The Expression And Function Of RSU1P2 In Cervical Cancer

Objective:Long non-coding RNAs(LncRNAs)are a class of endogenous non-coding RNAs of more than 200 nucleotides in length.It is reported that lncRNAs can regulate genes expression at different levels and are widely participated in various physiological and pathological processes.Emerging evidences have proved that a number of differentially expressed lncRNAs are associated with tumorigenesis,and involved with numerous cancer types.However the functional role for the vast majority of these unique RNAs is still in question.Previous studies in our lab have verified that RSU1P2 is up-expressed in cervical cancer tissues,indicating that it functions as oncogene.In this study,we aim to reveal the effects of RSU1P2 on cellular malignancy and to illustrate the regulation mechanism,which could shed light on identifying new molecular targets and designing novel treatment for cervical carcinoma.Methods:First,we enforced or blocked the expression of RSU1P2 in HeLa and C33A cells,and then evaluated the effects on malignant phenotypes using a series of assays,including MTT assay,colony formation,Transwell invasion and Transwell migration experiments and vasculogenic mimicry assay.And FACS and western blot were used to observe the effects of RSU1P2 on the cell cycle process and Epithelium-Mesenchymal transition(EMT),respectively.Second,according to bioinformatics analysis and known tumor suppressive function,let-7a was selected for our further study.The candidate genes targeted by let-7a were predicted by bioinformations,and verified by the fluorescent report assay,qRT-PCR and western blot.Then,after overexpression or suppression of RSU1P2,the mRNA or protein levels of candidate targets(IGF1R、MYCN、EphA4 and C14orf28)were measured using qRT-PCR or Western blot.Meantime,we used qRT-PCR to analyze the expression of let-7a and its candidate targets in 14 paired specimens of cervical carcinoma and their adjacent noncancerous tissues.Furthermore,the fluorescent report assay was used to investigate the microRNA dependency of RSU1P2-mediated regulation.At last,transcription factor MYCN was predicted to bind the promoter region of RSU1P2 by bioinformatics.Combining with qRT-PCR we detect whether MYCN could affect the expression level of RSU1P2 and let-7a.Results:we found that RSU1P2 could enhance the proliferation,angiogenesis,invasion and migration capacity,promote EMT process and the G1/S transformation of HeLa and C33A cells,thus functioning as an oncogene.RSU1P2 transcript contains many potential binding sites of various miRNAs.In the fluorescent report assay and qRT-PCR analysis,we vertified that let-7a directly binded this gene and negatively regulated the expression of RSU1P2.We screened and comfirmed that IGF1R、MYCN、EphA4 and C14orf28 were target genes of let-7a.When enhanced RSU1P2 level in HeLa cells,the mRNA and protein level of candidate targets can be positively regulated,and the level of let-7a was reduced.In cervical carcinoma tissues,RSU1P2 level is positively associated with target genes of let-7a,which comfirmed that RSU1P2 function as ceRNA.Moreover,we demonstrated that RSU1P2 exerts a role in post-transcriptional regulation by a microRNA dependent way.Then we predicted that MYCN could bind the promoter region of RSU1P2 by bioinformatics analyzing.Enforced the expression of MYCN could up-regulate the expression of RSU1P2 and down-regulate the level of let-7a.Conclusions:Our results indicate that RSU1P2 promotes the malignant phenotype of cervical carcinoma cells,and acts as a ceRNA through regulating the expression of IGF1R、MYCN、EphA4 and C14orf28 by competing for shared miRNA--let-7a.Transcription factor MYCN could form a positive feedback loop with RSU1P2 to enhance its oncogenic capacity.