Expression And Mechanism Of Hsa＿circ＿0005397 In Hepatocellular Carcinoma

Objective 1.To detect the expression of circular RNA(circ RNA)hsa＿circ＿0005397 in tissue,plasma and cell lines of patients with hepatocellular carcinoma(HCC),and to explore the value of plasma hsa＿circ＿0005397 in the clinical diagnosis and treatment of HCC;2.To study the function of hsa＿circ＿0005397 in HCC cells in vivo and in vitro;3.To study the effect of eukaryotic translation initiation factor 4A3(EIF4A3)regulating the expression of hsa＿circ＿0005397 on the function of HCC cells.Methods 1.Screening of differentially expressed circ RNAs in the Gene Expression Omnibus(GEO)database;2.Detection and evaluation of the hsa＿circ＿0005397 method by quantitative real-time PCR(q RT-PCR);3.To investigate the clinical value of plasma hsa＿circ＿0005397 in HCC by ROC curve,correlation analysis of pathological parameters and combined detection of serum AFP and AFP-L3;4.Expression of hsa＿circ＿0005397 in HCC cancer tissues and cell lines detected by q RT-PCR;5.The RNA interference vector and overexpression vector of hsa＿circ＿0005397 were constructed respectively,and their efficiency was verified by q RT-PCR;6.CCK-8,clone formation,Transwell and cell cycle assays to analyze the function of hsa＿circ＿0005397 in HCC cells;7.Nude mice xenograft model was constructed to analyze the effect of hsa＿circ＿0005397 on HCC growth in vivo;8.Bioinformatics prediction and RNA binding protein immunoprecipitation(RIP)test to verify the RNA-binding protein(RBP)of hsa＿circ＿0005397;9.The expression of EIF4A3 in HCC tissues was detected by q RT-PCR,and the correlation between it and the expression of hsa＿circ＿0005397 was analyzed;10.The RNA interference vector of EIF4A3 was constructed,and its efficiency was verified by q RT-PCR and Western blot;11.Reverse experiment to explore the effect of EIF4A3 regulating hsa＿circ＿0005397 expression on the proliferation,migration and invasion of HCC cells.Results 1.Screen out hsa＿circ＿0005397 which is highly expressed in HCC from GEO database;2.The melting curve of hsa＿circ＿0005397was detected by q RT-PCR with single-peak specificity,agarose gel electrophoresis and Sanger sequencing showed that the primers were designed correctly,and the method had good linearity;Actinomycin D and Rnase R enzyme tests confirmed that hsa＿circ＿0005397 had stable ring structure;3.The plasma hsa＿circ＿0005397 level in HCC patients was increased,and it was correlated with tumor size(P=0.02)and TNM stage(P=0.006);Its combined detection with serum AFP and AFP-L3 could improve the sensitivity for diagnosing HCC;The postoperative dynamic monitoring showed that the content of plasma hsa＿circ＿0005397 in patients after operation was significantly decreased;4.Hsa＿circ＿0005397 is highly expressed in cancer tissues of HCC patients and HCC cell lines;5.Two hsa＿circ＿0005397 RNA interference vectors(si1,si2)were constructed,and the expression of hsa＿circ＿0005397 decreased after transfection into BEL-7404 and LM3 cells respectively;hsa＿circ＿0005397 overexpression vector(pc DNA)was constructed,and hsa＿circ＿0005397 was expressed after transfection into SK-Hep1 cells rise;6.The proliferation,clone formation,invasion and migration of BEL-7404 and LM3 cells transfected with si1 and si2 decreased,and the cell cycle was arrested in G0/G1 phase;the proliferation,clone formation and invasion of SK-Hep1 cells after transfection with pc DNA,enhanced migration ability and accelerated cell cycle progression;7.In vivo experiments show that RNA interference hsa＿circ＿0005397 can inhibit the growth of transplanted tumors in nude mice;8.Bioinformatics predicted that EIF4A3 was the RBP of hsa＿circ＿0005397,and it was confirmed by RIP test;9.EIF4A3 m RNA was highly expressed in HCC cancer tissues,and was positively correlated with the expression of hsa＿circ＿0005397(P<0.001);10.The RNA interference vector of EIF4A3(sh EIF4A3)was constructed and transfected into BEL-7404 and LM3 cells,respectively,and the expression of EIF4A3 m RNA and protein decreased;11.The expression of hsa＿circ＿0005397 significantly decreased in LM3 after transfection with sh EIF4A3;the recovery test showed that sh NC+pc DNA could up-regulate the expression of hsa＿circ＿0005397,while sh EIF4A3+pc DNA co-transfected LM3 cells could significantly inhibit the expression of hsa＿circ＿0005397;the clone formation test and transwell test further showed that,sh NC+pc DNA can promote the proliferation,migration and invasion of HCC cells,while sh EIF4A3+pc DNA co-transfected LM3 cells can significantly inhibit the proliferation,migration and invasion of HCC cells.Conclusions Hsa＿circ＿0005397 is highly expressed in HCC tissues and can act as an oncogene to promote HCC growth and metastasis;EIF4A3 promotes the malignant progression of HCC by regulating the expression of hsa＿circ＿0005397;in addition,the high expression of hsa＿circ＿0005397 in plasma can be used as a potential biomarker for HCC diagnosis and treatment.