The Role And Mechanism Of Circular RNA Related With Hepatocellular Carcinoma Recurrence

Background and Aims:Liver cancer is one of the most common tumors in the world.It is reported that there were about 748300 new cases and 695900 deaths of liver cancer worldwide in 2008.However,in 2020,the number of new cases of liver cancer worldwide increased to 906 000 and the number of deaths increased to 830 000.Hepatocellular carcinoma is the most common type of liver cancer,accounting for 75%-85%,and half of these cases and deaths were estimated to occur in China^[1,2].Radical resection is the main choice for patients with early liver cancer stage.But 70%of patients relapse within 5years after surgery^[3,4].Preoperative AFP level（?400ng/ml）and R0 resection are associated with low recurrent.HCC grade and surgical margin are independent risk factors for early recurrence of HCC,and spleen stiffness is an independent risk factor for late recurrence of HCC^[[5-7].However,we do not fully understand the timing,mode and mechanism of recurrence of HCC.Circular RNA is a new kind of single strand,covalently closed-loop non-coding RNA,which was first discovered from pathogens by Sanger in1976^[8].Circular RNA is formed through back splicing.It is relatively stable since the lack of 3’and 5’ends,and they are highly conserved among different species,and they are spatiotemporal specificlly expressed^[9].Circular RNA enrolled in many aspects of genesis and development of liver cancer,such as the up regulation of circular RNA has-circ-0000038 and down regulation of has-circ-0000650 promoted HBV replication and survival^[10].Circular RNA regulate the proliferation,migration and invasion of liver cancer cells^[11-13].Circ ARNT2 and circ RNA-102272 reduce the sensitivity of HCC to cisplatin^[14,15].Based on the model established by Huai the recurrence risk of patients with stage II/III colon cancer can be evaluated by using circ RNA signature,and circ RNA can be used as a tool to predict the disease progression of colon cancer after surgery^[16].has-circ-0000066and has-0069707 play an important role in predicting postoperative recurrence of nonfunctional pituitary adenomas^[17].It has been reported that low expression of circ ZKSCAN1 and high expression of circ AKT3 in exosomes are associated with recurrence of HCC^[18,19].Compared with other tumors,we know little about the role of circ RNA in the recurrence of HCC.Because of the relative stability and spatiotemporal specificity of circ RNA,it is of great significance to explore the possibility of circ RNA as a biomarker to predict the recurrence of HCC.The aims of the study was to analyzing the differential expression of circ RNA between HCC and adjacent tissues and between recurrent and non-recurrent,and verifing the key circ RNA related with HCC recurrence.Detecting the expression level of candidate circ RNA and analyzing the correlation between its expression and prognosis.Furtherly,exploring the structure,function and its potential mechanism of candidate circ RNA.Methods:1.The circ RNome and transcriptome data were retrieved from Gene Expression Omnibus（GEO）under accession GSE129687 and GSE129689.Comparisons between tumor and adjacent tissues and between recurrent and non-recurrent tumors were performed by edge R.Taking each circ RNA candidate as a profile,we applied Gene Set Enrichment Analysis（GSEA）to predict the circ RNA’s potential functions.The phenotype was permutated1,000 times and gene sets with false discovery rate（FDR）less than 0.25 were considered significantly enriched.PCR and Sanger sequencing were used to identify the differentially expressed key circ RNA related with HCC recurrence（circ KCNN2）.2.The expression of candidate circ RNA was detected by quantitative real-time PCR（q RT-PCR）in HCC tissues and paired adjacent tissues;Kaplan Meier was used to analyze the progress of HCC patients.3.In vitro,the candidate circ RNA was up-regulated or down regulated by transfection of lentivirus or si RNAs.The proliferation of HCC cells was detected by CCK8（cell counting kit-8,CCK8）and clone formation assay;the migration of HCC cells was detected by Transwell assay;the distribution of cell cycle was analyzed by flow cytometer（FCM）;Western blotting（WB）was used to detect the expression of