The Mechanism Of Circular RNA Circ＿0001459 Promotes The Progression In Hepatocellular Carcinoma Via The MiR-6165/IGF1R Axis

As a common malignant tumor,primary liver cancer has seriously threatened human health.The incidence rate and mortality rate in China are the highest in the world and have been at a high level.Hepatocellular carcinoma（HCC）is the main type of primary liver cancer.The early onset symptoms of most patients are not obvious or only show atypical symptoms.When there are obvious symptoms such as liver pain and hepatomegaly,it means that the patients have been in the middle and late stages of cancer with varying degrees of liver cirrhosis and lost the opportunity of radical surgery.At present,the mechanism of HCC occurrence and metastasis is not clear,so there is a lack of cure.Although great progress has been made in clinical diagnosis and treatment technology at this stage,the therapeutic effect and prognosis of HCC are not ideal.At present,even after radical resection,the 5-year survival rate of HCC patients is still poor,mainly due to the high postoperative recurrence rate and metastasis rate.Therefore,it is of great significance to explore the molecular mechanism of HCC occurrence and metastasis and find new diagnostic and therapeutic targets.Circular RNA（circ RNA）widely exists in human normal tissues,cancer tissues and plasma.Moreover,more and more research reports show that circ RNA has a special regulatory role in the occurrence and development of tumors,which is expected to become a new biomarker for clinical diagnosis and prognosis,and provides new insights for the treatment of tumors.Therefore,studying the expression and biological function of circ RNA in HCC has important scientific significance and potential clinical application value.In this study,circ RNA sequencing was used to identify differentially expressed circ RNAs in HCC tissues and adjacent normal tissues.circ₀001459,which was highly expressed in HCC tissues,was taken as the research object and explored its function and related molecular mechanism in the occurrence and development of HCC.This study intended to clarify the role of circ₀001459 in the progression of HCC and provided a new theoretical and experimental basis for the diagnosis,prognosis and treatment of HCC.Part One: The expression of circ₀001459 in HCC tissues and its clinical significanceObjective: The expression profiles of circRNA in HCC tissues and corresponding adjacent tissues were detected,and the circ RNA with the highest expression was screened to study the relationship between its expression level and the clinical characteristics of HCC patients.Methods: The differential expression of circ RNAs in 4 paired samples of HCC tissues and corresponding adjacent tissues was analyzed by circ RNA sequencing.The 10 circ RNAs with the highest expression in the sequencing results were verified by q RT-PCR in another 10 paired samples of HCC tissues and corresponding adjacent tissues.According to the results of verification,the circ RNA with the most significant expression difference was selected as the target circ RNA.The sample size was expanded to 34 paired samples,and further q RT-PCR was used to detect the expression of the target circ RNA in HCC tissues and corresponding adjacent tissues.The expression level of the target circ RNA in the normal liver cell line LO2 and HCC cell lines（Huh7,Hep G2,SMMC7721,SK-HPE-1,and Hep3B）were detected by q RT-PCR.Nucleocytoplasmic separation experiment and FISH localization experiment of the target circ RNA were performed.The correlation between the expression level of the target circ RNA and the clinicopathological characteristics of HCC patients was statistically analyzed.Results: 183 differentially expressed circRNAs were screened by circRNA sequencing（fold change > 1.5,P < 0.05）,of which 94 circ RNAs were upregulated and 89 circ RNAs were downregulated in HCC.The circ₀001459 with the most significant upregulated expression was selected as the research target after further detection and screening by q RT-PCR.Compared with the normal liver cell line LO2,circ₀001459 was significantly higher expressed in the HCC cell lines（Huh7,Hep G2,SMMC7721,SK-HPE-1,and Hep3B）.Nucleocytoplasmic separation experiment and FISH localization experiment showed that circ₀001459 was mainly located in the cytoplasm.HCC patients with high expression level of circ₀001459 had higher TNM stage.Conclusion: Compared with the corresponding adjacent tissues,circ₀001459 was highly expressed in HCC tissues.Compared with the normal liver cell line LO2,circ₀001459 was highly expressed in HCC cell lines.The high expression of circ₀001459 may be related to the degree of differentiation of HCC.Part Two: The effects of circ₀001459 on the biological functions of HCC cellsObjective: To explore the biological behavior effects of circ₀001459 on the proliferation, migration and invasion of HCC cells and the tumorigenicity of xenografts in nude mice.Methods: HepG2 and Huh7 cells stably silenced and overexpressed circ₀001459 were constructed by lentivirus.Cell Counting Kit-8（CCK-8）cell proliferation assay,plate clone formation assay,and Ethynyldeoxyuridine（Ed