Effects Of EFEMP1 On The Proliferation, Invasion And Metastasis Of Ovarian Cancer

ObjectiveOvarian cancer is generally accepted as the lethal gynecological malignancy and ranks first in causes of gynaecological deaths. Its pathogenesis conceal, easy to abdominal widely disseminated and distant metastasis, is the key to delay dectection which lead to high mortality rate. As a result, the study on invasion and metastasis mechanism of ovarian cancer has important theoretical and clinical value. EFEMP1, a newly found extracellular matrix （ECM） glycoprotein, involved in the regulation of cell morphology, growth, adhesion and motion process and are closely related to the development of a wide variety of tumor invasion and metastasis. In previous work, we use limited dilution method to filter out high and low invasive subclones S1, S21. The result showed that they have a high degree of homology, and met the minimum heterogeneity requirements, and have different characteristics of invasion and metastasis. We use microarray analysis to test gene expression in both cell lines. The outcomes revealed that genetic differences of more than 1.5 times as many as 1596 genes, including EFEMP1 expression was up-regulated in highly invasive subclones-S1, and present low expression in the low invasive subclones-S21,11.739 times the difference in significant difference （P=0.003<0.001）. The slide of cells, real time RT-PCR and western blot all confirmed the over-expression of EFEMP1 in the high transfer cloned cell lines S1, and the expression is significantly higher than low transfer cloned cell lines of S21 expression. In previous studies, we found that EFEMP1 expression was up-regulated in ovarian carcinoma compared to normal ovarian tissue, and its over-expression was significantly associated with high stage, low differentiation, lymph node metastasis and poor prognosis of ovarian cancer. In conclusion, we speculated that EFEMP1 may have a close relationship with invasion and metastasis of ovarian cancer. Therefore, we further explored the effect of EFEMP1 on ovarian cancer invasion and metastasis mechanism in our research.1.To clarify the mechanism of EFEMP1 in the process of invasion and metastasis of ovarian cancer cells.2.To clarify the role of EFEMP1 in the development of ovarian cancer and to provide theoretical basis for ovarian cancer prognosis judgment, more effective inhibition of invasive ovarian cancer and the treatment of ovarian cancer.Methods（1）High transfer cloned cell lines S1 and low transfer cloned cell lines S21 was isolated from the same ovarian cancer cell line SKOV3 using limited dilution method; S1 and S21 were highly homologous, meeting the heterogeneity of minimum requirements.（2）We used microarray analysis to detect the two cell lines. At the same time using immunocytochemistry, real time RT-PCR and western blot to verify the gene expression.（3）The over-expression of EFEMP1 was achieved with recombining pGC-LV-GV287-GFP vector with the EFEMP1 （NM₀01039348） gene, while knock-down of EFEMP1 was achieved with cloning small hairpin RNAs （shRNAs） used self-inactivating lentivirus vector containing a CMV-driven GFP reporter and a U6 （GeneChem, Shanghai, China）. Cell function analysis technology were used to study cell proliferation, apoptosis and invasion mobile ability before and after RNA interference and EFEMP1 over-expression.（4）We built tumor xenografts in nude mice and simulate the process of cell growth, invasion and metastasis of ovarian cancer, and further study the mechanism of EFEMP1 in ovarian cancer cell proliferation, apoptosis, invasion, and movements in vivo.（5）To study the relationship between EFEMP1 and ovarian cancer invasion, metastasis in vitro and in vivo.Results1. The molecular expression differences of EFEMP1 in different transfer ability of cell lines(DMicroarray analysis show that, genetic differences more than 1.5 times were as many as 1596 genes, mainly involving the TRAIL, SIP, TNF, PI3K, EGFR1, TGFBR multiple signal transduction pathways.（2）EFEMP1 was high expressed in high invasive subclones SI and was low expressed in the low invasive subclones S21, and the significant difference were 11.739 times（P = 0.003< 0.001）.（3）Moreover, ICC, real time RT-PCR and western blot all confirmed different expression of EFEMP1 in high and low invasive subclones.2. The cell function change before and after EFEMP1 over-expression and knockdown（l）The transfection efficiency more than 80% after recombing vector with EFEMP1 gene. Real time RT-PCR and western blot were used to confirm the transfection efficiency.（2）Cell growth curve and soft agar colony formation assay cloning results show that knockdown of EFEMP1 significantly inhibited cell proliferation in the high invasive subclones, and EFEMP1 over expression significantly improved cell proliferation ability in low invasive subclones.（3）The cell cycle detection results:low EFEMP1 expression result in G1/G0 phase cells increased, S phase cells reduced; high EFEMP1 expression result in lossing of cells in the cell lines G1/G0 phase and more S phase cells.（4）In vitro transwell assays showed that high EFEMP1 expression promote cell invasion and mobile, while EFEMP1 silencing suppressed cell invasion and mobility.3. Established tumor xenografts in nude mice and observed the effect of EFEMP1 on ovarian cancer cell invasion and metastasis（1）RNA interference group, the tumor volume was small, the tumor growth is slow and the tumor rate is low; conversely, in EFEMPl over expression group, tumor growth is fast, and the tumor volume is big, tumor rate as high as 100%.（2）Tumor tissue have more multiple nodular in the EFEMPl over expression group and the negative control group. Clear coated incomplete around, closely connect with the surrounding tissue and hard to tear apart. In RNA interference group, tumor tissue is small, smooth oval, coated complete and esay to separate.（3）On one hand, results show that, EFEMPl silenced group contain more cancer cells in mice tumor tissues compared with high transfer cloned cell lines, and without pulmonary metastasis; on the other hand, compared with, EFEMPl over-expression cells contained more tumor cells than low invasive subclones and increased pulmonary metastasis. Besides, the rate of pulmonary metastasis in S1, S21, S21-exEFEMPl were 100%,40% and 80%, respectively.Conclusions（1）EFEMP1 molecules are closely associated with ovarian cancer development.（2）EFEMP1 molecules promote ovarian cancer cell growth, invasion, and mobility.（3）EFEMP1 molecules increased split phase cells, caused cell cycle arrest, so as to promoted cell proliferation, reduced apoptosis.