The Expression Of Circular RNA Hsa＿circ＿0000591 In Hepatocellular Carcinoma And Its Biological Function In Hepatocellular Carcinoma

Background and ObjectiveHepatocellular carcinoma（HCC,hereinafter referred to as liver cancer）is the most common type of primary liver cancer.It is a malignant and aggressive disease with a very poor prognosis.Usually accompanied by chronic underlying diseases,the main reasons are HBV infection,HCV virus infection,alcohol abuse and non-alcoholic fatty liver.The 5-year survival rate of liver cancer is 18%,which is the second most fatal tumor after pancreatic cancer.The worse situation occurs in Asian countries（such as China）,where the 5-year survival rate of patients is reported to be even lower than 12%.Make liver cancer become a severe medical burden on a global scale.The discovery of key molecules that affect the occurrence,development and prognosis of liver cancer is of great significance for targeted therapy and prognostic judgment of liver cancer.Circular RNA plays an important role in gene regulation in the body,and at the same time plays a key role in tumor diagnosis,treatment and prognosis judgment.Therefore,whether circular RNA plays an important role in diagnosis,treatment and prognosis of liver cancer is the focus of this study.The evaluation value of circular RNA as a molecular marker for the prognosis of liver cancer after surgery,to explore its effect on the proliferation,migration,invasion,circulation and apoptosis of liver cancer.Methods1.Screening and verification of circular RNAAccording to the principle of clinical baseline data matching,three postoperative pathological tissues were selected from each group of patients for high-throughput sequencing,and the expression of circular RNA in liver cancer tissues and adjacent tissues collected after liver cancer resection was detected,and Through trend analysis,the circular RNAs with obvious abnormal expression in the early postoperative recurrence were screened out.Repeated verification by qPCR（Quantitative Polymerase Chain Reaction,qPCR）technology resulted in consistent sequencing results.The structural characteristics,subcellular localization and structural stability of hsa_circ₀000591 were studied by Sanger sequencing,nuclear and cytoplasmic separation,PCR and other experiments.2.Identification and clinical significance of hsa_circ₀000591 in liver cancer The hsa_circ₀000591 molecule was studied in depth,human liver cancer tissue and its corresponding normal adjacent tissues were detected by qPCR technology,and the relative expression of hsa_circ₀000591 in liver cancer cell lines was analyzed.Finally,the expression level of hsa_circ₀000591 and the overall survival rate（OS,Overall Survival）of the patients were statistically analyzed,and whether there is a relationship between the expression level of hsa_circ₀000591 in the liver cancer tissue of the patients and clinicopathological indicators.3.The effect of hsa_circ₀000591 on the biological function of liver cancer cellsIn this paper,CCK-8 experiment,EDU experiment,plate clone formation experiment,scratch experiment,Transwell chamber invasion and migration experiment,flow cytometry and other methods were used to study the expression of silenced hsa_circ₀000591 gene in liver cancer cell lines after lentivirus infection.,And studied the effect of silencing hsa_circ₀000591 gene expression on hepatocarcinoma cell proliferation,migration,invasion,cycle,apoptosis and other biological functions through the above methods and techniques.Result1.Screening and verification of circular RNAWe confirmed that hsa_circ₀000591 is a circular RNA structure with good circular RNA stability.The subcellular localization results confirmed that hsa_circ₀000591 mainly exists in the cytoplasm.2.Identification and clinical significance of hsa_circ₀000591 in liver cancerWe have verified by qPCR experiments that the expression of hsa_circ₀000591 in 84 patients with liver cancer tissues is significantly higher than its corresponding adjacent tissues.According to the collected clinical data analysis,the hsa_circ₀000591 high expression group is related to the patient’s albumin,and glutamine.Transferase,cellulose,D-Dimer,CEA,CA199,ascites and tumor diameter are closely related.The RLog-rank test results show that there is a significant difference between the high expression level and low expression level of hsa_circ₀000591 in liver cancer tissues.This study suggests that the high expression level of hsa_circ₀000591 in cancer tissues of liver cancer patients is positively correlated with poor prognosis,and hsa_circ₀000591 has something to do The potential of effective biomarkers to assess the prognosis of liver cancer.The single factor and multivariate analysis of the prognostic risk factors of liver cancer found that the high expression of hsa_circ₀000591 was an independent risk factor for postoperative death and recurrence in patients with liver cancer.3.The effect of hsa_circ₀000591 on the biological function of liver cancer cellsThe experimental results show that silence hsa_circ₀000591 can significantly inhibit the in vitro proliferation of liver cancer cell lines.The results of scratch test,Transwell invasion,and migration experiments show that silencing hsa_circ₀000591 can significantly inhibit the migration and migration ability of liver cancer cells.The results of flow cytometry show that hsa circ 0000591 increases the number of liver cancer cells in G1/G2 after silencing.,Promote apoptosis.Conclusions（1）hsa_circ₀000591 is a stable ring structure and is mainly expressed in the cytoplasm.（2）hsa_circ₀000591 is highly expressed in liver cancer tissues than in normal adjacent tissues,and is related to the patient’s age,gender,albumin,transglutaminase,cellulose,D-Dimer,CEA,CA199,ascites,and tumor diameter Such clinical indicators are closely related.It can be used to predict and evaluate the overall survival rate after liver cancer surgery.（3）There is a positive correlation between hsa_circ₀000591 and the proliferation,invasion and migration capabilities of HCC cells.