The Role And Molecular Regulatory Mechanism Of Circular RNA CircVRK1 In The Malignant Progression Of Colorectal Cancer

1.Background & ObjectiveAccording to data released by the International Agency for Research on Cancer(IARC)of the World Health Organization(WHO)in 2020,colorectal cancer(CRC)ranked third in the world in terms of new cases and second in terms of deaths among all types of cancer.In 2020,the number of new cases of CRC in China ranked second among all types of cancer,and the number of deaths ranked fifth.It can be seen that the burden of CRC is very heavy in both the world and China,seriously affecting the lives of patients and their families.The onset of CRC is relatively insidious,undergoing the evolutionary process of normal intestinal epithelial cells-adenoma-cancer.Hereditary CRC is closely related to genetic factors,while the cause of sporadic CRC is not very clear and is related to factors such as inflammation and lifestyle,which may be the result of the combined effect of genetic susceptibility and environmental exposure.Currently,there are no specific molecular diagnostic markers for CRC,and the treatment effect of advanced and metastatic CRC is not ideal.Finding specific diagnostic markers or therapeutic targets for CRC is of practical significance.The main strategy for CRC screening in China is a combination of fecal occult blood test and colonoscopy.However,due to the low acceptance rate of colonoscopy and the existence of blind spots in the examination method,and 95% of resected adenomas are meaningless,it is urgent to predict molecular markers for the evolution of adenomas to CRC to assist in precise screening with colonoscopy.As an important non-coding RNA,circRNA is more stable due to its circular structure and has tissue and spatiotemporal expression specificity in the occurrence and development of diseases.CircRNA can act as a sponge to competitively bind miRNA with m RNA,thus regulating target genes.It has been found that circRNA is involved in the migration,proliferation,invasion and other processes of CRC,and also plays an important regulatory role in many cancer signaling pathways.In our previous study,we found differential expression of 25 circRNAs,including circVRK1,between cancer and adjacent tissues in paired CRC samples.GSEA enrichment analysis revealed that circVRK1 is significantly involved in important signaling pathways,such as DNA damage repair,suggesting that circVRK1 may play an important role in the occurrence and development of CRC.Therefore,this study aims to clarify the anti-cancer or pro-cancer role of circVRK1 in the malignant progression of CRC through in vitro and in vivo functional experiments and public data analysis,and to explore its upstream and downstream molecular regulatory mechanisms,and to draw an epigenetic regulatory network associated with circVRK1,providing a scientific basis for exploring molecular markers or therapeutic targets that can be used for diagnosis in the evolution process of CRC.2.MethodsBased on the transcriptome sequencing and bioinformatics analysis of 51 paired CRC samples(normal/adenoma/cancer tissues)from 19 CRC patients in our previous study,we selected circVRK1(circ Base ID: hsa＿circ＿0000566),a significantly enriched circRNA in important signaling pathways with differential expression,for functional phenotypic and molecular mechanism studies :(1)The background expression of circVRK1 in normal human colorectal cell lines(FHC)and CRC cell lines(HCT116,SW620)was determined by using qRT-PCR.(2)Linear and circular primers were designed for circVRK1.The existence of circVRK1 circular structure was identified by RNase R enzyme digestion experiment,and the correctness of the sequence was verified by Sanger sequencing.The location of circVRK1 in CRC cells was determined by fluorescence in situ hybridization.(3)Lentivirus packaging technology was used to construct circVRK1 knockdown and overexpression of CRC stable cell lines,and the effects of knockdown and overexpression of circVRK1 on the invasion,migration,proliferation,cell cycle and apoptosis of CRC cells were studied.Through the subcutaneous tumorigenesis experiment in nude mice,the effect of knocking down circVRK1 on the subcutaneous tumorigenicity and growth of CRC cells in nude mice was analyzed.(4)To explore the downstream mechanism of circVRK1,we constructed a ceRNA network.Candidate miRNAs that potentially bind to circVRK1 were predicted using public databases(circ BANK,reg RNA2.0,Circular RNA interactome),and target genes that potentially bind to the miRNAs were predicted using public databases(miRDB,Target Scan,miRTar Base).Further screening of the predicted target genes was conducted through literature keyword searches.Candidate miRNAs were determined by reverse deduction using the screened target genes.