| Background:Hepatocellular carcinoma(HCC)is well known as a malignant tumor of the digestive system.Although there are numerous measures and programs for the treatment of HCC,more than half of the patients are still in the middle and late stage and terminal stage when they are diagnosed,accompanied by intrahepatic and extrahepatic metastasis,involving other important tissues and organs.Due to the missed period of excellent diagnosis and treatment,the mortality rate of HCC patients is high and the prognosis is poor.Therefore,it is urgent to develop new targets for HCC diagnosis and treatment.Studies have shown that m6 A methylation(m6A)plays an important role in tumors and related diseases with a variety of mechanisms.Long noncoding RNA(Lnc RNA)acts as a regulatory factor on related genes and regulates their expression,and plays an important role in inhibiting the growth of tumor cells.Dachshund homolog 1(DACH1)plays the role of tumor suppressor gene in many tumor diseases,and also shows a significant influence in the development of related diseases.Therefore,from the perspective of epigenetics,the prognostic significance and immune correlation of m6A-associated Lnc RNA and DACH1 on HCC were studied.Objective:Based on the HCC genome data and its related clinical characteristic data in the human gene database,the scientific calculation methods of informatics and statistics were used,Combined with RT-q PCR,WB,scratch experiments and IHCrelated experiments,the effects of m6A-associated Lnc RNA and DACH1 on HCC prognosis and immune infiltration were explored,in order to provide valuable guidance for the diagnosis and treatment of HCC.Methods:The gene data of HCC were obtained from TCGA,and the Lnc RNAs associated with m6 A was determined by co-expression network.The prognostic risk model was constructed by univariate and multivariate survival analysis and LASSO regression analysis,and the independence and accuracy of risk score were evaluated.The relationship between DACH1 and m6 A associated Lnc RNAs,and the relationship between risk score,DACH1 and prognosis and immune infiltration were analyzed.The expression and distribution of DACH1 in HCC was discussed by RT-q PCR,WB,scratch and IHC related experiments.Results : It was determined that 56 Lnc RNAs associated with m6 A were significantly correlated with the overall survival of patients with HCC.There was a significant difference in the expression of 56 m6A-associated Lnc RNAs between HCC and normal tissues.The levels of immune cell infiltration were significantly different in the 56 Lnc RNAs(m6A-related)clusters.Compared with HCC tissues,the expression level of DACH1 in adjacent normal tissues was significantly upregulated,and there was no significant difference in DACH1 expression in the group,and the expression of DACH1 was closely related to the expression level of most of the 56 Lnc RNAs(m6Arelated).GSEA identified two signal pathways,namely m TOR and P53 signal pathways.Eleven Lnc RNAs associated with m6 A,namely AC027097.1,LINC01138,LINC01224,DDX11-AS1,MKLN1-AS,AL031985.3,AC098484.1,AL158166.1,POLH-AS1,WAC-AS1 and NRAV,were identified by univariate,multivariate and LASSO regression analysis.The calculated risk score can not only accurately predict the prognosis of HCC patients,but also serve as independent prognostic factors of HCC patients,but also related to the immune microenvironment of HCC.The expression of DACH1 in HCC was positively correlated with the infiltration of 6 kinds of immune cells related to prognosis.RT-q PCR showed that the expression of DACH1 m RNA in Hep G2 and Huh7 of HCC cells was lower than that of normal hepatocytes L-02.WB showed that the protein expression level of DACH1 in normal hepatocytes L-02 was significantly higher than that in Hep G2 and Huh7 HCC cells.WB showed that the expression level of DACH1 protein in normal tissues of 5 HCC patients was higher than that in HCC tissues.Scratch test showed that the migration distance of HCC cells treated with 10μmol/L 5-Aza-2d C on Hep G2 and Huh7 was significantly lower than that of the control group.The results of IHC showed that the positive staining of DACH1 protein was brown and distributed in the nucleus,and the expression of DACH1 in adjacent normal tissues was significantly higher than in HCC tissues.Conclusions:Using bioinformatics analysis,it is suggested that the risk score of m6A-related Lnc RNAs is independent and reliable in the prognosis of HCC patients.m6A-related Lnc RNAs may be involved in the regulation of HCC immune microenvironment through m TOR and P53 signal pathways,and has a certain regulatory effect on immune cell infiltration.The expression of DACH1 is closely related to the expression of m6A-associated Lnc RNAs associated with HCC prognosis and prognosis-related immune cells.The expression of DACH1 in normal hepatocytes,HCC cells,HCC tissues and adjacent normal tissues was verified by experiments,which was consistent with the predicted results in bioinformatics analysis.In a word,the above studies enrich the understanding of m6A-related Lnc RNAs and DACH1 in the occurrence,development and prognosis of HCC,and provide new references for its diagnosis and treatment. |
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