Study On The Expression And Mechanism Of STC1 Gene In Human Cervical Cancer

Objective:To investigate the expression of STC1 gene in cervical cancer tissues.To explored the potential role and mechanism of STC1 in the progression of cervical cancer by overexpressing STC1 gene in CaSki cells.Methods:(1) we first examined the expression level in normal and cancerous matched cervical tissues by RT-PCR. Then, another 60 cases of formalin-fixed, Paraffin-embedded specimens, including 15 cases of cervical cancer,15 cases of cervical intraepithelial neoplasia(CIN),15 cases of inflammation tissues,15 cases of normal cervical epidermis, were used to assess the expression status of STC1 protien by immunohi stochemi stryThe recombinant eukaryotic plasmid pcDNA3.1 (+)-STClwas transfected into cervical cancer cell CaSki by lipofectamine 2000. The control group was transfected with Empty vector pcDNA3.1(+).The cells stably expressing STC1 gene were screened by medium with G418. The expression of STC1 gene was determined by Western blot. The ratios of cell cycles and apoptosis rate were analyzed by flow cytometry. The influence of STC1 on the tumor development of cervical carcinoma in vivo was examined.Xenograft tumors tissues from nude mice were made into paraffin sections for HE staining,moreover, the expression of Ki67 and PCNA were detected by immunohistochemistry staining(2)After the CaSki cells were treated with TNF-α, pyrrolidine dithiocarbamate and thapsigargin, RT-PCR and Western Blot assessment were performed for alteration in expression of STC1, NF-κB, PARP,and Caspase-3 gene. Apoptosis was analyzed with flow cytometry.Chromatin immunoprecipitation (CHIP) was applied to detect the proteins combined to the promotor of STC1 gene.Result:(1)RT-PCR revealed that the expression level of STC1 in cervical cancer tissues was decreased compared with the normal ones. Then, immunohistochemistry analysis revealed that the protein level of STC1 was lowest in tumor tissues, relatively higher in CIN tissues, and further increased in inflammation and normal tissues, indicating its role in the progression of cervical cancer. The recombinant eukaryotic expression plasmid pcDNA3.1(+)-STC1 was successfully constructed, and it could be correctly expressed in the CaSki cells. Flow cytometry analysis illustrated that G1 phase proportion of CaSki/STC1 cells increased compared to control cells. In vivo tumor growth assay showed that the tumor growth of CaSki/STC1 cells reduced.(2) When the expression of STC 1 gene were enhanced in CaSki cells, thapsin, TNF-a readily suppressed the cell proliferation and induced the cell apoptosis. The expression of NF-κB increased and the activity of STC1, PARP and Caspase-3 were enhanced in response to TNF-αand thapsin. The expression of STC1 and NF-κB was positive correlation, which was testified by RT-PCR and Western blot. When CaSki cells were treated with Pyrrolidine dithiocarbamate (PDTC), an effective inhibitor of NF-κB, the activity of NF-κB decreased and the expression of STC1 down-regulated in a time-dependent manner.Chromatin immunoprecipitation (CHIP) showed that NF-κB protein directly bound to STC1 promoter in CaSki cells.Conclusion:STC1 inhibits cell proliferation and invasion of cervical cancer cells through NF-κB activation.