Preliminary Study On The Effect And Mechanism Of Silencing EIF5A2 Gene On The Proliferation And Migration Of Cervical Cancer Cell

Cervical carcinoma( CC) is the common tumor occurring in women worldwide,and ranking the second in mortality rate of various malignancies for women, just after breast cancer. There are approximately 500,000 new cervical cancer cases in the world each year, and more than 300,000 of these cases die annually. The Chinese incidence of cervical cancer ranks second in the world and the age tends to be younger, which seriously damages female health. At present, although the diagnosis and treatment for cervical cancer has made great progress, its prognosis and mortality have not been significantly improved. The early-stage patients treated by surgery could achieve good prognosis, however those patients with advanced cervical cancer 5 years of survival rate is only 50 %, the treatment is however not acceptable reasonably. Other treatments including chemotherapy and radiotherapy, have serious side effects as well as lacking specificity. Therefore it contains a great significance for cervical cancer to probe its origin and development pathological mechanism and seek its better and more effective treatments.EIF5A2 gene is one of the homologous genes e IF5 A family and locates in human chromosomes 3q26.2, which plays an importantly regulatory role in the process of cell growth, differentiation, proliferation and apoptosis. Abnormal expression of e IF5A2 was found in ovarian cancer, liver cancer, bladder cancer and esophageal squamous cell carcinoma, which was considered as a tumor marker with high malignant degree. Silencing e IF5A2 could effectively inhibit proliferation, invasion, metastasis and induce apoptosis of tumor cells. Recent studies found that expression of e IF5A2 in cervical cancer tissue was significantly increased, and was correlated with higher FIGO stage, lymphovascular space involvement and pelvic lymph node metastasis. However, the regulation mechanism of e IF5A2 in cervical cancer and the VI effects on biological function of cervical cancer cell are still not identified clearly, which needs further research.More and more studies during recent years discovered that Rho A/ROCK signal pathway was continuously activated in a wide variety of tumor and closely related to proliferation, apoptosis, invasion and metastasis of tumor cell. Another study confirms that the e IF5A2 gene mediated invasion and metastasis of pancreatic cancer through regulating the signaling pathway of the Rho A/ROCK. In addition, overexpression of e IF5A2 promoted the proliferation and migration of hepatocellular carcinoma, meanwhile significantly activating Rho A/Rac1 signaling pathway. These studies indicated that e IF5A2 gene regulated tumor cell proliferation and migration through Rho A/ROCK signaling pathway, but how its mechanism works is still not identified clearly in cervical cancer.Our study firstly, we analyzed the correlation between e IF5A2 expression and cervical cancer by real-time PCR and western blot, and applicated RNA interference technology to reduce e IF5A2 expression in Hela and observed the effect of e IF5A2 interference on proliferation, cycle and migration of Hela cell by MTT, flow cytometry and wound healing assay. Finally,we tested the expression of Rho A and ROCK in cervical cancer tissue, and study goes further to the effect of abnormal expression of Rho A on the proliferation and migration of cervical cancer cells through by upregulating or downregulating the Rho A expression,we transfected Rho A expression vector into Hela cell with e IF5A2-sh RNA to explore the effect of e IF5A2 gene on the regulation of Rho A/ROCK signaling pathway and clarify its mechanism and effect in the progress of cervical cancer. This study aims to further clarify the pathogenesis of cervical cancer and provide a new molecular target and more theoretical analyses for the clinical prevention. Part 1. Analysis the expression of e IF5A2 in cervical cancer tissueObjective: To analyze whether there remains a difference expression of e IF5A2 in cervical cancer and adjacent normal clinical tissue, and argument whether the expression of e IF5A2 is in correlation with cervical cancer.Method: 15 pairs cervical cancer and corresponding adjacent normal clinical tissue samples were collected and used to detect the m RNA and protein expression of e IF5A2, by real-time PCR and western blot. The relationship between e IF5A2 and cervical cancer was analyzed from the level of molecules and protein respectively.Result: The expression of e IF5A2 were significantly higher in cervical cancer tissues in comparison with adjacent normal clinical tissues(p<0.05).Conclusion: We successfully confirmed that the expression of e