MiR-1228 Promotes Metatasis And Proliferation In Hepatoma Cells By Joining In P53 Positive Feedback Regulation

Objective:Hepatocellular carcinoma(HCC)is one of the commonest solid tumor worldwide and the third leading cause of cancer-induced death,but the origin and developing process are complex and still obscure.Recent reports show that altered expression or activity of specific genes involve in the pathogenesis of HCC,including microRNA(miRNA).Recent studies show that a variety of miRNAs are involved in the regulation of cancer cell biology program via joining in the cell signalling pathway,exerting functions as oncogenes and tumor suppressors.p53,the acknowledged tumor suppressor,encoded by TP53 gene,regulates many genes by its function in regulation of cell-cycle progression,DNA repair,apoptosis,and angiogenesis.In p53 signal pathway,it joins in the regulation of many miRNAs,meantimes,directly regulated by some miRNAs as their downstream target.In this paper,we focus on looking for a miRNA and its host gene,which participate in p53 signal pathway,and explore their function in HCC,in order to provide a theoretical basis for the diagnosis and treatment of malignant transformation of HCC.Methods:First,we verified the funcion of p53 in QGY-7703 and HepG2 cells via colony formation,transwell migration and invasion assays.Second,p53 was found targeting the promoter region of LRP1 by bioinformatics,and there was a miRNA miR-1228 located in the intron region of LRP1.Meantime,according to resules of prediction of the same transcriptional start site of miR-1228 and its host gene LRP1,expression vectors with the two different-length fragments in promoter region were constructed basing on the vector of pGL3-basic-luc+,and the promoter activity was confirmed by luciferase reporter experiment in QGY-7703.Then,combining with Real-time RT-PCR we explored the effection on the expression of p53 to miR-1228.Third,we enforced or blocked expression of miR-1228 in QGY-7703 and HepG2 cells,we evaluated the effects on malignant phenotypes using a series of assays,including colony formation,transwell migration and invasion assays.Fourth,the candidate gene TP53 targeted by miR-1228 were screened by bioinformatics,and verified by EGFP report assay,real-time PCR and western blot.Then,overexpress p53 in rescue experiment furtherly demonstrated that p53 was a target of miR-1228.Real-time PCR was used to analyze the expression of miR-1228 and p53 in 20 paired specimens of HCC and their adjacent noncancerous tissues,and.Results:Overexpression of p53 in QGY-7703 and HepG2 cells suppressed cell proliferation,migration and invasion capacity.Subsequently we screened and validated the miRNA targeting p53,miR-1228.Meatime,We cloned the two promoters of miR-1228 and found the fragment of p1546 and p866 of promoter could active dual-Luciferase reporter gene transcription in QGY-7703 cell line,and the p866 showed higher luciferase activity.Then we predicted that p53 could binding the promoter region of miR-1228 after some related literature review and bioinformatics analyzing.Enforced the expression of p53 could down-regulate the expression of miR-1228.After further exploring the cell function of miR-1228,we found that miR-1228 could enhance the proliferation,migration and invasion capacity of QGY-7703 and HepG2 cells.Rescue experiment showed that the promotion of cell proliferation,migration and invasion capacity caused by miR-1228 were abrogated by the expression of p53.The same correlations among miR-1228 and p53 were observed in 9 of 20 paired specimens of HCC and their adjacent noncancerous tissues,and three hepatic cell lines.Conclusions:It is the first time we found that miR-1228 could form a positive feedback loop with p53 and play an oncogene role in HCC by promoting the metastasis and proliferation of HCC.As a tumor suppressor,p53 was shown to suppress the expression of miR-1228,indicating that it could repress its negative regulators to increase its expression.