| Hepatocellular carcinoma(HCC)is a very common primary malignant liver tumor.Despite much of our advances in HCC treatment,long-term survival in HCC remains low,mainly because most patients are only detected in advanced stages.Therefore,it is crucial to explore the molecular mechanisms of the occurrence,progression,invasion and metastasis of HCC and to find new biomarkers for the early diagnosis and treatment of HCC.Long non-coding RNA(long non-coding RNA,Lnc RNA)can play an important role in the development of diseases,many Lnc RNAs are differentially expressed in HCC,and these differentially expressed Lnc RNA are closely related to the development of HCC,and can be used as markers for liver cancer diagnosis,treatment response resistance,prediction of recurrence and prognosis.Several recent studies have shown that Lnc RNA can act as a competitive endogenous RNA(ce RNA)and play an important role in HCC.DSCAM-AS1 may play a role in the biological process of HCC.The effect of mi R-124 affects the proliferation of HCC and may interact with DSCAM-AS1 to affect the proliferation of HCC.Long non-coding RNA ADAM metalopeptidase and9 antisense RNA2(ADAMTS9-AS2)are involved in the development and development of various types of cancer including liver cancer.To explore the possible mechanism of Lnc RNA DSCAM-AS1 and Lnc RNA ADAMTS9-AS2 in HCC and provide theoretical support for the diagnosis and treatment of HCC.This study has the following four parts:Part One Differential expression and clinical significance of Lnc RNADSCAM-AS1 and mi R-124 in hepatocellular carcinomaObjective: To observe the expression level and differential significance of Lnc RNADSCAM-AS1 and mi R-124 in hepatocellular carcinoma.Methods:1.The expression levels of Lnc RNA DSCAM-AS1 and mi R-124 in HCC tissues were measured by quantitative polymerase chain reaction.2.The Pearson correlation coefficient was used for analyzing the expression correlation of DSCAM-AS1-and mi R-124 in HCC tissues.3.Differences in the expression levels of DSCAM-AS1 and mi R-124 in the HCC tissues was analyzed by One-way ANOVA and Tukey checkout.Results:1.The expression level of DSCAM-AS1 was significantly increased in HCC tissues compared with non-tumor tissues;instead,mi R-124 expression in HCC tissues was significantly lower in HCC tissues than in non-tumor tissues.2.A significant negative correlation occurred between DSCAM-AS1 expression levels and mi R-124 in HCC tissue samples,and no correlation between DSCAM-AS1 and mi R-124 in non-tumour tissues.3.With increasing clinical stage,the expression level of DSCAM-AS1 increased significantly,while mi R-124 was significantly decreased.HBV and HCV infection did not have significant effects on the expression levels of DSCAM-AS1 and mi R-124.Summary:1.DSCAM-AS1 and mi R-124 were differentially expressed in HCC tissues.2.DSCAM-AS1 and mi R-124 expression were inversely correlated in HCC tissues.3.Expression of DSCAM-AS1 and mi R-124 was affected by the clinical stage,but not by HBV and HCV infection.Part two Lnc RNA DSCAM-AS1 and mi R-124 negatively regulate and promote the proliferation of HCC cellsObjective: To study the interactions between DSCAM-AS1 and mi R-124 in HCC cells and its effects on HCC cell proliferation.Methods:1.SNU-449 and SNU-398 cells were transfected with the DSCAM-AS1 expression vector or mi R-124 mimic,and the interaction between DSCAMAS1 and mi R-124 was analyzed.2.The effect of the transfection on the proliferation of SNU-449 and SNU-398 cells was analyzed by the CCK-8 assay.3.The Inta RNA online program was applied to predict the direct interactions between DSCAM-AS1 and mi R-124.The RNA-RNA pulldown experiments confirmed a direct