Investigation Of The Mechanism Of Mir-146b Rugulating The Migration And Invasion Of Ovarian Cancer Cell Via Targeting Rac1

Objective:During our previous study,it has been found that miR-146b could cause disorder arrangement of actin cytoskeleton and inhibition of invasion and metastasis in ovarian cancer cells.In this study,we explored the target gene of miR-146b and investigated the mechanism of miR-146b changed the morphology of epithelial ovarian cancer?EOC?cell lines through disturbing the arrangement of cytoskeleton and cell tight junction which finally led to the decline of cell migration and invasionMethods:1.Bioinformatics methods were used to predict the target genes of miR-146b.Ras-related C3 botulinum toxin substrate 1?Rac1?which has a high score and is related to cell morphology was selected as the candidate target gene.The dual-luciferase reporter gene assay was used to verify it.2.Western blot was performed to determine the protein level of Rac1 when miR-146b was transfected into ovarian cancer cell transiently or stably.3.Two pLKO.1-puro Rac1 shRNA lentiviral vectors were constructed.Stable Rac1 knockdown was performed using a lentiviral-mediated transduction system in ovarian cancer cells.Western blot was used to determine the protein level of Rac1 gene.4.The inverted microscope was used to observed the cell morphology,and the protein expression of actin and zonula occludens-1?ZO-1?were detected by western blot and the location and distribution of them were visualized under the laser confocal microscopy.Wound healing and transwell assay were applied to detect the cell migration and invasion.5.The pCDH-NEO miR-146b sponge vector was constructed and a lentiviral vector-mediated transduction was performed to silence the expression of miR-146b in OVCAR3-miR-146b.Real-time quantitative PCR was used to quantify miR-146b level.Western blot,microscope,immune-fluorescence,wound healing and transwell assay were used to detect the Rac1 expression,cell morphology,actin cytoskeleton,migration and invasion of OVCAR3 cells,respectively.6.The pCDH-NEO Rac1 CDS vector was constructed and a lentiviral-mediated transduction was performed to rescue the protein expression of Rac1 in OVCAR3-miR-146b.Western blot was used to determine the protein level of Rac1.The microscope,immune-fluorescence,wound healing and transwell assay were applied to detect the cell morphology,cytoskeleton,migration and invasion,respectively.Result:1.The dual-luciferase reporter assay declared that the activity of luciferase in which co-transfected with pri-miR-146b and Rac1 3'-untranslated region?3'-UTR?was decreased compared with the control group co-transfected with pri-miR-146b and psiCHECK2 plasmids?P<0.05?,but the activity of luciferase recovered when the seed sequence of Rac1 3'-UTR was mutated?P>0.05?.2.Western blot showed that the protein expression of Rac1 was down-regulated after miR-146b was overexpressed transiently or stably.3.Western blot demonstrated that the protein level of Rac1 could be knocked down by Rac1 shRNA₁ instead of Rac1 shRNA₂ in ovarian cancer cells.4.It was found that the morphology of Rac1 knockdown ovarian cancer cells became smaller and epithelioid.The expression of F-actin and ZO-1 had no significant change,but the arrangement of F-actin was disturbed and ZO-1 relocated from the cytoplasm to intercellular cell tight junctions which strengthened the cell-cell junctions.The ability of cell migration and invasion was decreased after knocking down Rac1?P<0.01?.5.The pCDH-NEO miR-146b sponge vector was constructed successfully.Although it was not demonstrated that the expression of miR-146b was decreased by RT-qPCR,the Rac1 expression was increased,morphology of became mesenchymal,actin cytoskeleton rearranged in order,and cell migration and invasion ability was recovered.6.The pCDH-NEO Rac1 CDS vector was also constructed successfully.Western blot showed that the protein expression of Rac1 was up-regulated.The morphology,cytoskeleton,cell migration and invasion ability were partially rescued.Conclusion:miR-146b could suppress target gene Rac1 and disturb the arrangement of actin cytoskeleton and location of ZO-1 which cause the morphology of ovarian cancer cells become smaller and epithelioid,leading to the decline of cell migration and invasion ability.It may provide a new therapeutic target for EOC.