CircRNA ACTN4 Facilitates The Progression Of Intrahepatic Cholangiocarcinoma And Its Molecular Mechanisms

Background and aimsIntrahepatic cholangiocarcinoma（ICC）is a highly malignant liver disease which is second only to hepatocellular carcinoma（HCC）in morbidity.It is characterized by insidious onset and high aggressiveness.Patients are often in the median and late stages of the disease when they are diagnosed,and as a result,vast majority of patients lost the opportunity of surgical treatment.So,the overall prognosis of ICC is rather poor.In recent decades,the incidence of ICC has been increasing worldwide.The main common risk factors of ICC include hepatolithiasis,primary sclerosing cholangitis,liver schistosomiasis,cirrhosis,choledochal cyst,Caroli disease,hepatitis B virus infection,hepatitis C virus infection,obesity and smoking.At present,there is still a lack of effective treatments for ICC,and surgical resection is the most effective therapy.However,the overall 5-year survival rate of ICC patients is less than 10%due to the low proportion of patients receiving surgical treatment and the high postoperative recurrence rate.CircRNAs（circRNAs）are a rich and conserved class of novel non-coding RNA molecules,which are generated by direct reverse splicing of precursor m RNAs or by exon-skipping circularization.Currently,most circRNAs can act as molecular sponges of intracellular mi RNAs to regulate downstream gene expression.In addition,a small part of circRNAs can interact with RNA-binding proteins（RBPs）to regulate the transcription of targeted genes.A very few circRNAs can even serve as templates for protein translation to produce small peptides and proteins.An increasing number of studies have shown that circRNAs play an important role in human diseases.Abnormally-expressed circRNAs have been found to be closely associated with disease progression and prognosis in a variety of cancers.However,few studies have reported the role and mechanism of circRNAs in the tumorigenesis and development of ICC.In view of this,this study is based on gene chip sequencing technology to find the significantly up-regulated circRNAs in ICC,and explore the roles of circRNAs in the malignant progression of ICC through a series of molecular biology techniques.Methods1.In this study,the clinicopathological data of 67 patients with ICC who underwent hepatectomy in the Eastern Hepatobiliary Surgery Hospital from March 2016 to August2017 were collected retrospectively.Meanwhile,intraoperative fresh frozen carcinoma tissues and their corresponding adjacent normal liver tissues were also obtained from the67 patients.2.Seven pairs of the above samples were randomly selected to analyze the difference in circRNA expression between cancer and paracancerous tissues according to the Human circRNA Array（V2 version）guidelines of Array Star company,and the target molecule with significantly high expression in ICC tumor tissues,namely circACTN4,was finally screened out.3.The optimal cut-off value of circACTN4 expression in the remaining 60 ICC patients was determined by receiver operating characteristic curve（ROC）,and then these patients were divided into two groups with high and low circACTN4 expression.The survival was analyzed by Kaplan-Meier（KM）method and log-rank test.Cox proportional hazards model was used to analyze the risk factors affecting the prognosis of patients.4.The effects of circACTN4 on the proliferation and invasion of intrahepatic cholangiocarcinoma cells in vitro were tested by CCK-8,colony formation,Transwell,and endothelial tube formation assays.To detect the effect of circACTN4 on the proliferation and metastasis of intrahepatic cholangiocarcinoma cells in vivo,we also established a subcutaneous model by subcutaneous injection of FRH0201 cells into the flanks of male BALB/c nude mice（4-6 weeks of age）,and a lung metastasis model of intrahepatic cholangiocarcinoma by injecting FRH0201 cells into the tail vein of 6-week-old male BALB/c nude mice.5.To find significantly changed genes after circACTN4 overexpression in ICC,we first transfected overexpressed plasmid into the RBE cells,and then used overexpression sequencing technology to obtain the gene profiles with significantly different expression.Then,GO and KEGG analyses were used to further narrow down the target genes.Finally,the relationship between the above target genes and circACTN4 was further explored through q RT-PCR,Western blot and Chromatin isolation by RNA purification（Ch IRP）experiments.6.In the Pull down experiment of circACTN4,we incubated intrahepatic cholangiocarcinoma cell lysates with pre-synthesized biotin labeled probes,and then precipitated the corresponding proteins in the above cell lysates by binding with streptavidin magnetic beads.Subsequently,we analyzed the precipitated proteins by liquid chromatograph-mass spectrometer（LC-MS）/mass spectrometer（MS）,followed by immunoprecipitation,RNA immunoprecipitation（RIP）,RNA electrophoretic mobility shift assay（EMSA）and