Expression Of Notch1 In Cervical Cancer Tissues And Its Effect On Invasion And Metastasis Of Cervical Cancer Hela Cells

Cervical cancer is the second most common malignant tumor in women worldwide after breast cancer, with the first incidence in developing nations, while its incidence is much lower than that of breast cancer, Endometrial cancer and Ovarian Cancer.Important advances have taken place in the diagnosis and treatment of this cancer in recent years. Surgery or chemoradiotherapy can cure 80-95% of women with early stage disease (stagesⅠandⅡ) and 60% with stageⅢdisease; however, the 5-year overall survival rate (14%) remains poor in malignant tumors with higher levels. In the present time, early detection, early diagnosis and early treatment is an effective way to reduce the incidence and mortality rates of cervical cancer. Obviously, seeking a new molecular therperutic target and a better understanding of the pathogenesis of cervical cancer help to improve the prognosis of patients with cervical cancer.Notchl protein is a transmembrane receptor family that is structurally and functionally conserved from worms to human. Notchl was first identified in Drosophila as a gene involved in neuronal cell fate decision, but this family of receptors is now known to regulate the fate decisions of developing cells in various tissues during embryogenesis as well as in postnatal stages. Upon binding to its ligand, Notchl protein is protiolytically cleaved, releasing an NICD, which then translocates into the nucleus. The NICD associates with transcriptional factors known as Su (H)/CBF1, regulating the expression of target genes, and successively modulating the development and growth of cells. Constitutive expression of active NICD in targeted cells also results in an "activated" Notch phenotype.Previous studies revealed the Notch1 signaling pathway, as one of key signaling pathway regulating cell differentiation, played a key role in cell development, proliferation, differentiation and apoptosis that affect the development and function of many organs. It appears reasonable to assume that deregulation of Notch1 function is involved in the genesis of different types of cancers. This appears to be true, and insights into links between Notch signaling and cancer are rapidly expanding. The Notch1 signaling pathway has been associated with several human cancers, including lung carcinoma and neuroblastoma. Most notably, it has been linked to T-cell acute lymphoblastic leukemia (T-ALL), where activating mutations within Notch1 have been identified in>50% of tumors. Furthermore, growth arrest occurs in several T-ALL-derived cell lines when Notch1 signaling is blocked, suggesting that modulating the Notchl signaling pathway may be an effective treatment strategy for this tumor type. In addition, we now know that aberrant Notchl signaling plays a role in other types of leukemias and in mucoepidermoid tumors. In addition, Notchl can be a dominant oncogene as well as a tumor suppressor gene, depending on the cellular context. The rapidly increasing knowledge about Notchl signaling in cancer will hopefully provide a basis to explore the possibility of using Notch signaling as a therapeutic target in cancer.However, report about relationship between Notchl signaling pathway and cervical cancer remains elusive. In order to further understand their relationship between Notchl signaling pathway and the development of cervical cancer, in the current study, eukaryotic expression vector pcDNANICD was constrcted, the role of overexpression of Notchl in cervical cancer was investigated, and the effect of overexpression of Notchl on cell proliferation was studied, furthermore, effect of overexpression of Notchl on cell cycle, cell apoptosis, invasion and metastasis was analyzed by flow cytometry and Boyden chamber, these findings further show that Notch1 may be a potential molecular target in the treatment of cervical cancer.Methods1. Total RNA was extracted from placeta tissues, full-length of Notchl NICD was amplified by RT-PCR. In addition, PCR product and pcDNA3.1(+) were digested with BamHI和XbaI, respectively. PCR product and pcDNA3.1 were ligated with T4 DNA ligase, and tranformed into JM109 E.coli cells.2. Eukaryotic expression vector pcDNANICD was transfected into Hela cells, and Hela strains stably expressing Notch1 were screened.3. Notch1 and Hesl mRNAs and proteins analyzed by semi-quantitative RT-PCR and Western blotting methods.4. The effect of overexpression of Notch1 on cell proliferation was analyzed using CCK-8 kit.5. Cell cycle and apoptosis were detected by Flow cytometry.6. Each cell number was counted in Boyden chamber experiment and effect of Notchl gene cell invade capable was compared.7. The results of RT-PCR and Western blotting were analyzed using Gene Tools software. All experiments results were from at least three separate experiments. The data were performed by one-way analysis of variance using SPSS version 13.0. Summary statistics were expressed at means±standard deviations, except as otherwise stated. In all statistical analyses, a P value<0.05 was considered statistically significant, and all P values were two-sided.Results1. About 2.3 kb fragments were obtained by RT-PCR, which is accordance with predicted size. After sequencing, Notchl gene was analyzed by Blast. In addition, about 5.4kb and 2.3kb fragments were obtained by digesting pcDNANICD with BamHI and XbaI, suggesting that eukaryotic expression vector was constructed accurately.2. Expressions of Notch1 and Hes1 mRNAs in Hela cells transfected with pcDNANICD were markedly increased compared to that cells untreated and stably expressing pcDNA3.1 (P<0.05). However, there was no difference in Hes1 expression of Hela cells untreated and stably expressing pcDNA3.1, but evidently difference in Notchl expression, which may be due to the toxicity of lipofectamine 2000 and plasmids, or errors in experiment operation. 3. Expressions of Notch1 and Hesl proteins in Hela cells stably expressing Notch1 were markedly increased compared to that cells untreated and stably expressing pcDNA3.1 (P<0.05). However, there was no difference in Notch1 expression of Hela cells untreated and stably expressing pcDNA3.1, but evidently difference in Hesl expression, which may be due to the toxicity of lipofectamine 2000 and plasmids, or errors in experiment operation.4. Overexpression of Notch1 could markedly inhibit the proliferation of Hela cells.5. There was no difference in cell cycle and apoptosis in Hela cells untreated and stably expressing pcDNA3.1 (P>0.05). Overexpression of Notchl could arrest Hela cells at G0/G1 phase and induce cell apoptosis compared to that in Hela cells untreated and stably expressing pcDNA3.1 (P<0.05).6. Boyden chamber invasion experiment in vitro showed that cell numbers in invasing Matrigel membrane was profoundly decreased in Hela cells overexpressing Notch1. However, there was no difference in Hela cells untreated and stably expressing pcDNA3.1.Conclusion1. Overexpression of Notch1 can evidently inhibit the proliferation of Hela cells and arrest cell cycle at G0/G1 phase.2. Overexpression of Notch1 can induce cell apoptosis; further inhibit invasion and metastasis of Hela cells.