| Background:Liver cancer is a significant malignancy worldwide,particularly in Asian regions such as China where high incidence and mortality rates pose a serious threat to population health.Hepatocellular carcinoma(HCC),the primary type of liver cancer,accounts for around 85% of all liver cancer cases.Despite advances in medical technology,the prognosis for HCC remains poor due to its asymptomatic early stages,resulting in late diagnosis and missed opportunities for efficient treatment.Even patients with early HCC who underwent radical surgery face a high risk of recurrence.Therefore,the overall 5-year survival rate of HCC is extremely low.Effectively solving the problems of low early diagnosis rate and high recurrence rate after surgery is the key to improve the long-term quality of life of HCC patients.Circular RNAs(circ RNAs)is a unique type of non-coding RNA that is widely present in eukaryotes with tissue specificity and developmental stage dependency.Its circular structure,formed by covalently connecting the ends,ensures stable chemical properties of the molecule.Advances in RNA sequencing technology have led to further recognition of various biological functions of circ RNA,including its role as a competing endogenous RNA(ce RNA).By directly binding to micro RNA molecules,circ RNA competes to regulate the downstream target genes of micro RNA.In recent years,circ RNA has been explored as a therapeutic target and detection biomarker for cancers,particularly in liver cancer,where studies have shown its importance in the disease’s onset and progression.However,the role of circ RNA in liver cancer recurrence and the regulatory mechanisms involved still require further invistigation.This study aims to investigate the molecular mechanisms underlying HCC recurrence.By conducting circ RNA sequencing on tumor and adjacent tissue samples from patients who experienced recurrence and those who did not within five years after surgery,circ RNAs significantly associated with HCC recurrence were identified,among which circ CAR(circ RNA Constitutive active/androstane receptor,circ RNA▁03654)was further analyzed.The expression level of circ CAR in HCC tissues was confirmed using clinical cohort samples,and its regulatory mechanisms in the occurrence,development,and recurrence of HCC were explored,as well as its potential as a molecular biomarker and therapeutic target for HCC recurrence.Methods:1.Transcriptomes,circ RNAs and micro RNAs sequencing were performed on tumor and paracancer tissue samples from the first and second surgeries from 4 patients with HCC recurrence within 2-5 years after the first surgery,and 9 patients with no recurrence within 5 years after the surgery;Differential analysis of sequencing data was performed to screen out circ RNAs that were differentially expressed in paired tumor and paracancer tissues,and differentially expressed in tumors from first surgery of patients with recurrent HCC and those without recurrence.Combined with Gene Set Enrichment Analysis(GSEA),circ CAR with high gene enrichment level was screened out.NCBI-Primer was used to design specific primers across back-splicing sites,and gel electrophoresis and Sanger sequencing were used to verify the specificity of primers and back-splicing junction sites.The cyclic properties of circ CAR were verified by RNase R enzyme tolerance assay.2.We used a follow-up cohort of 144 HCC patients who underwent radical resection of HCC to detect the expression level of circ CAR in tumor and adjacent tissue samples,and analyzed the correlation between the expression level of circ CAR in tumors and clinical detection indicators.Through R language survminer package,Kaplan-Meier method was used to analyze the correlation between circ CAR expression level in tumor tissues and prognosis.3.Hep G2,Huh7,SK-Hep-1 and SNU387 liver cancer cell lines were selected for further study.Lentivirus-mediated circ CAR and Sh-circ CAR plasmids were used to construct over-expression cells of Hep G2 and SK-Hep-1 cells with low circ CAR expression level and circ CAR knockdown cells of Huh7 and SNU387 with high circ CAR expression level.Four groups of the cells were constructed,and the effects of circ CAR on the proliferation of HCC were detected by Cell Counting Kit-8(CCK8)and flow cytometry.The effects of circ CAR on the migration and invasion of HCC were detected by scratch healing assay and Transwell assay.The effect of circ CAR on cell cloning of HCC was determined by plate cloning assay.4.SK-Hep-1 with stable over expression of circ CAR and Huh7 with stable Sh-circ CAR expression were used to carry out subcutaneous tumor bearing experiments on NOD-SCID mice,and the effect of circ CAR on the growth of liver tumors in vivo was investigated by timing measurement of tumor growth.5.circ CAR specific biotin labeled probe was designed and RNA Pull-down experiment was conducted.Then,we sequenced the drop-down micro RNAs,combining with the micro RNAs sequencing data of patients’ surgical tissue samples,to