(5)We used qRT-PCR to detect changes in the expression levels of downstream target genes after overexpression or knockdown of circVRK1.We also used RNA interference technology to knock down downstream target genes and investigate the effects of knockdown on functional phenotypes of CRC cells,such as proliferation and apoptosis.We performed RNA purification chromatin isolation(Ch IRP)experiments,dual-luciferase reporter gene experiments,and RNA pull-down assays to detect the interaction between circRNA and miRNA,as well as the interaction between miRNA and m RNA,and their binding sites.(6)We predicted the upstream molecules that regulate the expression of circVRK1 using public databases(miRDB)and validated their regulatory effects on circVRK1.We used RNA immunoprecipitation(RIP)experiments to demonstrate the binding between upstream genes and the pre-m RNA of circVRK1’s parental gene.We analyzed the expression of upstream and downstream genes in paired CRC and adjacent normal tissue samples using public databases and evaluated the predictive effect of these genes on the prognosis and survival of CRC patients.3.Results(1)Compared with normal colorectal cell lines(FHC),the expression of circVRK1 in CRC cell lines(HCT116,SW620)was significantly higher(P<0.05).(2)After the RNA of CRC cells was digested by RNase R enzyme,the relative expression level of the products amplified by circular primers was significantly higher than that of the products amplified by linear primers,indicating that circVRK1 was more tolerant to the digestion of RNase R enzyme than that of linear RNA,and the products were not amplified by circular primers in genomic DNA,but by linear primers,which further proved the circular characteristics of circVRK1.The Sanger sequencing results of the products amplified by the circular primer showed that the sequencing sequence was completely matched with the sequence of circVRK1 in the circ Base database.The results of fluorescence in situ hybridization showed that circVRK1 was mainly distributed in the cytoplasm of CRC,around the nucleus,and a small amount in the nucleus.(3)Compared with the control group,knockdown of circVRK1 enhanced the invasion,migration,and proliferation ability of CRC cells(P < 0.05),reduced the proportion of apoptotic cells(P < 0.05),and increased the proportion of cells in the S phase of the cell cycle(P < 0.05).In contrast,overexpression of circVRK1 weakened the invasion,migration,and proliferation ability of CRC cells(P < 0.05),increased the proportion of apoptotic cells(P < 0.05),and decreased the proportion of cells in the S phase of the cell cycle(P < 0.05).Knockdown of circVRK1 increased the growth ability of subcutaneous tumors in nude mice(P < 0.05).(4)Through public database and literature searches,we screened six candidate miRNAs(hsa-miR-583,hsa-miR-612,hsa-miR-558,hsa-miR-578,hsa-miR-615-5p,hsa-miR-1208)that potentially bind to circVRK1,as well as 15 candidate target genes(RASAL2,DDX5,PNN,POLE3,TFAP4,NEDD8,PLAGL2,JAK1,WNT5 A,TP63,YTHDF1,TP53,APC2,DR1,HMGB2)that potentially bind to the miRNAs.(5)Preliminary screening was conducted based on public database and literature screening,as well as qRT-PCR to verify the relative expression level of downstream target genes.The physical combination of circVRK1 and hsa-miR-583 was confirmed by chirp experiment,dual luciferase reporter gene experiment,and RNA pull down technology.At the same time,the qRT-PCR detection found that after knocking down or overexpressing circVRK1,only RASAL2 expression level of candidate downstream genes showed a consistent trend of decreasing or increasing.The dual luciferase reporter gene experiment and RNA pull down technology proved the physical combination of hsa-miR-583 and RASAL2.After knocking down RASAL2,the proliferation ability of CRC cells increased and apoptosis decreased(P<0.05).(6)According to miRDB database predictions,there are six potential binding sites between EIF4A3 and the pre-m RNA of circVRK1’s parental gene VRK1.RIP experiments confirmed their physical binding.Knockdown or overexpression of EIF4A3 resulted in a decrease or increase in the relative expression level of circVRK1,respectively(P < 0.05).According to the data analysis results from the public database GSCA,the expression of EIF4A3 and RASAL2 in CRC tissue was higher than that in adjacent normal tissue in paired samples(P < 0.05).According to the analysis results of the human protein atlas,GEPIA,and TCGA public databases,patients with high expression of EIF4A3 in CRC tissue have a longer prognosis and survival period.4.ConclusionThe circular RNA circVRK1,regulated by EIF4A3,inhibits the invasion,migration,and proliferation of CRC cells and promotes CRC cell apoptosis through the circVRK1/hsa-miR-583/RASAL2 axis.As a tumor suppressor,circVRK1 plays a regulatory role in the occurrence and development of CRC and has potential clinical applications as a diagnostic biomarker or therapeutic target in CRC.