MiRNA-125a-5p Inhibits Retinoblastoma Cell Proliferation,Invasion And Metastasis Through TAZ-EGFR Pathway

Retinoblastoma(RB)is the most common intraocular malignant tumor in infants,which threaten the life and vision of retinoblastoma patients.Along with clinical management of retinoblastoma was gradually increased,the main purpose of retinoblastoma resolution was changed from saving lives of the patients to keep the vision to improve the quality of life.Most retinoblastoma patients cannot be diagnosed during the early period of retinoblastoma,and delayed diagnosis is the greatest issue for clinical treatment.MicroRNAs(miRNAs)is an endogenous non-coding RNA of 21-25 nucleotides that regulates post-transcriptional gene expression by affecting the translation and stability of complementary mRNAs,Growing numbers of evidences described that miRNAs can function as oncogenes and tumor suppressor genes by playing multiple roles in cancer development.Accumulating studies have shown that miR-125a-5p expression was significantly decreased in a variety of tumors and plays an important role in suppressing the proliferation and metastasis of different cancers,including ovarian cancer,hepatocellular carcinoma and breast cancer.However,the role and the mechanism of miR-125a-5p in retinoblastoma need further elucidations1.Materials and Methods1.1 Clinical Samples and cell cultureAll the retinoblastoma samples and the corresponding matched adjacent noncancerous retinal tissues were collected during surgery.All the samples were divided into two parts.After surgical enucleation,one part tissues were immediately frozen in liquid nitrogen and stored at-80?.Another part was for pathology analysis.Retinoblastoma cell lines Y79 and WERI-Rb-1 were purchased from the American Type Culture Collection(ATCC,Rockville,MD,USA)and the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences(Shanghai,China)respectively.1.2 miRNA microarray analysis and quantitative real-time PCR(qRT-PCR)MiRNA microarray analysis was evaluated to obtain expression profile of miRNAs in retinoblastoma tissues and adjacent noncancerous tissues.MiRNA with significantly profile changes were analyzed by qRT-PCR.Analysis the relationship between miR-125a-5p and retinoblastoma TNM stages.1.3 The role of miR-125a-5p in retinoblastoma cell proliferation,migration and invasionRetinoblastoma cells were transfected with miR-125a-5p mimic,antagomiR-125a-5p or the corresponding controls.Proliferation of retinoblastoma cells was evaluated by cell counting and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.Transwell assay and Matrigel assay were evaluated to analyze migration and invasion of retinoblastoma cells.1.4 The role of miR-125a-5p in colony formation and Xenograft tumor growthSoft-agar assay was evaluated the effect of miR-125a-5p on retinoblastoma colony formation.Y79 cells transfected with miR-125a-5p mimic,antagomiR-125a-5p or the corresponding controls were injected subcutaneously into the dorsum of mice.Tumor diameter was measured by a vernier caliper weekly.Tumor volume was calculated using the following formula:V =(length × width2)/2.Tumors were collected four weeks after injection,weighed and stored for the further experiments.1.5 Prediction and analysis of miR-125a-5p downstream targetBy using the TargetScan,TAZ was considered to be a direct target of miR-125a-5p.Luciferase activity assay and immunohistochemistry staining were performed to evaluate the relationship between miR-125a-5p and TAZ.1.6 The mechanism of TAZ,the direct downstream target of miR-125a-5p,onretinoblastoma proliferation and tumor formation The effect of TAZ on retinoblastoma cell proliferation,migration and invasion were performed by cell counting,MTT assay,Transwell assay and Matrigel assay.Western blot and qRT-PCR were used to analysis the potential mechanism of TAZ in retinoblastoma development.2.Results2.1 miR-125a-5p expression was significantly down-regulated in retinoblastoma tissues and retinoblastoma cell lines compared with adjacent non-tumor retina tissues.Statistical results showed that the expression level of miR-125a-5p was significantly correlated with TNM stage and differentiation of retinoblastoma.2.2 Overexpression miR-125a-5p significantly decreased proliferation,migration,and invasion of retinoblastoma cells,and inhibit tumor growth of retinoblastoma.Opposite outcome were observed in downregulation miR-125a-5p group in retinoblastoma.2.3 TAZ was a direct downstream target of miR-125a-5p.The expression of TAZ in retinoblastoma tissues and cell lines were significantly higher compared with adjacent noncancerous retina tissues.Overexpression of miR-125a-5p significantly reduced the expression of TAZ in retinoblastoma.The expression levels of MiR-125a-5p and TAZ in retinoblastoma were negatively correlated.2.4 Knockdown of TAZ expression levels decreased the proliferation,migration and invasion of retinoblastoma cells,which similar to pattern observed in overexpression of miR-125a-5p retinoblastoma tissues and cells.Furthermore,upregulation of TAZ reversed the effect of miR-125a-5p in decreasing proliferation,migration,invasion and growth of retinoblastoma in overexpression miR-125a-5p cells.3.Conclusion3.1 miR-125a-5p expression was significantly down-regulated in retinoblastoma and negatively correlated with TNM stage of retinoblastoma,suggesting that miR-125a-5p may function as a tumor suppressor in the development of retinoblastoma.3.2 Overexpression of miR-125a-5p significantly reduced the proliferation,migration and invasion of retinoblastoma cells.A similar pattern was found in TAZ knockdown retinoblastoma cells.The effect of miR-125a-5p on reducing development of retinoblastoma was reversed by TAZ.These finding suggested that miR-125a-5p may be involved in the development of retinoblastoma by directly targeting the expression of TAZ in retinoblastoma.3.3 The expression level of miR-125a-5p can significantly differentiate retinoblastoma tissue and non-tumor tissue.Meanwhile,miR-125a-5p expression was inversely correlated with TAZ expression level in retinoblastoma.Therefore,miR-125a-5p may be a potential treatment target for use as part of a novel chemotherapy strategy for patients with retinoblastoma.