Low Expression And Functional Analysis Of Hsa＿circ＿0002980 In Hepatocellular Carcinoma

Objective:Primary liver cancer is the most common malignant tumor of the digestive system,which endangers the health of people in many countries in the world.China has one of the highest morbidity and mortality rates of primary liver cancer,with hepatocellular carcinoma(HCC)as the main tissue subtype.Although there has been great progress in the diagnosis and treatment of primary hepatocellular carcinoma(HCC)in recent years,most patients were diagnosed in the middle and late stage,resulting in no significant improvement in prognosis,with a 5-year survival rate of less than 37%.The main reason is that HCC has no obvious clinical symptoms in the early stage and there is no simple and non-invasive early diagnosis method.Therefore,the search for a simple and feasible biomarker with high sensitivity and specificity,which is non-invasive,can effectively improve the early diagnosis rate of HCC and improve the prognosis.CircularRNA(circRNA)is a large class of endogenous non-codingRNA(ncRNA)with regulatory ability.As a closed-loop structure formed by reverse splicing,it is not vulnerable to the destruction ofRNA exonuclease.It has extracellular stability and evolutionary conservatism,and plays an important role in the regulation of gene expression.Studies in recent years have shown that circRNA is associated with the occurrence of a variety of diseases and has potential as a marker for diagnosis and prognosis of diseasesMaterials and methods: In the first stage,HCC-related circRNA high-throughput chips(GSE78520,GSE94508,GSE97332)in NCBI and GEO were collected,and circRNA differentially expressed between HCC and its matched normal tissue samples were screened byRobustRank Aggreg.The screening criteria were: fold change(FC)≥1,p < 0.05.Heat map was drawn and the candidate circRNA with great difference was selected.qRT-PCR(real-time quantitative reverse transcription polymerase chain reaction)was used for verifying the expression leve of candidates circRNA in 16 pairs of HCC and adjacent normal tissue samples.The second phase,circRNA expression vector was bulilded,and transfection into two kinds of liver cancer cells(Huh7,Hep3B).Subsquently,observe the effect of circRNA in cell apoptosis,cell cycle,proliferation,migration,invasion,etc.In the third stage,bioinformatics technology was used to predict the potential miRNA and circRNA binding sites,double luciferase reporter gene detection method was used to verify the binding sites and analyze the potential mechanism of action.Results:1.By usingRobustRank Aggreg to integrate three HCC chip data sets,it is found that there were a total of 6 significantly differentially expressed circRNAs(2 up-regulated circRNAs,hsa＿circRNA＿103510 and hsa＿circRNA＿100542,and 4 down-regulated circRNAs,hsa＿circRNA＿102166,hsa＿circRNA＿0002980,hsa＿circRNA＿105031 and hsa＿circRNA＿100291).In addition,q PCR was used to verify that hsa＿circRNA＿0002980 was significantly lower expressed in HCC tissues compared with non-cancer tissues(P < 0.05).2.In vitro cell function experiments showed that the overexpression of hsa＿circRNA＿0002980 could significantly promote the proliferation of liver cancer cells,enhance the migration and invasion ability of cancer cells,and interfere with cell cycle,resulting in a significant increase in the proportion of cells in the DNA synthesis stage(S stage).3.We predict that hsa-miR-1303,hsa-miR-877-5p,and hsa-miR-142-5p,all has conservative binding sites with hsa＿circ＿0002980 by bioinformatic.And meta-analysis shows that the expression level of miR1303 in hepatocellular carcinoma is higher than normal liver tissue.The area under curved surface of sROC(summary receiver operating characteristics)reachs 0.81,which is of good diagnostic value for hepatocellular carcinoma.However,dual-luciferase reporter assay do not support the interaction between hsa-miR-1303 and hsa＿circ＿0002980.Conclusion: Hsa＿circ＿0002980 was significantly lower expression in hepatocellular carcinoma tissues than in normal non-cancerous liver tissues.In vitro cell function experiments showed that overexpression of hsa＿circ＿0002980could significantly promote the proliferation of hepatocellular carcinoma cells,promote the ability of cell migration and invasion,and interfere with cell cycle,resulting in significantly increased proportion of cells in the DNA synthesis stage(S stage).There may be no interaction between hsa-miR-1303 and hsa＿circ＿0002980.