The Study Of Diagnostic Value Of Circular RNA And Homer2 In Hepatocellular Carcinoma And The Molecular Mechanism Of CircSMAD2 In Hepatocellular Carcinoma

Background:Hepatocellular carcinoma?HCC?is a common malignant tumor worldwide.Owing to lacking typical clinical symptom and sensitive diagnostic biomarker,HCC is ussualy diagnosed at the advanced stage.Therefore,identifying novel diagnostic biomarkers and therapeutic targets is urgent.Objective:Circular RNAs?circRNA?,a class of non-codling RNAs,have been found to be involved in various diseases.The aims of this study are to measure the expression and access the diagnostic value of hsa_circ₀001445 and circSMAD2 inHCC.Next,we further explored the biological function of circSMAD2 and investigated the underlying molecular mechanism.Moreover,the aims of the last part are to investigate the expression and diagnostic value of tissue Homer2 in HCC and to prepare the anti-Homer2 monoclonal antibody.Methods:The expression levels of the circRNA hsa_circ₀001445 and circSMAD2 in HCC and adjacent non-tumor tissues and plasma of HCC,cirrhosis,hepatitis B and healthy control were measured by quantitative Real-Time polymerase chain reaction?qRT-PCR?.Then,the diagnostic value of plasma hsa_circ₀001445 was accessed using receiver operating characteristic?ROC?curve.Next,we first reconstructed the circSMAD2 formation plasmid and over-expressed circSMAD2 in HCC cell line HepG2.Then,the biological function of circSMAD2 was observed by proliferation,apoptosis,migration,invasion,and western blot assays.Furthermore,mimics of top ten ranked target miRNA of circSMAD2 were synthetized,and psiCHECK-circSMAD2-wt and its mutation type?psiCHECK-circSMAD2-mut?were reconstructed.Next,the dual-luciferase reporter assay verified the directly binding of circSMAD2 and miRNAs.In addition,we over-expressed and knockdown circSMAD2 in HCC cell and measured the influence on target miRNAs' expression.Finally,we co-transfected circSMAD2 and target miRNA to investigated their relationship on HCC regulation.Finally,The expression of Homer2 in HCC tissues was measured using qRT-PCR,and the diagnostic value of Homer2 in detecting HCC was accessed using ROC curve.Then,the Homer2 antigen protein and the screening peptide were reconstructed,expressed,and purified.Next,the BALB/c mice were immunized by the antigen protein.The cell fusion technique was applied to obtain the hybridoma cells,and the monoclonal antibody was purified from the ascites.Indirect ELISA and western blot were used to identify its specificity.Results:hsa_circ₀001445 expression level was significantly down-regulated in HCC tissues?P<0.001?and markedly associated with the number of tumor foci?P=0.014?.Meanwhile,circSMAD2 level was significantly down-regulated too?P=0.014?,but its expression level was associated with the differentiation degree of the HCC tissues?P<0.001?.In addition,we found that the plasma hsa_circ₀001445 transcription levels in HCC patients was lower than in cirrhosis?P<0.001?and hepatitis B?P<0.001?patients as well as in healthy controls?P<0.001?.In fact,ROC analysis indicated that plasma hsa_circ₀001445 could be a fairly accurate marker to distinguish HCC cases from healthy controls as well as patients with cirrhosis or hepatitis B.Next,we reconstructed the pcDNA3.1?+?-circSMAD plasmid,which could effectively over-express circSMAD2 in HCC cells,and the biological assays demonstrated that over-expressed circSMAD2 inhibited the proliferation,migration,invasion,and epithelial-to-mesenchymal transition?EMT?of HCC cells.Next,the dual-luciferase reporter assay verified that miR-629 directly bound circSMAD2.In addition,we found that over-expression of circSMAD2 suppressed the expression of miR-629 in HCC cells,whereas knockdown of circSMAD2 up-regulated the expression of miR-629.Furthermore,co-transfection of miR-629 mimics with circSMAD2 reversed the circSMAD2 effects of inhibiting the migration,invasion,and EMT in HCC cells.Finally,the Homer2 expression in HCC was significantly down-regulated and associated with the differentiation degree of the HCC tissues?P=0.012?,and the tissue Homer2 expression level is a valuable biomarker in HCC detection?AUC=0.660?.In additon,we obtained Homer2 antigen protein and screening peptide.Furthermore,we prepared the anti-Homer2 monoclonal antibody via the hybridoma technique,andthe new prepared monoclonal antibody was named as anti-Homer2-2B7.ELISA and Western blot assay verified that the antibody is specific.Conclusions:circRNA hsa_circ₀001445 and circSMAD2 were both down-regulated in HCC tissues,and plasma hsa_circ₀001445 might be a fairly good biomarker in HCC detection.In addition,circSMAD2 might inhibit the migration,invasion,and EMT in HCC cells by targeting miR-629.Finally,the Homer2 expression was down-regulated in HCC tissues.Then,the anti-Homer2 monoclonal antibody was prepared,and it will be a useful tool in the Homer2 related researches.