U）assay were used to detect cell proliferation.Flow cytometry was used to detect cell cycle changes.Cell scratch assay and Transwell assay were used for the detection of cell migration and invasion ability.Subcutaneous tumorigenesis assay in nude mice was to detect the role of silenced circ₀001459 on transplantation tumor growth in vivo.Results: Compared with the control group,the absorbance of cells in the silenced circ₀001459 group was decreased.The ability of colony formation was weakened.The percentage of Ed Upositive cells was decreased.The proportion of cells in G1 phase was increased,but the proportion of cells in S phase was decreased.Cell migration and invasion abilities were significantly inhibited.Compared with the control group,the absorbance of cells in the overexpression circ₀001459 group was increased.The ability of colony formation was enhanced.The percentage of Ed U-positive cells was increased.The proportion of cells in G1 phase was decreased,but the proportion of cells in S phase was increased.Cell migration and invasion abilities were significantly enhanced.The results of animal experiments showed that the average volume and weight of subcutaneous tumors in nude mice in the silenced circ₀001459 group were lower than those in the control group.Conclusion: circ₀001459 can accelerate HCC cell cycle progression and promote HCC cell proliferation,migration and invasion in vitro.Silenced circ₀001459 in animal experiments can inhibit tumor growth.Part Three: The circ₀001459/mi R-6165/IGF1 R axis promotes the molecular mechanism of HCC progressionObjective: To investigate and verify the mi RNA adsorbed by circ₀001459 and its downstream target gene,and to clarify the mechanism of circ₀001459 affecting HCC progression through the ce RNA network.Methods: The mi RNAs that circ₀001459 might bind were analyzed by bioinformatics screening.Changes in the expression levels of previously screened mi RNAs were detected in Hep G2 and Huh7 cells which were silenced circ₀001459.Dual-luciferase reporter assay,RNA immunoprecipitation（RIP）and RNA pull-down assay further verified the targeting binding relationship between circ₀001459 and the target mi RNA.RNA fluorescence in situ hybridization（FISH）experiment was detected the co-localization of circ₀001459 and the target mi RNA in Hep G2 and Huh7 cells.The expression level of the target mi RNA in the normal liver cell line LO2 and HCC cell lines was detected by q RT-PCR.q RT-PCR was used to detect the expression of the target mi RNA in HCC tissues and corresponding adjacent tissues.The downstream target gene possibly regulated by the target mi RNA was analyzed by bioinformatics screening,and the binding relationship between the target mi RNA and the downstream target gene was verified by the dual-luciferase reporter experiment.The expression level of the downstream target gene in the normal liver cell line LO2 and HCC cell lines was detected by q RT-PCR.q RT-PCR was used to detect the expression of the downstream target gene in HCC tissues and corresponding adjacent tissues.The expression levels of circ₀001459 and the target mi RNA in Hep G2 and Huh7 cells were regulated,and the expression of downstream target gene and EMT-related molecular markers E-cadherin,N-cadherin and vimentin were detected by western blot.The recovery experiment of cell function further verified the above experimental results.Results: Among the mi RNAs that circ₀001459 might bind to by bioinformatics screening,mi R-6165 was most significantly altered in Huh7 and Hep G2 cells which were silenced circ₀001459.Compared with the normal liver cell line LO2,mi R-6165 was significantly downregulated in HCC cell lines.Compared with the corresponding adjacent tissues,the expression of IGF1 R was downregulated in HCC tissues.Dual-luciferase reporter experiments,RIP experiments and RNA pull-down experiments showed that circ₀001459 could target mi R-6165.RNA FISH experiments showed that circ₀001459 co-localized with mi R-6165 in Huh7 and Hep G2 cytoplasm.Through bioinformatics and dual-luciferase reporter experiments,IGF1 R was a downstream target gene regulated by mi R-6165,and the expression level of IGF1 R was regulated by circ₀001459 and mi R-6165.Compared with the normal liver cell line LO2,IGF1 R was significantly overexpressed in HCC cell lines.Compared with the corresponding adjacent tissues,the expression of IGF1 R was increased in HCC tissues.Western blot analysis showed that after circ₀001459 knockdown,the expression levels of IGF1 R,pAkt,Bcl-2,N-cadherin and vimentin were significantly decreased,and the expression levels of p27 and E-cadherin were markedly increased.In contrast,overexpression of circ₀001459 had the opposite effect on the expression of these proteins.The recovery experiments further showed that both mi R-6165 and IGF1 R could block circ₀001459-mediated changes in HCC cell proliferation,migration and invasion.Conclusion: circ₀001459 can act as a ce RNA and indirectly upregulate the expression level of IGF1 R by competitively binding to mi R-6165,thereby enhancing the proliferation,migration and invasion ability of HCC cells and promoting the progression of HCC.