| BackgroundCervical cancer is the second most common female malignant tumor ranking after breast cancer. In 2012 years, there were estimated 527,600 new cervical cancer cases and 265,700 deaths cases worldwide, nearly 90% of cervical cancer deaths occurred in developing parts of the world, and showed the tendency of the young; it was also a serious threat to women’s health. Although mortality of cervical cancer was reduced according to the screening of cervical cytology and the improvement of cervical cancer treatment, it was still a leading cause of tumor related death in the world. Therefore, it is necessary to seek a novel biomarker or a new treatment for cervical cancer.It is generally known that the immune system is responsible for eliminating tumor cells and infected cells with microorganisms or foreign antigens. Many immune cells play an important role in removing tumor according to the production of cytokines. Interferon exerts antitumoral effects on various tumor cells, which are needed to the participation of interferon induced genes. The human guanylate-binding protein 1 (GBP1) is composited with 593 amino acids glycoprotein belonging to interferon-inducible GTPases super family. There was not the expression of GBP 1 in normal human skin tissue, but GBP1 was enhanced when the skin was affected by inflammation, which suggested that GBP1 may be a potential warning sign of inflammation. GBP1 was strongly induced by inflammatory cytokines and inhibited spreading and migration of endothelial cells in inflammatory diseases. In recent years, the antitumoral effect of GBP1 has become a hot spot of attention, although many experiments have confirmed that the GBP1 could inhibit the formation and development of colon cancer, breast cancer and prostate cancer, the role of GBP1 in the progression of cervical cancer remains unclear.ObjectivesThe purposes of this study were to analyze the expression of GBP1 in cervical cancer, determine its biological functions in cervical cancer lines, CaSki and HeLa cells, elucidate its correlation with the IFN-γ-mediated tumor defense.Methods1. GBP1 expression was examined by immunohistochemical staining in 150 human cervical tissue specimens. Those tissue samples contained 98 patients with cervical cancer,32 patients with intraepithelial neoplasia (CIN), and 20 patients with normal cervical tissues. Meanwhile, we also analyzed the relationship between GBP1 expression and clinical pathological features of cervical cancer samples by statistical software.2. Two cervical cancer lines, CaSki and HeLa cells, were used in our study. GBP1 protein was measured by western blot analysis in the presence of INF-γ or not.3. Immunofluorescence analysis was used to determine GBP1 distribution in CaSki and HeLa cells.4. One pair of siRNA sequences targeting GBP1(GBP1-siRNA) were designed and synthesized to deplete endogenous GBP1 expression in CaSki cells, and the GBP1 isoform expressing lentivirus plasmid(Lenti-GBP1) was employed to study the effects of GBP1 enforced expression in HeLa cells. Western blot were used to detect the interference or reconstruction efficiency of GBP1-siRNA or Lenti-GBP1.5. IFN-γ treatment:After transfected by GBP1-siRNA, CaSki cells were treated or not by IFN-γ.6. The experiment was conducted in six groups in CaSki cells-blank group, blank+IFN-γ, scramble-siRNA(NC) group, scramble-siRNA+IFN-γ(NC+IFN-γ) group, GBP1-siRNA group, GBP1-siRNA+IFN-γ group, The experiment was divided three groups in HeLa cells-blank group, Lenti-NC group, Lenti-GBP1 group. Proliferation and migration were determined by CCK-8 assay and transwell chamber assay, respectively. Apoptosis was measured by flow cytometry.Results1. The expression of GBP1 was gradually increased from normal cervical tissues to CIN and cervical cancer tissues. Positive expression rate of GBP1 in normal cervical tissues was 30% (6/20) and was 62.5% in CIN (20/32). The expression of GBP1 in CIN was higher than that in normal cervical tissues (p=0.0448,p<0.05). In cervical cancer tissues, the positive expression rate of GBP1 was 87.8%(86/98), which was significantly higher than that in CIN and normal cervical tissues(p<0.01), the differences have statistical sense.2. High expression of GBP1 in cervical cancer was significantly related to early FIGO stage (I), good histological grade(G1-G2), shallow depth of infiltration, non-metastasized(p<0.05).3. The expression of GBP1 was found in CaSki cells by western blot in absence of IFN-y, but not in HeLa cells. GBP1 protein was evidently induced in CaSki and HeLa cells after the treatment of IFN-y, but its expression was higher in CaSki cells than that in HeLa cells.4. Immunofluorescence assay demonstrated there was a weaker layer of green fluorescent staining of the cytoplasm in CaSki cells, but not any green fluorescent staining in Hela cells in absence of IFN-y. After the stimulation of IFN-y, green fluorescent staining of the cytoplasm was markedly promoted in CaSki and HeLa cells.5. The GBP1-siRNA sequence resulted in a significant inhibition to GBP1 expression in CaSki cells, and Lenti-GBP1 markedly upregulated GBP1 expression in HeLa according to western blot.6. Knockdown of the expression of GBP1 by GBP1-siRNA improved cell proliferation, migration and inhibited cell apoptosis. IFN-y inhibited proliferation, migration and increased apoptosis in CaSki cells. Additionally, knockdown of GBP1 in CaSki cells confined the capacities of inhibiting proliferation and migration, inducing apoptosis of IFN-y. Enforced expression of GBP1 by Lenti-GBP1 in HeLa cells remarkably inhibited proliferation, migration and promoted apoptosis.CCK-8 results revealed that knockdown of GBP 1 by GBP1-siRNA evoked a ignificant improvement on the proliferation of CaSki cells compared to control group (p<0.001). Conversely, treatment with IFN-y markedly inhibited cell growth compared to control group (p<0.001). However, the antiproliferative effect of IFN-y significantly declined when CaSki cells were transfected by GBPl-siRNA (P<0.05). Additionally, the cell viability in the lenti-GBP1 transfection group producted a marked inhibition compared to control groups in HeLa cells (p<0.01).Transwell results revealed that the ability of migration was promoted in the GBP1-siRNA group compared with that in control group (p<0.001), but was inhibited in the IFN-y group compared with that in control group (p<0.001), while the effect of IFN-y in GBP1-siRNA-transfected group was decreased (p<0.001). In addition, migration of transfected HeLa with Lenti-GBP1 was significantly lower than the migration of control group (p<0.001).Flow cytometry shown that the total apoptosis rate of the GBP1-siRNA group was significantly lower than that of control group in CaSki cells (p<0.01). On the contrary, in treatment of CaSki cells with IFN-y markedly increased the total cell apoptosis rates compared with control group (p<0.001), while the IFN-γ-mediated proapoptosis was almost abrogated in the presence of GBP-siRNA (p<0.001). Furthermore, enhanced expression of GBP 1 by Lenti-GBP1 significantly promoted cell apoptosis in HeLa cells (p<0.001).Conclusions1. The expression of GBP1 was high in cervical cancer, which may be induced by inflammatory cytokines of tumor microenvironment. Its expression was associated with good clinical pathological features of cervical cancer, which may provide a line of defense for the damage of inflammation so that slow down the occurrence and development of cervical cancer.2. GBP1 and IFN-y could suppress proliferation, migration and promote apoptosis of cervical cancer cells.3. Knock down of GBP1 by GBPl-siRNA weakened the antitumoral role of IFN-y in cervical cancer, which illustrated that IFN-y-mediated antitumoral effects may be partly achieved by GBP1.4. GBP1 is expected to become a new suppressor in cervical cancer. |
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