The Role Of MiR-205Mediated VEGF Promoting Invasion Of Human Ovarian Cancer Cell

Background and Objective:Ovarian cancer (OVCA) is the most lethal gynecologic malignancy. The high mortality of OVCA is mainly due to late-stage diagnosis with the nature that this disease is asymptomatic in the early onset, and the OVCA cells are highly metastatic and invasive. Understanding of the mechanism associated with metastatic ability in OVCA is of important theoretical and clinical significance. Vascular endothelial growth factor (VEGF) is one of the most important proangiogenic factors. Many studies have proved that VEGF is involved in tumor cell growth, invasion and metastasis. Our previous comparative proteomics studies had found that VEGF had inducible effect on two differential expression proteins:Ezrin and Lamin A/C, and they were highly related with the invasion of OVCA. We speculate that the two proteins are likely to be involved in the invasion and metastasis of OVCA regulated by VEGF. Recent studies have found that miRNAs, a class of small molecule and non-coding RNA, are playing an important role in the development of OVCA, so the functions of miRNA have attracted many researchers’attention. We have predicted the existence of potential binding sites of miR-205in the3’UTR of Ezrin and Lamin A/C by bio-informatics, which showed the clue that there may be a close relationship between miR-205and the two differentially expressed proteins induced by VEGF. Is miR-205involved in the regulation of invasion and metasta-sis of OVCA? To answer this question, we carried out a preliminary study on the role of miR-205in VEGF-promoted invasion of OVCA cells.Methods:(1) Four human OVCA cell lines were cultured under same conditions:HO-8910(low-metastatic), HO-8910PM (high-metas-tatic), SKOV-3(low-metastatic) and SKOV-3ip (high-metastatic).(2) HO-8910was tested for VEGFR (including VEGFR1and VEGFR2) expression by immunocytochemistry.(3) In vitro, effect of VEGF on invasion of low-metastatic cells was measured by cell counts that penetrated through Matrigel-coated transwell chambers.(4) Effect of VEGF on cell apoptosis of low-metastatic cells was detected by flow cytometric analysis.(5) Effect of VEGF on proliferation of low-metastatic cells was measured by MTT assay.(6) After treated with VEGF for24hours, HO-8910was tested for miRNAs expression profile by microarray analysis, and the IPA software was performed to construct the interaction network diagram among differential proteins and miRNAs.(7) All four cell lines were detected for Ezrin, Lamin A/C mRNA and miR-205expression by quantitative real time-PCR.(8) Ezrin and Lamin A/C expression in four cell lines were detected by Western blot.Results:(1) Under the same culture condition, the number of seeding and culture time, the cells of HO-8910PM and the SKOV-3ip showed faster growth than that of HO-8910and SKOV-3.(2) The expression of VEGFR1and VEGFR2were found to be strong positive in HO-8910.(3) After treated by VEGF, the low-metastatic OVCA cells showed higher invasive ability (P<0.05).(4) Less apoptotic cells were found in VEGF treated OVCA cells (P<0.05).(5) Less proliferation were observed in VEGF treated low-metastatic OVCA cells (P<0.05).(6) After treated with VEGF,32miRNAs showed significant differentially expressed (P<0.05), among which18miRNAs were up-regulated and14miRNAs were down-regulated. There were11direct interacting pathways between differential miRNAs and17previously identified differential proteins by the IPA software analysis and there are four pathways related to miR-205.(7) Without VEGF, the expression of Ezrin mRNA in the high-metastatic OVCA cells were lower than that in the low-metastatic OVCA cells (P<0.05). The expression of Lamin-A/C mRNA was also lower than that in HO-8910PM (P<0.05), and higher than that in SKOV-3ip (P<0.05). After treated with VEGF, mRNA level of Ezrin and Lamin A/C decreased significantly (P<0.05), while miR-205expression increased significantly in HO-8910and SKOV-3cells (P<0.05).(8) After treated with VEGF for24hours, protein levels of the Ezrin and Lamin-A/C were down-regulated in HO-8910and SKOV-3cells.Conclusions:(1) VEGF can promote the invasion of OVCA cells and inhibit their apoptosis.(2) VEGF can down-regulate the levels of Ezrin and Lamin-A/C mRNA and protein expression levels in OVCA cells, respectively.(3) VEGF can up-regulate the expression of miR-205in OVCA cells.(4) The promotion of OVCA cells invasion by VEGF may partially due to down-regulation of Ezrin, Lamin-A/C caused by miR-205up-regulation.