Expression And Function Of E2-EPF In Cervical Cancer And Its Molecular Mechanism

E2-EPF ubiquitin carrier protein is a 24-kDa protein that is a member of the E2 family of ubiquitin-conjugating enzymes, which has been showed to be highly expressed in many tumor tissues. E2-EPF, E1 ubiquitin-activating enzyme and an E3 ubiquitin ligase, catalyze the addition of ubiquitin to substrate proteins. Polyubiquitinylation of the substrate protein can target that protein for proteasome dependent destruction. Von Hippel-Lindau (VHL) tumor suppressor gene product is a part of an active E3 ubiquitin ligase complex. In the presence of oxygen, hypoxiainducible factor(HIF) is targeted for destruction by the E3 ubiquitin ligase complex containing the VHL tumor suppressor protein (pVHL).E2-EPF target pVHL for proteosomal degradation under normoxic conditions resulting in the stabilization of hypoxia inducible factor 1α(HIF-1α). Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that controls the expression of a battery of more than 100 target genes whose protein are expressed in responses to cellular and systemic hypoxia. HIF-1αhas been showed to be associated with increased tumor growth, angiogenesis and tumor metastasis. Thereafter, increased expression of E2-EPF in multiple cancers was reported to promote tumor growth, invasion and metastasis by the pVHL-HIF-1αpathway.Role of E2-EPF in cervical tumors has not been reported until now. In this study, our objectives are to explore the expression level of E2-EPF in cervical tumor and its function. The followings are studied:E2-EPF is overexpressed in cervical tumor specimensTotally,13 normal cervical tissues, and 75 cervical carcinoma tissues were analysed. Relative expression levels of were determined by a Taqman real-time PCR system. E2-EPF was significantly overexpressed in tumors relative to normal tissues. The average relative expression level in tumors is 4.08 folds of the normal tissue and expression level of E2-EPF in tumor tissues of clinical stage ofⅡ-Ⅳis 2.72 folds of tumor tissues in stage I. Both are statistically significant. Therefore E2-EPF is also high expressed in cervical cancer, and its expression level is associated with the clinical stage of tumor.Establishment of E2-EPF low expression cloneCommercial available E2-EPF shRNA vector was used to down-regulate the expression of E2-EPF. E2-EPF shRNA plasmid vector was transfected into the SIHA cell line with lipofectamine reagent and the low expression clone was screened by puromycin selection.15 low expression clones were obtained. The lowest expression level of E2-EPF of the obtained clones was 18% of the normal in mRNA and 20% of the normal in protein respectively.Decreased expression of E2-EPF resulting in lower expression of HIF-1αBecause HIF-1αis extremely unstable in normoxia, it is hard to detect its protein level by routine method. In this section, we use the HIF1 Luciferase Reporter Vector to detect the presence of HIF1. Results showed that the expression level of E2-EPF was positively correlated with the HIF-1αexpression level. In the E2-EPF low expression clone, HIF-1αexpression was significantly lower than the normal and control cells.Effects of E2-EPF knock down on cell cycle, cell growth and invasionThe cell growth of E2-EPF low expression clone was significantly lower than the normal and control cells detected by MTT cell proliferation kit. In the soft agarose clone formation assay, the clones formed by the E2-EPF low expression clone cell were much lower than the normal and control cells. Cell cycle analysis by flow cytometry showed that the percentage of G0-G1 phase cells of E2-EPF low expression clone was increased and the percentage of S, G2-M phase (particularly S phase) cells was decreased. Using the classic transwell cell invasion assay, reduced cell invasion ability of E2-EPF low expression clone was observed. These results showed that E2-EPF knock down has significant negative effects on the cell growth, cell cycle and invasion.Effects of E2-EPF knock down on cell sensitivity to radiotherapy and chemotherapy Radiotherapy of OGy,4 Gy,7 Gy,10 Gy dosage were given to cells and MTT cell proliferation assay kit was used to evaluate the cell number. Results showed that the relative survival cell number of the E2-EPF low expression clone was not significant different from the normal and control cells. Annexin-V,PI kit combined with flow cytometer were used to detect the apoptosis level of each dosage, a similar results of the apoptosis and necrosis cell number were showed.Different kinds of chemotherapeutic drugs (Cisplatin, Doxorubicin, Paclitaxel, Doxetaxel, Topotecan, Etoposide)were added to the cell culture media to observe the effect of E2-EPF knock down on the sensitivity of cells to these agents. MTT assay was used to evaluate the relative cell number. Results showed that E2-EPF knock down increased the cell sensitivity to the topoisomerase 1 inhibitor (Topotecan) andⅡ(Etoposide and Doxorubicin) and no effect on the sensitivity to Cisplatin, Paclitaxel, Doxetaxel. These results indicate that down regulation of E2-EPF can increase the cell sensitivity to topoisomerase inhibitors and can be used in combination to improve therapeutic effect of chemotherapy.E2-EPF knock down showed decreased tumorigenesis abilityCertain no. of tumor cells were s.c. inoculated into nude mice to monitor the tumor development. Weight of tumors from E2-EPF low expression clone cells was about 1/4 of the control cells. These results indicate that E2-EPF play an important role in tumor development in vivo. Targeting E2-EPF pathway may be an effective biotherapy method of late stage cervical cancer.In conclusion:E2-EPF is overexpressed in cervical tumor specimens and its expression level positively correlated with the clinical stage. Down regulation of E2-EPF by RNAi results in a decreased expression level of HIF-1α. E2-EPF knockdown decreases the growth, clone formation, cell invasion and tumorigenesis of SIHA cell line and increases the chemosensitivity to topoisomerase inhibitors. E2-EPF plays an important role in the tumorigenesis and development. Targeting of E2-EPF pathway may be a new therapeutic way of cervical tumor treatment.