| Primary liver cancer is the third most common cause of cancer death worldwide,and hepatocellular carcinoma(HCC)is the most frequent type of primary liver cancer.The HCC patient’s 5-year survival rate is extremely low mainly because of its insidious onset,difficulty in early diagnosis,high degree of malignancy,high recurrence rate and high metastasis rate,making it a serious public health threat.CircularRNAs(circRNAs)are a class of non-codingRNAs that have attracted much attention in recent years.They are characterized by high stability and tissue-specific expression and high abundance.A large number of studies have shown that circRNA plays an important role in the disease progression of various diseases such as tumors.Although some circRNAs related to the occurrence and development of HCC have been found,the number of circRNAs is large,and many potential functional circRNAs have not been discovered yet.The present study used high throughputRNA sequencing(RNAseq)technology to screen differentially expressed circRNAs in HCC tissues and adjacent normal tissues,then a series of molecular and cell experiments were conducted to find circRNAs with important biological functions in HCC,and preliminary explore the mechanism of circRNAs in HCC.The purpose of this study is to provide a research basis for further research on the pathogenesis and progression of HCC,as well as the search for new diagnostic markers and therapeutic targets.This study is divided into the following four parts:Part I Screening of differentially expressed circRNAs in hepatocellular carcinoma tissuesObjective: To explore the expression profile of circRNAs in HCC tissues and adjacent normal tissues,and screening significantly differentially expressed circRNAs in HCC tissues and adjacent normal tissues.Methods: Five cases of newly diagnosed HCC patients who had not received anti-tumor therapy were selected.HCC tissues and adjacent normal tissues were collected during surgery.TotalRNA was extracted for theRNA-seq of circRNAs,and obtained the circRNAs expression profile.Significantly differentially expressed circRNAs should meet the following criteria: Fold change(FC)≥2,P<0.05.Results: A total of 23,217 circRNAs were detected in five pairs of HCC tissues and adjacent normal tissues,among which 114 circRNAs were significantly up-regulated and 144 circRNAs were significantly down-regulated.Conclusion: There are a large number of differentially expressed circRNAs in HCC tissues.Part Ⅱ Validation of differentially expressed circRNAs in HCC tissues and cell lines.Objective: To verify the expression level of candidate differentially expressed circRNAs in tissue samples and cell lines,analyze the relationship between the expression level of differentially expressed circRNAs and clinicopathological characteristics of HCC patients,and explore the diagnostic value of differentially expressed circRNAs in HCC patients.Methods: Among the top 20 significantly up-regulated circRNAs and the top 20 significantly down-regulated circRNAs obtained byRNA-seq,three upregulated and three down-regulated circRNAs were selected as candidate circRNAs.First,reverse primers were designed for six candidate circRNAs.The circular structures of circRNAs were verified by Ribonuclease R(RNase R)digestion test,and the specificity of the primers was verified by TA clone sequencing.Then,quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect the expression levels of candidate circRNAs in 71 paired HCC tissues and adjacent normal tissues,one normal liver cell line HL-7702 and six hepatocellular carcinoma cell lines(HCCLM3,MHCC97-H,MHCC97-L,Hugh-7,Hep G2,and Hep3B).The 71 HCC patients were divided into low expression group and high expression group according to the median expression level of each circRNAs in HCC tissue.The chi-square test was used to examine the relationship between the expression level of each circRNAs and clinicopathological characteristics.Receiver Operating Characteristic(ROC)curve was used to evaluate the diagnostic value of differentially expressed circRNAs in HCC.Results: The q RT-PCR results showed that circ GNAO1,circ RNF180,and circ MERTK were significantly downregulated in HCC tissues compared to their adjacent normal tissues(all P < 0.001).In contrast,circ SNX6 was significantly upregulated in HCC tissues compared to the adjacent normal tissues(P <0.05).However,circ RHOBTB3 and circ UGGT2 showed no statistical difference between HCC and adjacent normal tissues(P > 0.05).The expression levels of circ GNAO1 were significantly higher