interaction between them.Results:1.Overexpression of DSCAM-AS1 significantly downregulated mi R-124,moreover,mi R-124 overexpression also downregulated DSCAMAS1..2.Overexpression of mi R-124 inhibited proliferation of SNU-449 and SNU-398 cells compared to normal controls,whereas overexpression of DSCAM-AS1 promoted proliferation of SNU-449 and SNU-398 cells.Overexpression of DSCAM-AS1 reduced the effect of mi R-124 overexpression.3.The predictions revealed that a strong base pairing may form between DSCAM-AS1 and mi R-124.The RNA-RNA pulldown experiments showed a significantly higher mi R-124 levels in the DSCAM-AS1 pull-down group when compared to the NC group,confirming the direct interaction between them.Summary:1.DSCAM-AS1 and mi R-124 negatively regulate each other in HCC cells.2.The interaction between DSCAM-AS1 and mi R-124 is involved in regulating the proliferation of HCC cells.3.There is a direct interaction between DSCAM-AS1 and mi R-124.Part three Mechanism of ce RNA regulation of Lnc RNA DSCAM-AS1 in hepatocellular carcinomaObjective: The regulatory network of lnc RNA-mi RNA-m RNA-pathway was constructed by bioinformatics methods,subsequently focusing on targeting m RNA and proliferation-related pathways.Methods:1.Download the level3 RNA-seq data for the UCSC TCGA LIHC,and then extract the clinical information.2.The m RNA of tumor(Tumor)and besides tumor(Normal)were differential analyzed using the classical Bayesian method provided by the limma package.3.The mi RNA-m RNA relationship predicted by mi RDB and Targetscan7.2 was integrated with the target genes that can be regulated by mi R-124-3p obtained by differentially expression.The obtained key genes were KEGG PATHWAY-analyzed using the DAVID 6.8 tool.4.The mi RNA-m RNA relationship pairs obtained above were screened for DSCAM-AS1-mi RNA-m RNA-pathway relationship pairs.Network construction was performed using the cytoscape 3.6.1 software.5.Expression and prognostic analysis of key m RNA in ce RNA based on the online tool GEPIA.Results:1.Differential expression analysis of the m RNA of TCGA was performed.Differentially expressed genes were selected using a threshold of |log FC |> 1 & adj.P <0.05,There are a total of 1283.2.The 78 target genes significantly regulated by mi R-124-3p in HCC.3.The ce RNA network includes 1 lnc RNA,1 mi RNA,and 78 m RNA.4.The m RNA in ce RNA is mainly involved in proliferation,apoptosis,and cancer-related pathways.For example,proliferation-related pathways include cell cycle pathway(CDK4,ANAPC7),PI3K-AKT pathway(CDK4,COL4A1,LAMC1,etc.),and MAPK pathway.5.ANAPC7 was highly expressed in HCC,negatively associated with overall survival(OS)and disease-free survival(DFS);CDK4 was highly expressed in HCC,negatively with OS and DFS.Summary:Lnc RNA DSCAM-AS1 / mi R-14-3p / ANAPC7 / CDK4 /cell cycle networks were constructed by bioinformatics methods to predict the possible mechanism of DSCAM-AS1 in HCC proliferative phenotype.Part four Mechanism of ce RNA regulation of Lnc RNA ADAMTS9-AS2 in hepatocellular carcinomaObjective: The regulatory network of lnc RNA-mi RNA-m RNA-pathway was constructed by bioinformatics methods,subsequently focusing on targeting m RNA and proliferation-related pathways.Methods:1.The gene expression RNA-seq expression matrix data(m RNA /lnc RNA)for hepatocellular carcinoma(LIHC)was downloaded from the UCSC Xena database.Mature mi RNA expression matrix data and sample clinical phenotype data and survival information were also downloaded.The RNA-seq data were annotated according to the gtf gene annotation files provided by GENCODE.The mi RNA data were transformed against the mature mi RNA ID using the mi RBase database.2.Differential