Circular RNA Hsa_circ₀001306 Functions As A Competing Endogenous RNA To Regulate FBXW7 Expression By Sponging MiR-527 In Hepatocellular Carcinoma

Hepatocellular carcinoma?HCC?is one of the most common types of primary liver cancerr worldwide,accounting for about 75-85%.The early symptoms of hepatocellular carcinoma are not obvious.The rate of 5-year survival is still low in HCC patients.At present,the main treatment is the comprehensive treatment based on surgery.However,due to the lack of better biological markers for early diagnosis,a large number of patients have lost the opportunity of surgery when they are found to have hepatocellular carcinoma.Therefore,the search for new biomarkers and therapeutic targets have become urgent clinical needs.As a newly discovered non-coding RNA,circRNA is widely found in biology.Unlike the canonical linear RNAs,circRNAs form a covalently closed continuous loop structure with neither 50 caps nor 30 polyadenylated tails,which makes them more stable than linear RNAs.There is a growing body of literature showing that circRNAs are differentially expressed in different tissues.Besides,circRNAs can act as endogenous competitors of mRNA to regulating miRNAs for biological functions.These evidences indicate that circRNA plays an important biological role in the life process.In this study,we found a HCC related circRNA,hsa_circ₀001306,which was significantly downregulated in HCC specimens.The expression of hsa_circ₀001306 is closely related to tumor size.In addition,we found that hsa_circ₀001306 may act as a ceRNA of miR-527,thereby reducing levels of its endogenous targets,f-box containing 7,and WD repeat domain?FBXW7?.Therefore,hsa_circ₀001306 may be a new target for HCC.Part I The expression level and role of hsa_circ₀001306 in HCCObjective:Study on the expression level and role of hsa_circ₀001306 in HCC.Methods:We firstly examined differentially expressed circRNAs in eight HCC and paired normal tissues obtained from the first hospital affiliated to suzhou university by high-throughput RNA sequencing.The downregulation of hsa_circ₀001306 was identified and further verified by qRT-PCR using 50 HCC specimens and paired adjacent normal tissues obtained from the First Affiliated Hospital of Soochow University.Secondly,we evaluated the effects of hsa_circ₀001306 on HCC cell proliferation,invasion,apoptosis,and cell cycle.Finally,we used an animal model to validate the in vitro experimental results.Results:?1?40 upregulated circRNAs and 56 downregulated circRNAs were identified between HCC and adjacent normal specimens by high throughput RNA sequencing.?2?The expression of hsa_circ₀001306 was significantly downregulated in HCC specimens compared with paired adjacent normal tissues.?3?The expression of hsa_circ₀001306 was closely related to tumor size.?4?The effects of hsa circ₀001306 on HCC cell proliferation,invasion,apoptosis,and cell cycle was evaluated.Conclusions:?1?The expression of hsa circ₀001306 was significantly downregulated in HCC specimens compared with paired adjacent normal tissues.The expression of hsa circ₀001306 was closely related to tumor size.?2?Knockdown of hsa_circ₀001306 promotes HCC proliferation in vitro and vivo.Knockdown of hsa_circ₀001306 promotes the invasion,reduces the ratios of Hep-1 and Hep-G2 cells in G0/G1 and impedes apoptosis.Part II The mechanism of low expression of hsa circ₀001306 promoting the progression of HCCObjective:Study on the expression level and role of hsa_circ₀001306 in HCC.Methods:Bioinformatics was used to analyze and predict the possible downstream miRNA and mRNA of hsa_circ₀001306.The downstream miRNA of hsa_circ₀001306 was verified by fluorescence in situ hybridization,double luciferase reporter gene technique and real-time quantitative PCR.The functional recovery experiment of hsa_circ₀001306 were used for verfying the results.Finally,Western blot was used to detect the protein expression level of the downstream target gene.Results:hsa_circ₀001306 functions as a ceRNA to sponge miR-527 in HCC cells.?2?hsa_circ₀001306 knockdown could downregulate F-box and WD repeat domain containing 7?FBXW7?,the target of miR-527,thereby promoting HCC cell proliferation and invasion.?3?Mechanistic studies indicated that hsa_circ₀001306 acts as a ceRNA for miR-527,which resulted in the reduction of its endogenous target,FBXW7.The negative relationship between hsa_circi₀001306 and miR-527 was validated by qRT-PCR and Spearman's rank correlation analysis in 50 pairs of specimens.?4?Interfering with hsa_circ₀001306 could promote subcutaneous tumor growth in vivo.Conclusions:Hsa_circ₀01306 is significantly downregulated in HCC,and the hsa_circ₀001306/miR-527/FBXW7 axis plays an important role in HCC progression.Our findings suggest a potential new biomarker for the diagnosis and treatment of hepatocellular carcinoma.