Survivin Regulates NF-κB P65 In Esophageal Squamous Cell Carcinoma

Objective: Esophageal carcinoma is the fourth most common malignant tumor in the world, with a high mortality rate and a poor prognosis. It has seriously endangered people’s health and life safety, and the 5 year overall survival rate is only about 10%. China is one of the countries with high incidence of esophageal cancer. Each year nearly half of newly diagnosed esophageal carcinoma occured in China. Esophageal squamous cell carcinoma(ESCC) is the most common pathological type of esophageal cancer in China. Although the treatment of esophageal carcinoma has been greatly improved in recent years, the long-term survival of patients is still not optimistic. Studies showed that early diagnosis and treatment could improve overall survival, and some biomarkers changed significantly before tumorigenesis of ESCC, detecting these molecular markers is helpful in early diagnosis. Survivin is a member of the inhibitor of apoptosis protein family, which promotes tumorigenesis, progression and has connection with clinical stage and prognosis in ESCC by regulating cell apoptosis. The NF-κB family of transcription factors, especially the subunits NF-κB p65 affect cell apoptosis, proliferation and differentiation through regulating genes expression, play an important role in carcinogenesis. Our previous study showed that reduction of Survivin expression dephosphorylated NF-κB p65 in esophageal carcinoma cells, which indicated that Survivin may activate NF-κB p65 in ESCC. However, Survivin’s regulatory mechanism of NF-κB p65 and its action site remained unclear. In this study, we use YM155, LV3-Survivin shRNA recombinant plasmid to downregulate Survivin, and GV142-Survivin overexpression recombinant plasmid to upregulate Survivin in ESCC, then detect the expression of NF-κB p65 and its upstream gene IKK α and IKKβ to investigate whether Survivin could regulate the expression of NF-κB p65 through binding to IKKα and/or IKKβ promoter region, we also conduct Flow cytology assay to assess Survivin’s influence on cell cycle andapoptosis. Methods: 1) Human esophageal squamous cell carcinoma cell line ECA109 and KYSE150 were cultured. Different concentrations of Survivin inhibitor-YM155 were added into culture medium as experimental group, cells treated with complete medium as blank control group. Cells were harvested and counted 48 hours after to assess the influence of YM155 on cell survival. Total RNA and protein were also extracted 48 hours after for RT-qPCR and Western boltting assay. Expression of Survivin, IKKα, IKKβ and NF-κB p65 were detected at mRNA and protein level to verify the regulation relationship between Survivin and NF-κB p65. 2) LV3-Survivin shRNA plasmid and LV3-control plasmid were constructed and transiently transfected ECA109 and KYSE150 cells. Cells were divided in to three group: LV3-Survivin shRNA group, LV3- control group and cells treated with complete medium as blank control group. Cells were harvested 48 hours after transfection. Total RNA and protein were extracted for RT-qPCR and Westernboltting assay. Expression of Survivin at mRNA and protein level were detected to assess the transfection efficiency. The expression of IKKα, IKKβ and NF-κB p65 were detected at mRNA and protein level to verify the regulatory relationship between Survivin and NF-κB p65. Cell cycle and apoptosis were detected by Flow cytometry to assess Survivin’s influence on biological behaviour of cells. 3) GV142- Survivin over-expression recombinant plasmid and GV142-control plasmid were constructed and transiently transfected ECA109 and KYSE150 cells. Cells were divided in to three group,included: GV142-Survivin over-expression group,GV142-control group and cells treated with complete medium as blank control group.Cells were harvested 48 hours after transfection. Total RNA and protein were extracted for RT-qPCR and Western boltting assay. Expression of Survivin at mRNA and protein level were detected to assess the transfection efficiency. The expression of IKKα, IKKβ and NF-κB p65 were detected at mRNA and protein level to verifu the regulatory relationship between Survivin and NF-κB p65. Cell cycle and apoptosis were detected by Flow cytometry to assess Survivin’s influence on biological behaviour of cells. 4) Chromatin immunoprecipitation experiments: To investigate weather Survivin protein could bind to IKKα and/or IKKβ promoter directly or indirectly in vitro, ECA109 and KYSE150 cells were transiently transfected with GV142-Survivin overexpression recombinant plasmid, Survivin monoclonal antibody was used to immunoprecipitate the DNA segment combined with Survivin protein. DNA segment was identified by specificity primers of IKKα or IKKβ promoter. 5) Dual luciferase reporter gene assay: IKKβ promoter luciferase reporter gene plasmid were constructed, termed pGL3- IKKβ promoter plasmid. ECA109 and KYSE150 cells were co-transfected with pGL3- IKKβ promoter plasmid, pRL-TK plasmid and/or GV142-Survivin over-expression plasmid. Survivin’s regulation of transcriptional activity of IKKβ promoter were investigated. Results: 1) The specific inhibitor of Survivin-YM155 could inhibit cell proliferation in both ECA109 and KYSE150 cells and down-regulate the expression of Survivin, IKKα, IKKβ and NF-κB p65 at transcriptional and posttranscriptional level. 2) LV3-Survivin shRNA plasmid could down-regulate the expression of IKKα, IKKβ, NF-κB p65 at mRNA level, and the expression of IKKα, IKKβ, p-NF-κB p65 at protein level in ECA109 and KYSE150 cells. Downregulated expression of the Survivin promoted cells apoptosis and induced cell cycle arrested in G2/M phase in ECA109 and KYSE150 cells. 3)GV142-Survivin over-expression plasmid could up-regulate the expression of IKKα, IKKβ, NF-κB p65 at mRNA level, and the expression of IKKα, IKKβ, p-NF-κB p65 at protein level in ECA109 and KYSE150 cells. Overexpression of Survivin could inhibit cell apoptosis and shorten the G2/M phase. 4) Chromatin immunoprecipitation assay showed that Survivin protein could directly or indirectly bind to IKKβ promoter fragment in ECA109 cells at region of 0 to-700 bp. 5) Dual luciferase report gene assay showed that Survivin enhanced the transcriptional activity of IKKβ promoter in ECA109 cells. Conclusions: 1) In ECA109 and KYSE150 cell, reduction of Survivin expression downregulates the expression of NF-κB p65, IKKα, IKKβ at transcription level and inhibits activation of NF-κB p65 at protein level; Survivin overexpression upregulates the expression of NF-κB p65, IKKα, IKKβ at transcription level and promotes activation of NF-κB p65 at protein level; 2) In ECA109 cell, Survivin binds with IKKβ promoter at 0 to-700 bp region; 3) Survivin induces the transcriptional activity of IKKβ promoter and activates NF-κB p65; 4) Survivin overexpression promotes tumorigenesis and progression of esophageal squamous cell carcinoma by inhibiting cell apoptosis and promoteing cell proliferation.