expression analysis was performed by using the T test for ADAMTS9-AS2.Association analysis of ADAMTS9-AS2 and clinical factors was performed using the chi-square test.Expression values of ADAMTS9-AS2 and prognostic information of samples were calculated by Kaplansurvival analysis by the Logrank test.3.The m RNA and mi RNA of the tumor(Tumor)VS besides tumor(Normal)were also analyzed differently by using the classical Bayesian method provided by the limma package.4.Functional enrichment and pathway analysis were performed using the local GSEA software with the KEGG pathway gene set as the enrichment background.5.Pearson correlation coefficients for ADAMTS9-AS2 and m RNA were calculated separately,and Lnc RNA and m RNA relationship pairs were obtained by correlation test for coexpression analysis.6.Based on the m RNA and differential mi RNA in the co-expression network Lnc RNA A-m RNA,the final mi RNA-m RNA relationship pair was selected,combining the results of mi RWalk,mi Randa,the RNA22 and Targetscan.7.Network construction was performed using the mi RNA-m RNA-m R N A pairs and ADAMTS9-AS2-mi RNA relationships obtained above,and node connectivity(degree)analysis was performed using the Cytoscape plugin Cyto NCA.8.GO BP and KEGG pathway enrichment analysis of m RNA in the network using the online database metascape.Results:1.ADAMTS9-AS2 was not significantly different between normal and cancer groups,and analysis using subgroups showed that the expression of ADAMTS9-AS2 in HCC in male patients,G1,M0,T1,T3,T4,and tumor grade I and II compared with the normal group.2.The relationship between ADAMTS9-AS2 and prognosis was not significant in all cancer samples,so we drew K-M survival curves of ADAMTS9-AS2 in early samples(stage i-ii)and late samples(stage iii-iv),respectively.The results showed that the optimal cut-off group and the relationship between ADAMTS9-AS2 expression and prognosis in early and late samples were significant.3.Positive correlated KEGG pathway contained 36 and 8 negative correlated KEGG pathway,the pathway with positive correlation was hypertrophic cardiomyopathy,and the pathway with negative correlation was steroid metabolism pathway.4.A total of 124 ADAMTS9-AS2-m RNA co-expression relationship pairs were obtained,14 ADAMTS9-AS2-mi RNA relationship pairs,78 mi RNA-m RNA relationship pairs,and the ce RNA network included 1lnc RNA,9 mi RNA,and 11 m RNA.5.Functional enrichment analysis of the m RNA in the network yielded a total of 8 GO BP enrichment results,and the first-ranked enrichment results showed a negative regulation of cell-cell adhesion.Summary:Through the bioinformatics analysis,We predicted three ce RNA networks involving ADAMTS9-AS2: the ADAMTS9-AS2 /mi R-10b-5p / DUSP5 / MARK signaling pathway;ADAMTS9-AS2 /mi R-9-3p,mi R-500a-5p,mi R-501-5p,mi R-452-5p / LIFR / JAK-STAT signaling pathway;The ADAMTS9-AS2 / mi R-182-5p,and mi R-196b-5p /CXCL12 / NF-kappa B signaling pathway.Conclusion:1.DSCAM-AS1 and mi R-124 can mutually negatively-regulate their expression to regulate the proliferation of HCC cells.2.The Lnc RNA DSCAM-AS1 / mi R-14-3p / ANAPC7 / CDK4 / cell cycle networks were constructed by bioinformatics methods to predict the possible mechanism of action of DSCAM-AS1 on the proliferative phenotype of liver cancer.3.Through the bioinformatics analysis,We predicted three ce RNA networks involving ADAMTS9-AS2: the ADAMTS9-AS2 / mi R-10b-5p /DUSP5 / MARK signaling pathway;ADAMTS9-AS2 / mi R-9-3p,mi R-500a-5p,mi R-501-5p,mi R-452-5p / LIFR / JAK-STAT signaling pathway;The ADAMTS9-AS2 / mi R-182-5p,and mi R-196b-5p / CXCL12 /NF-kappa B signaling pathway. |
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