Coordinated Targeting Of MMP-2/MMP-9 By MiR-296-3p/FOXCUT Exerts Tumor Suppressing Effects In Choroidal Melanoma

Background:Choroidal melanoma is the most common intraocular tumor in adults,and overexpression of matrix metalloproteinase-2 or matrix metalloproteinase-9(MMP-2/MMP-9)is associated with angiogenesis and tumor metastasis of the choroidal malignant melanoma(CMM).mi R-296-3p and FOXCUT are the potential non-coding RNA targeting on MMP family,while there is not sufficient data for their effect.Objective:This study aims to investigate the functions and mechanisms of microRNA or long non-coding RNA-targeted MMP-2/MMP-9 in CMM,and to explore the potential of coordinated targeting of MMP-2/MMP-9 by mi R-296-3p and FOXCUT.Method : Tissue Micriarray was performed to examine 6 clinical samples of normal choroid membranes and 18 clinical samples of choroidal melanoma from CMM patients.Human-derived choroid cells and choroidal melanoma C918 cells were prepared for experiments in vitro.m RNA and total proteins were extracted from clinical samples for further q PCR and Western Blot to assess expressions of MMP-2/MMP-9.In situ hybridization histochemistry(ISH)and immunohistochemistry(IHC)were performed to assess space distribution of MMP-2/MMP-9.Affinity was examined between MMP-2/MMP-9 and mi R-296-3p or FOXCUT.Luciferase reporter gene vector of mi R-296-3p and lentiviral vector of FOXCUT were established for transfection on C918 cell line.PCR and Western Blot were performed to examine transcription and translation of MMP-2/MMP-9 in C918 cells.To explore detailed mechanisms,CCK-8 was performed to assess proliferation of C918 cells;The colony-formation assay was performed to assess cloning of C918 cells;Scratch assay was performed to assess migration of C918 cells;Transwell experiment was performed to assess invasion of C918cells;Flow cytometry was performed to assess cell cycles and apoptosis of C918 cells.ISH and IHC were performed to assess space distribution of mi R-296-3p and FOXCUT.To assess coordinated targeting of MMP-2/MMP-9 by mi R-296-3p and FOXCUT,CCK-8 and flow cytometry were performed for further detection.Result:We demonstrated that expressions of MMP-2/MMP-9 were increased in CMM tissues and C918 cells in comparison with normal choroidal melanocytes.Bio-informatics prediction and our experiments validated that MMP-2 and MMP-9 were simultaneously targeted by mi R-296-3p and FOXC1 promoter upstream transcript(FOXCUT);Furthermore,significant down-regulations of mi R-296-3p and FOXCUT were found in C918 cells compared with choroidal melanocytes from the unaffected eyes,and a positive correlation was observed between their levels in three cases of eye malignant melanomas.Compared with blank transfected C918 cells,mi R-296-3p or FOXCUT overexpression transfected C918 cells had a significantly higher rate of proliferation,verified by CCK-8.Compared with blank transfected C918 cells,mi R-296-3p or FOXCUT overexpression transfected C918 cells had a significantly higher rate of cloning,verified by the colony-formation assay.Compared with blank transfected C918 cells,mi R-296-3p or FOXCUT overexpression transfected C918 cells had a significantly higher rate of S phase but decreased rate of G0/G1 phase in cell cycle,verified by flow cytometry.Compared with blank transfected C918 cells,mi R-296-3p or FOXCUT overexpression transfected C918 cells had a significantly higher rate of apoptosis,verified by flow cytometry.Compared with blank transfected C918 cells,mi R-296-3p or FOXCUT overexpression transfected C918 cells had a significantly shortened distance of migration,verified by scratch assay.Compared with blank transfected C918 cells,mi R-296-3p or FOXCUT overexpression transfected C918 cells had a significantly lower rate of invading cells,verified by Transwell experiment.Furthermore,significant downregulations of mi R-296-3p and FOXCUT were found in C918 cells compared with choroidal melanocytes from the unaffected eyes,and a positive correlation was observed between their levels in three cases of eye malignant melanomas,verified by CCK-8 and flow cytometry.Conclusion:Our data indicated that MMP-2/MMP-9 was coordinately targeted by two non-coding RNAs,mi R-296-3p and FOXCUT,which were decreased,and tumor-suppressing factors in CMM.Further study will show the possibility of developing them as therapeutic candidates for CMM.