| Backgruond & AimsHepatocellular carcinoma(HCC)is the most common pathological type of primary liver cancer and accounts for about 80% of primary liver cancer in China.HCC is highly invasive,which leads to a high recurrence rate after radical resection.The recurrence rate of HCC within 5 years of radical resection can reach 60%.However,there are few studies investigating the early molecular events of HCC invasion,and it is not clear how HCC gradually acquires and enhances its invasive potential.Our research team put forward the basic thesis of Cancer Evo-dev theory,indicating that chronic inflammation could promote cancer evolution and development.Cancer Evo-dev theory indicates that the repeated “necrosis-proliferation” process in chronic inflammatory environment promotes “mutationselection-malignant” transformation of precancerous cel s.Interaction between environmental exposure and genetic susceptibility in immune moleculars can trigger immune imbalances,which al ows persisted inflammatory responses and leads to precancerous lesions.Long-term stimulation of inflammatory mediators,cytokines,and chemokines can increase the expression of nucleic acid-editing enzymes(like cytosine deaminase),which will increase genome unstability and activate somatic mutatio n mechanisms.Driver somatic mutations will gradually alter the expression of important moleculars in cancer cel s and will confer cancer cel s with advantages in prolieration and invasion.Non-coding RNA plays an important role in all stages of HCC evolution.Circular RNA is an important catagory of non-coding RNA.Through a special loop-forming mechanism,the circular RNA constitutes a stable circular structure.Researchers have found that the expression of circular RNA is characterized by high tissue specificity and high time specificity.Compared to microRNAs and lncRNAs,circular RNAs are more stable and more conserved in mammalian cel s.Circular RNA can take effort in various ways.One of wellstudied function of circular RNA is the competitive endogenous RNA(ceRNA)functio n.By adsorbing miRNA,the circular RNA can prevent miRNAs from hindering the translatio n of mRNA,so as to play an important role in the epigenetic regulation of gene expression.Analysis on the function of circular RNA on HCC invasion will help elucidate the epigenet ic regulatory network involved in HCC invasion regulation and contribute to the developme nt of an important diagnostic and preventive effect on early HCC recurrence.The aim of this study was to explore HCC evolution and development,and to discover important molecular events in early stages of HCC invasion,and to explore the dynamic processes of key signaling pathways during HCC evolution.This study will screen important circular RNA associated with HCC recurrence by high-throughput sequencing and to investigate its mechanism.The target of important circular RNA will be explored following ceRNA network theory.This research will analyze expression profile and somatic mutatio n profile,so as to explore the dynamic changes of signaling pathways during HCC evolutio n.By implementing this project,we hope that we can find new biomarkers and therapeutic targets for the diagnosis and treatment of HCC recurrence.Methods1.Patient involvementThis study involved HCC patients who had no HCC recurrence within 60 months after radical resection(control group)and HCC patients who had HCC recurrence after radical resection(case group).HCC tissues and adjacent tissues of involved patients were collected for sequencing.2.Screening and discovery of circular RNAs associated with HCC recurrence:(1)Circular RNA sequencing and mRNA sequencing were performed in included HCC and adjacent tissues.Differentially expressed circular RNAs between the HCC tissues of the initial operation of case group(PT)and the HCC tissues of the control group(CT) was found.Gene enrichment analysis(Gene Sets Enrichment Analysis,GSEA)was performed by combining the mRNA expression data and circular RNA expression data. Finally,circ39630 was found to be a potential marker for HCC recurrence;(2)Specific primers for circ39630 was designed.The presence of circ39630 in HCC and adjacent tissues was verified and back-splicing site of circ39630 was confirmed by sanger-sequencing;(3)High tolerance of circ39630 to RnaseR digestion was verified.3.Regulatory role of circ39630 in HCC cell lines(1)A circ39630 over-expression plasmid was constructed.HCC cell lines stably overexpressing circ39630 was established using lentivirus.SiRNA transfection was performed to knock-down circ39630;(2)HCC cell lines over-expressing circ39630,HCC cell lines with knocked-down circ39630 and control HCC cell lines were utilized in proliferation assay,transwell assay,and soft agar colony formation assay.Also,the proportion of apoptosis of these cell lines were also measured by flow cytometry.4.The mechanism investigation of circ39630(1)MiRNA sequencing of the included HCC and adjacent tissues was performed.Data of circular RNA sequencing,mRNA sequencing,and miRNA sequencing were combined for co-expression network analysis.Potential miRNA binding sites of the circ39630 was analyzed utilizing Miranda software.A circular RNA-miRNA-mRNA regulation axis [circ39630-(miR-222-3p)-GLS2] was found;(2)The expression levels of miR-222-3p and GLS2 were verified in HepG2 cells overexpressing circ39630 and HepG2 cells with knocked-down circ39630.5.Discovery and validation of genes associated with HCC recurrence(1)Analyze the mRNA sequencing data,find the genes differentially expressed in the