downstream target genes of candidate circ RNA;q RT-PCR was used to detect the expression of target of candidate circ RNA in HCC tissues and adjacent tissues,and the correlation between the expression and prognosis was analyzed.In vivo,we constructed the cell lines with up-regulated candidate circ RNA by transfecting lentivirus.To observe the growth of tumor we subcutaneously planted the cell lines into NOD-SCID;4.The target gene of circ KCNN2,miR-520c-3p,was predicted and verified by dual-luciferase reportor assay and circular RNA immunoprecipitation（circ RIP）.The target gene of miR-520c-3p,MBD2,was predicted and verified by dual-luciferase reportor assay.The relationship between circ KCNN2,miR-520c-3p and MBD2 was further detect by q RT PCR and WB.5.The effects of lenvatinib on cancer growth was detected in cells and organoids that up-regulated circ KCNN2.Results:1.Comparisons between tumor and adjacent tissues and between recurrent and non-recurrent tumors were performed by edge R.Fifty-three differentially expressed circ RNA were obtained by fold change≥2 and false discovery rate less than 0.05;taking each circ RNA candidate as a profile,we applied Gene Set Enrichment Analysis（GSEA）to predict the circ RNA’s potential functions.The phenotype was permutated 1 000 times and gene sets with false discovery rate（FDR）less than 0.25 were considered significantly enriched.According to the level of abundance,fold changes between recurrent and non-recurrent tumors,and the number of enriched gene set simultaneously,circ KCNN2 ranked highly and was chosen for further study.2.The expression of circ KCNN2 is down regulated in HCC tissues,and the down regulation of circ KCNN2 is related with poor prognosis of HCC patients.3.By transfecting lentivirus the expression of circ KCNN2 was up-regulated,up-regulating of circ KCNN2 inhibits the proliferation and migration of HCC cells,and cell cycles were arrest in G0/G1 phase.On the contrary,knocking down the expression of circ KCNN2 by si RNAs transfection promotes the proliferation and migration of HCC cells and arrest the cell cycle in S phase;in vivo,up regulation of circ KCNN2 inhibits tumor growth.4.The putative targets of miRNAs of circ KCNN2 was predicted by Reg RNA2.0.Among the candidates,miR-520c-3p ranked the highest,so we chose it for further validation.Dual-luciferase reporter assay and circ RIP showed that circ KCNN2 could bind miR-520c-3p.CCK8,clone formation and FCM cell cycle detection assay indicated that up regulation of miR-520c-3p promoted the proliferation and cell cycle arrestted at S phase.Down regulation of miR-520c-3p inhibited the proliferation and cell cycle arrestted at G0/G1 phase.miR-520c-3p mimics can reverse the up-regulation effects of circ KCNN2,and miR-520c-3p inhibitors can reverse the down-regulation effects of circ KCNN2.5.GPC3 has been reported as a downstream target gene of miR-520c-3p,but we did not observe the effects of miR-520c-3p mimics and inhibitors on GPC3 m RNA expression.So we searched the database（miRDIP and mir Tarbase）to find the potential targets of miR-520c-3p.We found that MBD2 was one of the candidates and verified by dual-luciferase reporter assay and WB and q RT-PCR.6.By transfecting lentivirus,the HCC organoid could express labeled fluorescence and circ KCNN2 expression in HCC organoids were successfully up-regulated by q RT-PCR assay.In order to explore the effect of circ KCNN2 on drugs used in HCC patients with advanced stage,we found that the up regulation of circ KCNN2 in cells and organoids enhances the anti-tumor effect of lenvatinib.The expression of MBD2 was elevated by lenvatinib.Conclusion:The expression of circ KCNN2 is down regulated in HCC and related with short relapse free survival.Up regulation of circ KCNN2 inhibited the proliferation and migration of HCC,and arrest cell cycle in G0/G1 phase;down regulation of circ KCNN2 promoted the proliferation and migration of HCC,and arrest cell cycle in S phase.circ KCNN2 sponge miR-520c-3p,partially through miR-520c-3p/MBD2 signaling pathway.circ KCNN2coordinated with lenvatinib to inhibit tumor groth.Our study demonstrated that circ KCNN2 plays an anti-tumor role in HCC.circ KCNN2 is a candidate for predicting HCC recurrence and a potential target for therapies of HCC.