| Background and Objective:Hepatocellular carcinoma(HCC)is one of the most common pathological types in primary liver cancer and one of the most common malignant tumors in the world.The characteristics of high recurrence and high metastasis of HCC make itself a major medical burden worldwide and a major bottleneck that affects the treatment effect.In the postoperative recurrence of HCC,because the prognosis of early recurrence is worse,exploring the key molecules in patients with early recurrence of HCC has important values for the targeted therapy and prognosis assessment.Circular RNA(circ RNA)plays an important role in gene regulation in vivo,and plays a key role in tumor diagnosis,treatment and prognosis judgment.Whether the circ RNA also has a crucial influence in the early recurrence of HCC is the focus of this study.We will focus on the evaluation of the value of circ RNA as a molecular marker for early recurrence of HCC after hepatectomy,and explore its impact on proliferation,migration,invasion,cycle,apoptosis and other aspects of HCC,and further investigate the potential mechanism,which will help in the preparation of individualized adjuvant therapy for hepatectomy,and provide new and more promising therapeutic targets.Methods:1.Screening and verification of circ RNA associated with early recurrence of HCC after hepatectomyAccording to the principle of clinical baseline data matching,3 cases of postoperative pathological tissue were selected from each group of patients for high-throughput sequencing to detect circ RNA in HCC tissues,nontumorous tissues of patients with early recurrence and HCC tissues of patients without early recurrence.For the expression situation,try to screen out the circ RNA that may be obviously abnormally expressed in the early recurrence of HCC through trend analysis,and use q PCR(Quantitative Polymerase Chain Reaction,q PCR)technology to further verify,in order to confirm the reliability of the sequencing results.2.Identification and clinical significance of circ＿0005218 in HCCSelect circ＿0005218 as a representative molecule to conduct in-depth exploration.Using PCR,gel electrophoresis,Sanger sequencing,ribonuclease R digestion and other experiments,the structural characteristics,stability and subcellular localization of circ＿0005218 were studied.Then,the relative expression levels of circ＿0005218 in human HCC tissues and corresponding nontumorous tissues,HCC cell lines and human normal liver cell line were detected by q PCR technology.Finally,statistically analyze the relationship between the expression level of circ＿0005218 and the prognosis of patients with early recurrence,overall survival and disease-free survival,and analyze the correlation between the clinical pathological indicators of patients and the expression level of circ＿0005218.3.The effect of circ＿0005218 on the biological function of HCC cellsLentiviral transfection technology was used to stably overexpress or silence circ＿0005218 in hepatocellular carcinoma cell lines,and then to detect circ＿0005218 expression by means of CCK-8 experiment,EDU experiment,plate clone formation experiment,scratch experiment,transwell experiment,flow cytometry,etc.The effect of changes in quantity on the biological functions of HCC cell proliferation,migration,invasion,cycle and apoptosis.4.Mechanism study of circ＿0005218 in HCCStarting from bioinformatics analysis,the dual luciferase experiment and RNA pulldown experiment were used to screen and verify the miRNA bound by circ＿0005218,and then to perform functional verification at the cell level,and finally dig deep into the m RNA regulated by miRNA downstream.Results:1.Screening and verification of circ RNA associated with early recurrence of HCC after hepatectomyIn the sequencing results,based on the analysis of the relationship between the expression of the HCC tissues with early recurrence,the nontumorous tissues with early recurrence,and the HCC tissues without early recurrence,and statistical correlation analysis,a total of 8 sets of circ RNA sets were obtained,of which two groups had statistics significance,only one group is more likely to have clinical significance.This group includes 57 differentially expressed circ RNAs,showing a trend of highest expression in HCC tissues with early recurrence,moderate expression in HCC tissues without early recurrence,and low expression in nontumorous tissues with early recurrence.Some molecules were selected for q PCR detection,which verified the reliability of the sequencing results.2.Identification and clinical significance of circ＿0005218 in HCCWe confirmed that circ＿0005218 is a circ RNA structure and has the characteristics of good stability of circ RNA.Subcellular localization confirmed that circ＿0005218 is mainly located in the cytoplasm.The expanded sample size test found that the expression level of circ＿0005218 in HCC tissues and HCC cell lines was significantly higher than that of the corresponding nontumorous tissues and normal liver cell lines,and once again confirmed that the expression level of circ＿0005218 in the early recurrence HCC tissues was significantly higher than the early nonrecurrence HCC tissues.Clinical data analysis shows that the high expression group of circ＿0005218 is closely related to the number of tumors,microvascular invasion,alpha-fetoprotein level and early recurrence.ROC curve found that circ＿0005218 has predictive value for early recurrence of HCC after operation.Log-rank test showed that the overall survival rate and disease-free survival rate of the circ＿0005218 high expression group and the low expression group were significantly different,indicating that the high expression of circ＿0005218 in patients with HCC is associated with poor prognosis,and can be used as a new effective biomarker for evaluating the prognosis of HCC.Univariate and multivariate analysis of prognostic risk factors showed that high expression of circ＿0005218 is an independent risk factor for postoperative death and recurrence of HCC patients after hepatectomy.3.The effect of circ＿0005218 on the biological function of HCC cellsOverexpression of circ＿0005218 significantly promoted the proliferation of HCC cell lines in vivo and in vitro.In contrast,circ＿0005218 inhibited the proliferation of HCC cell lines in vivo and in vitro significantly after silencing.Through scratch experiment,transwell migration and invasion experiments,we found that circ＿0005218 overexpression and silence significantly promoted and inhibited the migration and invasion ability of HCC cell lines,respectively.The results of flow cytometry showed that overexpression of circ＿0005218 caused the S-phase cells of HCC cells to increase significantly and inhibited apoptosis;silencing circ＿0005218 caused the G2/M-phase cells of HCC cells to increase significantly and promoted apoptosis.4.Mechanism study of circ＿0005218 in HCCmiR-31-5p was the target of circ＿0005218,and circ＿0005218 affected the activity of mir-31-5p by adsorption.miR-31-5p partially reversed the effect of intervention on the biological behavior of HCC cells after intervention circ＿0005218,and overexpression or silencing circ＿0005218 affected the expression of the downstream target molecule CDK1 of miR-31-5p.The above showed that the specific mechanism of circ＿0005218 regulating HCC was through inhibiting the activity of miR-31-5p and thereby up-regulating the expression of CDK1 ultimately affected the progression of HCC.Conclusions:(1)circ＿0005218 was highly expressed in HCC tissues and HCC cell lines,and was significantly higher in HCC tissues of patients with early recurrence than patients without early recurrence.(2)The expression of circ＿0005218 in tumor tissues of HCC patients was significantly related to the number of tumors,microvascular invasion,alpha-fetoprotein,and early recurrence.It can be used to predict the early recurrence of HCC after hepatectomy,as well as the evaluation of overall survival rate and disease-free survival rate.(3)circ＿0005218 was positively correlated with the ability of HCC cells to proliferate,migration,and invasion.(4)circ＿0005218 competitively bound miR-31-5p to regulate the important downstream molecule CDK1. |
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