HCC tissue of the initial surgery group(PT)and the HCC tissue of the control group(CT);(2)Data of Hepatocellular Carcinoma Cohort(LIHC)in the Cancer Genome Atlas(TCGA)were downloaded and survival analyses were performed to determine the relationship of interested gene with the recurrence free survival time of HCC.6.Investigation on dynamic changes of expression profile during HCC evolution(1)Calculate gene expression dynamic changes of each patient(Primary tumor vs.recurrent tumor;adjacent tissue of primary tumor vs.adjacent tissue of recurrent tumor);(2)Single-sample GSEA analyses were performed for each patient to summarize the change of important pathways.7.Study of somatic mutations associated with HCC recurrence(1)Exon sequencing was performed in each HCC tissue and adjacent tissue,and Mutect software was utilized to obtain somatic mutations in each HCC tissue;(2)Somatic genes of tumors in control group,primary tumors in case group,and recurrent tumors in case group were analyzed.8.Study of somatic mutation during the evolution of HCC(1)Mutation signatures of tumors in control group,primary tumors in case group,and recurrent tumors in case group were analyzed;(2)Dynamic changes of tumors in control group,primary tumors in case group,and recurrent tumors in case group were analyzed,and pathway analyses were performed.Results1.The expression of circ39630 in adjacent tissues was lower than that in HCC tissues, and the expression in HCC tissues of the first operation in case group was lower than that in the control group(FDR<0.001);2.GSEA analysis showed that the expression level of circ39630 was negatively correlated with epithelial-mesenchymal transition(FDR<0.1);3.The cyclic structure and back-splicing point of circ39630 was consistent with the expectation.Circ39630 tolerated RnaseR digestion and the content of circ39630 did not change significantly after RnaseR digestion(P>0.05),while its linear product decreased significantly after RnaseR digestion(P=0.008);4.The proliferative capacity of circ39630-overexpressing SK-HEP-1 cells was significantly lower than that of control SK-HEP-1 cells(P=0.005)and circ39630-knockdown SK-HEP1 cells(P=0.001);The proliferative capacity of circ39630-overexpressing HepG2 cells was significantly lower than that of control HepG2 cells (P=0.043)and circ39630 knckdown HepG2 cel s(P=0.004);5.The migration ability of SK-HEP-1 cells overexpressing circ39630 was significantly lower than that of control SK-HEP-1 cells(P<0.001)and SK-HEP-1 cells knocked down circ39630(P<0.001),the migration ability of circ39630 knocked-down SK-HEP-1 cells was significantly higher than that of control SK-HEP-1 cells(P<0.001).The migration ability of HepG2 cel s overexpressing circ39630 was significantly lower than that of control HepG2 cells(P<0.001)and circ39630 knocked-down HepG2 cells (P<0.001),the migration ability of HepG2 cells knocking down circ39630 was significantly higher than that of control HepG2 cells(P<0.001);6.After knocking down circ39630,the proportion of late apoptotic cel s in HepG2 was significantly lower than that in control HepG2 cells(P=0.049)and HepG2 cells overexpressing circ39630(P=0.004);7.Co-expression network analysis indicated that module expression where circ39630 existed was associated with HCC recurrence(P=0.014)and the GLS2 gene had the highest correlation with circ39630 expression in this module;8.ceRNA analysis indicated that has-miR-222-3p might be absorbed by circ39630 and this miRNA could bind to 3'UTR region of GLS2 mRNA.GLS2 was positively correlated with circ39630 expression(P<0.001).Circ39630 was negatively correlated with has-miR-222-3p(P<0.001).Hsa-miR-222-3p was negatively correlated with GLS2(P<0.001);9.Overexpression of circ39630 in HepG2 cel s decreased the expression of hsa-miR-222-3p(P=0.008)and knocking-down of circ39630 in HepG2 cells increased the expression of hsa-miR-222-3p(P=0.008);10.Overexpression of circ39630 in HepG2 cel s increased the expression of GLS2(P=0.008)and knocking-down of circ39630 in HepG2 cells did not alter the GLS2 expression significantly;11.HCC Patients with higher circ39630 expression had longer overall survival time(P=0.04)and longer recurrence free survival time(P=0.01);12.Multivariate Cox regression indicated that MEX3 A was an independent risk factor for HCC early recurrence(P<0.005);13.In the evolution process of HCC,the adjacent tissues showed a malignant trend;14.TP53 is the gene with the highest mutation rate,and the most common type of TP53 mutation is R249S;15.The TP53 mutation rate in HCC tissues in the initial operation of the case group is higher than that of control group;16.Recurrent tumors in HCC patients are highly similar to primary tumors in the mutational profile;17.During the evolution of HCC,somatic mutations in TP53 were retained,and somatic mutations that were eliminated during HCC evolution were significantly enriched in the Ras signaling pathway and the MAPK signaling pathway.Conclusion This study found that circ39630 played an important role in the recurrence of HCC and demonstrated that circ39630 might function through circ39630-miR-222-3p-GLS2 axis.This study demonstrated that circ39630 could inhibit HCC recurrence through restraining the proliferation,migration,and clone formation of HCC cells.This study also found a novel prognostic marker for recurrence after HCC: MEX3 A.This study also summarized the evolution pattern of HCC and found that TP53 might play an important role in the evolutio n of HCC. |
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