was significantly decreased(P<0.05), the expression ofcyclin D1, Notch1 and Hes1 were up-regulated(P < 0.05), while the other three groups had no significant changes(P>0.05). CCK-8 proliferation assay demenstrated that the inhibition rate of si RNA-SIRT1 group was 37%, while the negative control group was8.7%, the inhibition rate of transfection reagent control group was 7.3%. The inhibition rate of si RNA-SIRT1 group was significantly higher than other groups. The Transwell experiments indicated that the si RNA-SIRT1 interference group, the numbers of Si Ha cells across the artificial basement membrane was significantly decreased compared to the control groups. Flow cytometry displayed 16% in si RNA SIRT1 group, compared with only 2.6% in si RNA negative control group. Hoechst33258 staining revealed that the nucleus of Si Ha cells decreased, the chromatin condensed, and apoptotic bodies appeared,while the cell nuclei in the control groups were pale blue uniform.Conclusion:Chemically synthesized small interference RNA targeting SIRT1 gene not only restrained the proliferation, migration and invasion of Si Ha cells but also induced apoptosis in Si Ha cells, which was associated with Notch-hes signaling pathways.SIRT1 might play an important role in the carcinogenesis of cervical cancer through Notch-Hes signaling pathway.