| Background:Hepatocellular carcinoma(HCC)is one of the most common malignant tumour in China,the incidence of HCC ranks the fourth and the mortality rate ranks the third among all malignant tumors.The onset of HCC is hidden,and there are often no symptoms in the early stage.Many patients have entered the middle and late stages at the first diagnosis and have lost the opportunity for radical treatment.And metastasis,recurrence and drug resistance of HCC are more common than other solid tumors due to the high degree of heterogeneity.Therefore,it is particularly important to find new molecular markers in the occurrence and development of HCC and to explore the molecular mechanisms in the process of recurrence and metastasis.Nuclear enriched abundant transcript 1(NEAT1)is a recently discovered nuclear-restricted long noncoding RNAs(lnc RNA),which is located in the subnuclear structure and is called paranuclear plaque.Its expression is stimulated by many activated inflammasomes,mainly through the MAPK pathway to regulate the expression of cytokines and chemokines.Such as interleukin 6(IL-6)and chemokine 10(CXCL10),which participate in tumor immune response.Then they regulate the immune effect of tumors and promote the occurrence of tumors.NEAT1,as an oncogene,has been reported to be involved in the occurrence and development of many tumors,including lung cancer,colorectal cancer,gastric cancer,and esophageal cancer.The mechanism of competing endogenous RNA(ceRNA)is one of the important mechanisms for lnc RNA to play a regulatory role.Some lnc RNA and m RNA have miRNA response element(MRE),which can act as a molecular sponge to adsorb miRNA.Since the role of NEAT1 as ceRNA in the development of HCC has not been studied in depth,the role of NEAT1 as ceRNA in the development and metastasis of HCC will be further discussed here.Objective:The purpose of this study was to explore the following questions:1.To determine whether NEAT1 has ceRNA characteristics,the miRNAs which may bind to NEAT1 were screened;2.The expression levels of NEAT1 and miR-148a-3p in HCC samples were detected to analyze their relationship with clinicopathological characteristics and prognosis;3.Experiments in vivo and in vitro were performed to explore the effects of RREB1/NEAT1/miR-1247-5p on the proliferation,EMT and lung metastasis of HCC.Methods:1.Verification of NEAT1 characteristics: We downloaded the HCC miRNA data in the TCGA database,and used the online software cbioportal for analysis,and took log2FC> 1,P adj(corrected p value)<0.05 as the inclusion criteria to screen for miRNA with significantly low expression.We used the Star Base database to predict miRNAs that cloud potentially interact with NEAT1.The data obtained by TCGA and the data predicted by Star Base were intersected to obtain the miRNA for the final study.We used the Target Scan 7.2,Lnc Base v.2,miRDB online database to predict its potential target genes,and used Go EAST and DAVID online software to analyze the biological functions of the target genes.Cell lines that overexpressed and knocked down NEAT1 were constructed.The changes in miRNA expression were verified by q RT-PCR,and the binding of NEAT1 and screened miRNA was further verified by the dual luciferase reporter gene experiment.2.Verification of NEAT1/miR-148a-3p regulatory axis: The expression levels of NEAT1 and miR-148a-3p in HCC and paracarcinoma were analyzed by q RT-PCR.The expression levels of NEAT1 and miR-148a-3p in tissue microarray and cell lines were detected by FISH experiment.The relationship between NEAT1,miR-148a-3p expression and the prognosis of HCC patients was analyzed with Kaplan-Meier method.The relationship between NEAT1,miR-148a-3p and clinicopathological characteristics of HCC patients was analyzed by Chi-square test.The diagnostic value of NEAT1,miR-148a-3p for HCC was analyzed with ROC curve.The effects of NEAT1/miR-148a-3p axis on the proliferation,migration and invasion of HCC cell lines were verified by Ed U experiment,wound healing experiment,and Transwell experiment.3.Verification of the RREB1/NEAT1/miR-1247-5p regulatory axis: We used the GEPIA database to verify the correlation between the expression of RREB1 and NEAT1,and then used the q RT-PCR data to calculate the Pearson correlation coefficient.We analyzed the correlation between the RREB1,NEAT1,and miR-1247-5p in HCC tissues.We used JASPAR database to verify that RREB1 cloud bind to the promoter region of NEAT1,and the changes in the protein and m RNA levels of EMT marker proteins were analyzed by Western Blot and q RT-PCR after changing RREB1 and NEAT1.The changes in EMT marker proteins were analyzed by immunohistochemical.Lung metastasis was analyzed by HE staining.Results:1.The results of dual luciferase reporter gene experiments showed that miR-148a-3p,miR-1247-5p,miR-511-3p cloud significantly reduce the expression value of luciferase activity,indicating that NEAT1 cloud bind miR-148a-3p miR-1247-5p and miR-511-3p.The above indicates that NEAT1 may have the characteristics of ceRNA.2.According to the analysis of HCC clinical tissue samples,NEAT1 was overexpressed in HCC,while miR-148a-3p was low expression.Patients with overexpressed NEAT1 and low expression of miR-148a-3p had significantly worse OS,both it cloud be used as an indicator for prognosis of HCC.3.The results of in vivo and in vitro experiments indicated that overexpression of miR-148a-3p cloud inhibit the proliferation,migration and invasion of HCC cells,and the proliferation,migration and invasion behavior of liver cancer cells caused by overexpression of NEAT1 cloud be partially affected by miR-148a-3p offset.At the same time,in vivo and in vitro experiments had also verified that RREB1 cloud bind to the promoter region of NEAT1 gene.After RREB1 was knocked down,the expression level of NEAT1 decreased,while the expression level of miR-1247-5p and E-cadherin increased.The expression levels of N-cadherin,Vimentin and Twist decreased,and lung metastases were reduced.The biological effect of knocking down RREB1 cloud be offset by overexpression of NEAT1.The RREB1/NEAT1/miR-1247-5p axis regulated the epithelial-mesenchymal transition and lung metastasis of HCC cells.Conclusion:1.NEAT1 had the characteristics of ceRNA,which cloud adsorb a variety of related miRNAs.The characteristics cloud form a ceRNA regulatory network,and promote the proliferation,invasion and migration of HCC.NEAT1 cloud become a potential target for HCC treatment.2.The expression levels of NEAT1 and miR-148a-3p were important factors affecting OS and DFS in patients with HCC.High expression of NEAT1 was a risk factor,while high expression of miR-148a-3p was a protective factor.At the same time,overexpression of NEAT1 was also an independent risk factor affecting OS and DFS in patients with HCC.The expression of NEAT1 and miR-148a-3p had certain value in the diagnosis of HCC.Compared with miR-148a-3p,NEAT1 cloud have better diagnosis of HCC.3.NEAT1/miR-148a-3p axis cloud regulate the proliferation,migration and invasion of HCC cells,and this regulation axis may become a combined therapy target for HCC treatment.4.The RREB1/NEAT1/miR-1247-5p axis cloud regulate the epithelial-mesenchymal transition and lung metastasis of HCC cells.At the same time,RREB1 and NEAT1 cloud form a positive feedback loop to promote the epithelial-mesenchymal transition and lung metastasis of HCC cells.This regulatory axis may become the therapeutic target of HCC and lung metastasis. |
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