Preliminary Study For The Function And Molecular Mechanism Of CircADAMTS13 In Hcc Cell Proliferation By Targeting MiR-484

Hepatocellular carcinoma(HCC)is one of the most life-threatening and prevalent malignancies in the world.The prognosis of HCC remaines unsatisfactory due to the high recurrence rate and the difficulty in early diagnosis.Currently,the main treatment for HCC is comprehensive treatment mode based on surgical resection,still lacking effective therapeutic targets.Therefore,further screening potential diagnostic/prognostic biomarkers and therapeutic targets,as well as in-depth investigation of possible molecular mechanisms are crucial for optimizing HCC treatment.In recent years,advance on high-throughput sequencing technology and bioinformatic analysis,studies on non-coding RNA(nc RNA)have been continuously deepened.Among them,circular RNA(circ RNA)can participate in tumorigenesis,through their micro RNA response elements.However,alterations in circ RNA profiles during hepatocellular carcinoma(HCC)progression and their clinical significance remain large unclear.Here,we present extensive analysis of circ RNA profiles in tumor and matched peritumor tissue collected from 10 HCC patients,circ ADAMTS13 derived from Exon 13–14 of the ADAMTS13 gene was carefully investigated to explore the function and clinical value of circ RNA in HCC.Objective1.To screen out differential circ RNAs expression in tumor and matched peritumor tissues and clarify the correlation between circ ADAMTS13 expression level and clinicopathological characteristics of HCC patients for evaluating its prognostic value.2.To illuminate the biological function and molecular mechanisms of circ ADAMTS13 in HCC proliferation by targeting mi R-484.Conduct to provides insights for circ ADAMTS13 as a potential target for the treatment of HCC.Methods1.Total RNA were extracted from tumor and matched peritumor tissue collected from 10 HCC patients and subjected to RNA sequencing and bioinformatic analysis for identifying the differential circ RNAs expression pattern in HCC progression.2.Real-time quantitative PCR was used to validate the expression of circ ADAMTS13 in HCC tissue and matched peritumor.SPSS software was used to analyze the correlation between the expression level of circ ADAMTS13 and the clinical characteristics of HCC.3.Actinomycin D and RNase R treatment were conducted to verify the stability of circ ADAMTS13.The CCK-8 assay and flow cytometric analysis were performed to analysis the role of circ ADAMTS13 in cell proliferation and apoptosis on circ ADAM13-overexpressing HCC cells.4.Three independent mi RNA databases(Targetscan,RNAhybir and Ciranda)were used to predict circ ADAMTS13/mi RNA signal network.Luciferase reporter assay was performed to validate the interaction between circ ADAMTS13 and mi R-484.Colony formation assays was performed using PLC/PRF/5 and Hep G2 cells to confirm up-regulation of mi RNA could effect the function of circ ADAMTS13.Result1.A total of 92204 circRNAs were identified in 10 HCC tissue and matched peritumor;81.9% of them were found in exons.the length of those identified circ RNA was mostly around 100–450 nucleotides.A total of 42 circ RNA were dysregulated in all HCC tumor tissue samples when compared with matched peritumor tissues(fold change ≥ 2 and P < 0.05);most of them(38/42)were downregulated in HCC tissue samples.Three circ RNA(circ ADAMTS13、circ DPF3 and circ CASP8AP2)with high number of back-spliced reads were selected to validate their expression in another 26 HCC patients.The results show that the expression of circ ADAMTS13 and circ DPF3 were significantly downregulated in HCC tissues,which were well consistent with the sequencing results.2.We found that low expression of circ ADAMTS13 in HCC tumor tissues was associated with the larger tumor size(P=0.001)and more severe BCLC stage(P=0.030).Kaplan-Meier analysis showed that HCC patients with low circ ADAMTS13 expression in tumor tissue had a significant shorter RFS time(P=0.044).3.We examined the stability of circ ADAMTS13 by actinomycin D intervention and RNase R treatment.The q RT-PCR assays revealed that circ ADAMTS13 was highly stable with a half-life > 24 h,whereas linear ADAMTS13 m RNA exhibited a half-life of < 4 h.Digestion resistance experiments further confirmed that circ ADAMTS13 could be resistant to RNase R,whereas linear ADAMTS13 m RNA was easily degraded.CCK-8 assay and colony formation assay both showed that circ ADAMTS13 overexpression strongly suppressed cell proliferation of PLC/PRF/5 and Hep G2 cells.4.Luciferase reporter assay showed that circ ADAMTS13 could directly bind to mi R-484.Rescue experiments showed that mi R-484 mimics can reverse the tumor-suppressing roles of circ ADAMTS13 in HCC.Conclusions1.Low expression of Circ ADAMTS13 in tumor tissue is significantly associated with poor prognosis of HCC patients.2.Circ ADAMTS13 can serve as a tumor suppressor during HCC progression via the functional pathway of sponging mi R-484.and It might serve as a potential therapeutic target for HCC.