screen the potential downstream micro RNAs of circ CAR.The binding sites of circ CAR and micro RNAs were predicted by RNA22v2,and verified by double luciferase reporter gene assay.Micro RNA mimics(mimics)and inhibitors(inhibitors)were used to verify the functional interaction between circ CAR and mi R-183-5p through rescue experiments.6.Mi RDB,mi RTarbase and mi RWalk database were used to predict m RNAs which could be targeted by mi R-183-5p,and combined with Kyoto Encyclopedia of Genes and Genomes(KEGG)for further screening.And phosphatase and tension Homolog deleted on Chromosome(PTEN),which were highly correlated with liver cancer,were picked out.RNA22v2 predicted the binding sites of mi R-183-5p to PTEN that were verified by dual luciferase reporter assay.The functional correlation between circ CAR and PTEN was verified by rescue experiments.In addition,quantitative real-time polymer chain reaction(q RT-PCR)was used to analyze the relationship between the expression levels of the molecules.Results:1.Through differential analysis,we obtained 4 simultaneous differential expression circ RNAs in paired HCC cancer and adjacent tissues,and primary cancer tissues of recurrent patients and non-recurrent patients with fold change(FC)≥2 and false discovery rate(FDR)< 0.05.After GSEA enrichment analysis,with FDR < 0.25 as the standard,circ CAR with higher enrichment,smaller FDR and larger FC was finally selected for further study.The back-splicing sites of circ CAR were predicted by Sanger sequencing,and the RNase R tolerance experiments confirmed that circ CAR could withstand the degradation of RNase R.2.q RT-PCR showed that circ CAR expression was down-regulated in HCC tissues in the follow-up cohort,and further down-regulated in cancer tissues of patients with microvascular invasion,and down-regulated circ CAR expression was associated with shorter overall survival time,recurrence free and metastatic free time.3.In vitro experiments,up-regulation of circ CAR can significantly inhibit the proliferation,migration,invasion and clonal formation of Hep G2 and SK-Hep-1;The down-regulation of circ CAR promoted the proliferation,migration,invasion and cloning of Huh7 and SNU387.4.In subcutaneous tumor carrying experiments in mice,up-regulation of circ CAR can significantly inhibit tumor growth;Down-regulation of circ CAR can accelerate the growth of HCC tumors.5.In RNA Pull-down experiment,a total of 22 micro RNAs with FC≥2 and Tag counts≥50 in the experimental group were obtained.By combining the sequencing results with tumor and adjacent samples,three common potential downstream micro RNAs(mi R-183-5p,let-7d-5p,let-7e-5p)were obtained.Circ CAR can be directly bound to mi R-183-5p which was verified by dual luciferase reporter assay.Cell phenotype experiments showed that mi R-183-5p mimics can promote the proliferation,migration,invasion and cloning of HCC.Mi R-183-5p inhibitors have the opposite effect.Secures experiments demonstrated that mi R-183-5p mimics could partially recover the anticancer effect of overexpression of circ CAR.mi R-183-5p inhibitors reversed the oncogenic effects of circ CAR knockdown.6.A total of 44 potential downstream m RNAs were predicted by mi RDB and mi RTarbase database,and 9 potential downstream m RNAs were further screened by mi RWalk verification.Then,PTEN was finally identified as a candidate molecule by KEGG Pathway database for experimental verification.Dual luciferin reporter assay confirmed that mi R-183-5p could bind to PTEN.Knockdown of PTEN can significantly promote the proliferation,migration and invasion of HCC,which has the same effect as knockdown of circ CAR,and can restore the up-regulation of the tumor inhibitory effect produced by circ CAR.q RT-PCR showed that the change of circ CAR expression would not cause the change of mi R-183-5p expression,but could regulate the RNA expression of PTEN in the same direction.Mi R-183-5p could negatively regulate the RNA expression of PTEN,but could not affect the expression of circ CAR.Knockdown of PTEN did not affect the expression of circ CAR and mi R-183-5p.Conclusions:This study identified a significant downregulation of circ CAR in hepatocellular carcinoma(HCC)tissues,with a further decrease in HCC tissues with microvascular invasion,indicating that low expression of circ CAR can predict poor prognosis in patients.Upregulation of circ CAR expression inhibited HCC cell proliferation,migration,invasion,and colony formation,while downregulation had the opposite effect.circ CAR acts as a competing endogenous RNA(ce RNA)by binding to mi R-183-5p,which,in turn,inhibits the anti-cancer effect of PTEN via the phosphatidylinositol-3-kinase(PI3K)/AKT signaling pathway,thereby promoting malignant phenotypes such as proliferation,migration,and invasion.These findings demonstrate that circ CAR has the potential to become a biomarker for HCC prognosis and a therapeutic target to inhibit the occurrence and recurrence of HCC. |
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