IF5A2 was increased in cervical cancer tissue, and confirmed the potential of e IF5A2 as an important bio-marker for the prognosis and a potential molecular target for clinical treatment. Meanwhile, in cervical cancer pathological diagnosis, we successfully provided the theoretical analysis of the real-time PCR or immunohistochemical technology for the expression of e IF5A2 in cervical biopsy samples. Part 2 The effect of silencing e IF5A2 gene on proliferation and migration of cervical cancer cellsObjective: To explore the effect of silencing e IF5A2 gene on proliferation and migration of cervical cancer cellsMethod: constructing e IF5A2 interference carrier, transfecting Hela cells and screening stability cell by G418. The experiment was divided into three groups: Control group(Cont), negative control group(NC) and e IF5A2-sh RNA group(she IF5A2). The effect of e IF5A2 gene silencing on cell proliferation was detected by MTT assay. The effect of e IF5A2 gene silencing on cell cycle was examined by flow cytometry. The cell cloning efficiency in vitro was measured by colony formation assay. The cell migration ability was tested by transwell and wound healing assay.Result: The sh RNA-mediated e IF5A2 gene significantly affects the cell-growth inhibition in she IF5A2 group(p<0.05) in MTT. There came an accumulation in cells, in the G0 phase; and there also came a reduction, in S and G2 in she IF5A2 group, compared with NC group(p<0.05 or p<0.01). The cell cloning efficiency in vitro and cell migration ability was both significantly inhibited after e IF5A2 silencing(p<0.05).Conclusion: EIF5A2 gene silencing induces cervical cancer cell cycle arrest in G0 phase and effectively inhibits cell proliferation and migration. VIII Part 3 The molecular mechanism of e IF5A2 gene silencing on cell proliferation and migration of cervical cancerObjective: i)To analyze the correlation of Rho A with clinical and pathological parameters of cervical cancer and to confirm the effect of abnormal expression of Rho A on the proliferation and migration of cervical cancer cells. ii)To clarify the effect of e IF5A2 gene silencing on Rho A/ROCK signaling pathway in cervical cancer Hela cells, and to clarify the mechanism of Rho A/ROCK on cell proliferation and migration of Hela cells.Method: i)46 specimens of human cervical cancer and 34 cervicitis tissues(as control) were selected and used to detect the expression of Rho A and ROCK by western blot. We analyzed the association of Rho A expression with the age, tumor size, HPV infection, FIGO stage, vascular invasion, lymph node metastasis, expression of estrogen receptor and progesterone receptor. Constructing overexpression vector and interference fragment of Rho A and be transfected into Hela respectively. The experiment was divided into five groups: Hela group, Hela-Con group, Hela-Rho A+ group, si RNA-Con group and si RNA-Rho A group. The effect of abnormal expression of Rho A on cell proliferation was detected by CCK-8 assay, and the cell migration ability was tested by wound healing assay. ii)The effect of e IF5A2 gene silencing on Rho A/ROCK signaling pathway was testified by western blot. Building Rho A overexpression vector which was transfected into she IF5A2 Hela cells. The experiment was divided into four groups: NC group, she IF5A2 group, she IF5A2+Vector group(Vector) and she IF5A2+Rho A group(Rho A).The proliferation of Hela cells was detected by MTT assay. The migration of Hela cells was examined by wound healing assay.Result: i)The expression of Rho A, ROCKâ… and ROCK â…¡was significantly higher in cervical cancer tissues in comparison with control group(p<0.05 or p<0.01). And the Rho A was associated with FIGO stage, vascular invasion and lymph node metastasis, whose overexpression resulted in significant cell growth and migration from the results of cell CKK-8 and wound healing assay(p<0.05 or p<0.01). However, interfering Rho A could effectively inhibit proliferation and migration of tumor cells. ii)The protein expression of Rho A, ROCK and ROCK â…  â…¡ were decreased in Hela cells with e IF5A2 gene silencing, which was reversed after it transfected with Rho A overexpression vector. The ability to proliferate and migrate the Hela cells was significantly improved in Rho A group compared with the she IF5A2 group(p<0.05).Conclusion: i)The expression of Rho A was obviously increased in cervical cancer specimens and was closely related with clinical stage and metastasis of tumor, whose overexpression significantly promoted the proliferation and migration of cervical cancer cells. ii)The e IF5A2 gene mediated proliferation and migration of cervical cancer cells through activating Rho A/ROCK signaling pathway and so as to increase the expression of Rho A, ROCK and ROCKâ…  â…¡.