in HL-7702 cells than in HCCLM3,MHCC97-H,MHCC97-L,Hugh-7,Hep G2,and Hep3 B cells(all P < 0.01).However,we did not observe similar trends for circ RNF180,circ MERTK,and circ SNX6.The results of clinicopathological analysis showed that the microvascular invasion(MVI)incidence was significantly higher in the circ RNF180 low-expression group than in the circ RNF180 high-expression group(P=0.024).The results of ROC curve analyses showed that circ GNAO1,circ RNF180,and circ MERTK had good performance in discriminating HCC tissues from normal tissues(circ GNAO1: area under the curve [AUC] = 0.978,95% confidence interval [CI] 0.952–1.000,P < 0.001;circ RNF180: AUC = 0.903,95% CI 0.848–0.958,P < 0.001;and circ MERTK: AUC = 0.783,95% CI 0.705–0.862,P < 0.001).Conclusions: Circ GNAO1,circ RNF180,circ MERTK and circ SNX6 may be important circRNAs in HCC.Circ GNAO1,circ RNF180 and circ MERTK may serve as potential diagnostic markers for HCC.Part Ⅲ Effects of circ RNF180 on biological behavior of hepatocellular carcinoma cellsObjective: To investigate the effect of circ RNF180 on the biological behavior of hepatocellular carcinoma cells.Methods: Circ RNF180 overexpression plasmids were transfected into Hep G2 cells to generate a circ RNF180 overexpression cell line.The efficiency of circ RNF180 overexpression was verified by q RT-PCR.The Cell Counting Kit-8(CCK-8)experiment was used to detect the influence of circ RNF180 overexpression on the proliferation ability of HCC cells,and the clone formation experiment was used to detect the influence of circ RNF180 overexpression on the clone formation ability of HCC cells.Transwell migration and invasion assay was used to detect the effect of circ RNF180 overexpression on the migration and invasion ability of HCC cells.Results: The results of q RT-PCR showed that the expression level of circ RNF180 in circ RNF180 overexpression group was 3489.54±322.33,significantly higher than that of 1.00±0.31 in negative control(NC)group(P<0.01).The CCK-8 assay showed that the proliferation activity of circ RNF180 overexpression group was significantly lower than that of NC group(all P<0.05).The results of clone formation assay showed that the number of clones formed in circ RNF180 overexpression group was significantly less than that in NC group(P<0.01).Transwell migration and invasion assay showed that the number of cells migrated and invaded in circ RNF180 overexpression group was significantly lower than that in NC group(P<0.01).Conclusions: Circ RNF180 significantly inhibit the proliferation,migration and invasion of HCC cells,circ RNF180 may be a tumor suppressor in HCC.Part Ⅳ Bioinformatics prediction and preliminary exploration of the mechanism of differentially expressed circRNAsObjective: To construct a circRNAs-miRNAs-mRNAs regulatory network and preliminarily explore the molecular mechanism of differentially expressed circRNAs in HCC.Methods: Targeted miRNAs of circ GNAO1,circ RNF180,circ MERTK,and circSNX6 were predicted using the cancer-specific circRNA database(CSCD),circ Bank,and circRNA interactome(Circ Interactome).The Encyclopedia ofRNA Interactomes(ENCORI)was used to predict the expression levels of targeted miRNAs in HCC tissues and normal liver tissues.The mRNAs targeted by miRNAs were predicted by mi RWalk 2.0,and mRNAs differentially expressed in HCC and liver normal tissues were obtained by The Cancer Genome Atlas(TCGA)database.The circRNAs-miRNAs-mRNAs regulatory network was constructed using Cytoscape 3.60 software.The expression levels of hsa-mi R-182-5p and hsami R-1301-3p in 35 HCC tissues were detected by q RT-PCR.The correlation between the expression levels of circ GNAO1 and hsa-mi R-182-5p,and The correlation between the expression levels of circ NF180 and hsa-mi R-1301-3p in HCC tissues was examined by Pearson correlation analysis.Results: The miRNAs and mRNAs targeted by circ GNAO1,circ RNF180,circMERTK,and circ SNX6 were predicted by bioinformatics method,and 7 most likely targeted miRNAs and 386 most likely targeted mRNAs were obtained,thus a circRNAs-miRNAs-mRNAs regulatory network was constructed.The results of q RT-PCR showed that there was a significant negative correlation between the expression level of hsa-mi R-182-5p and circ GNAO1 in HCC tissues(P<0.01),however,there was no significant correlation between hsa-mi R-1301-3p and the expression level of circ RNF180 in HCC tissues.Conclusions: Circ GNAO1 may play a molecular regulatory role by targeting hsa-miR-182-5p. |
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