finally screened the target RBP molecule.7.To find the downstream target genes of circACTN4 and RBP,we first used Ch IP-seq technology to explore the downstream genes of the RBP.Second,we overlapped the overexpression sequencing results of circACTN4 and the Ch IP-seq results of RBP to obtain the genes which were the common targets of both circACTN4 and RBP.8.To find targeted mi RNAs of circACTN4,we used three database,such as Circular RNA interact database（https://circinteractome.nia.nih.gov/index.html）,Starbase database（http://starbase.sysu.edu.cn/index.php）and Circbase database（http://circbase.org/）for prediction.After RT-PCR,Western blot,RIP and luciferase reporter gene assays,we finally identified the mi RNA binding to circACTN4.Results1.According to the results of circRNA microarray analysis,a total of 202 circRNAs were significantly up-regulated in ICC tissues（Fold change≥2.0,P value≤0.05）.We selected the top 7 circRNAs with the largest log₂（Fold change）values.Then,through functional verification in cancer cells and database comparison,hsa＿circ＿0050898 was finally determined as our target molecule.Since hsa＿circ＿0050898 was derived from its parent gene ACTN4,we named it circACTN4.2.High expression of circACTN4 was an independent risk factor for the prognosis of patients with ICC.KM curve analysis showed that high expression of circACTN4 in ICC was significantly associated with worse overall survival（OS）and higher postoperative tumor recurrence.3.Functional experiments,such as colony formation assay,Transwell assay and endothelial tube formation assay,showed that the high expression of circACTN4significantly enhanced the proliferation,metastasis and invasion of intrahepatic cholangiocarcinoma cells in vitro.The results of animal model also showed that the volume of subcutaneous tumor and the number of metastastic lung foci in mice were significantly reduced after we knockdown the circACTN4.4.Three times of overexpression sequencing of circACTN4 showed that 103 genes were differentially expressed in RBE cells.The GO analysis revealed that molecular function item of transcription factor activity and protein binding was significantly overrepresented.On the other hand,the KEGG pathway analysis showed that part genes related to Hippo and Wnt signaling pathways were enriched with circACTN4overexpression.Among them,FZD7 attracted our attention.PCR and Western blot confirmed that FZD7 was significantly upregulated after circACTN4 overexpression.Ch IRP analysis further demonstrated that circACTN4 could be enriched in the upstream promoter region of FZD7,suggesting that circACTN4 was closely related to the initiation of FZD7.5.Pull down experiments showed that 19 proteins could specifically bind to circACTN4.The subsequent protein-protein interaction（PPI）analysis revealed that YBX1was a focus because of its high degree of centrality and direct interaction with several other genes.Subsequently,a series of experiemts and tissue sample validations assays confirmed that circACTN4 could interact with YBX1 protein.6.By Ch IP-seq experiment,we found that the number of target genes that YBX1could bind to the upstream promoter region was 631.Then we overlopped the results of Ch IP-seq experiment results with the 103 genes from the overexpression sequencing of circACTN4,and finally only obtained FZD7 and LRP1.We further confirmed that the downstream target gene of YBX1 was FZD7 in ICC by Ch IP-q PCR because YBX1 could be enriched in the upstream promoter region of FZD7,and this region was consistent with the results of circACTN4.Finally,a series of experiments confirmed that circACTN4recruited YBX1 to stimulate FZD7 transcription.7.CircACTN4 overexpression sequencing results have indicated that YAP1 gene of Hippo pathway was significantly up-regulated in ICC.Therefore,we wanted to explore the possibility that circACTN4 acting as a molecular sponge to regulate a related mi RNA and its corresponding downstream YAP1 gene.So,we further predicted 12 candidate mi RNAs that could interact with both circACTN4 and YAP1 through Starbase and Circbase.Screened by TCGA database and functional verification experiments,we finally found that only mi R-424-5p could bind to circACTN4 and YAP1 simultaneously.Furthermore,the luciferase reporter gene assay and RIP assay also confirmed that circACTN4 could upregulate the expression of YAP1 gene by adsorbing mi R-424-5p in ICC.ConclusionsThe high expression of circACTN4 in ICC is closely associated with poor postoperative long-term prognosis.CircACTN4 promotes the proliferation,metastasis and invasion of ICC cells in vitro and in vivo.Mechanism studies have shown that circACTN4promotes the progress and deterioration of ICC by recruiting YBX1 to transcriptionally activate FZD7.In addition,circACTN4 can also act as a molecular sponge of mi R-424-5p to upregulate the expression of YAP1,and promote tumor progression.Our results suggest that circACTN4 can be used as a potential prognostic marker and therapeutic target for ICC.