The Prognostic Value And Mechanism Of RNA M6A Modification And Its Regulatory Gene YTHDF2 In Hepatocellular Carcinoma

PART Ⅰ Analysis of differential expression and prognostic value of m6 A related regulators in hepatocellular carcinoma based on TCGA and ICGC databasesObjective: To investigate the expression of m6 A RNA methylation regulators in hepatocellular carcinoma and its relationship with clinicopathology and prognosis.Methods: First,using data from The Cancer Genome Atlas(TCGA)and the International Cancer Genome Consortium(ICGC)database,the expression levels of 13 major m6 A RNA methylation regulators in hepatocellular carcinoma(HCC)and normal liver tissues were compared.Secondly,based on the TCGA database,we explored the copy number variation(CNV)and single nucleotide polymorphism(SNP)mutations of regulators;and further analyzed the correlation between the expression of regulators and CNV.Thirdly,using univariate Cox with least absolute contraction and selection operator(Lasso)regression analysis,we constructed a risk model for the regulators that affect overall survival(OS)in both TCGA and ICGC and explored the correlation between the risk model and clinicopathological prognosis.Fourthly,further explored the relationship between the regulators used to construct the model and the clinicopathology of HCC;univariate and multivariate Cox regression were used to explore the prognostic value of each gene expression in HCC patients.Results:(1)Compared with normal tissues,the expression of 11 major m6A RNA methylation regulators in HCC tissues was significantly increased(P<0.001).(2)The difference in CNV between 13 regulators in HCC tissues and normal liver tissues reached statistical significance(P=0.000),and the SNP mutation rates of regulators in liver cancer tissues were all very low(all≤1.9%);(3)The four genes YTHDF1,YTHDF2,METL3 and KIAA1429 were selected to construct a risk model,which is related to clinicopathology and prognosis,and is an independent prognostic factor for evaluating OS in patients with HCC(P<0.001);(4)The expression of four genes was obviously correlated with pathological grade respectively(P<0.01);and YTHDF2 could be used as an independent prognostic factor for evaluating OS in HCC patients(P<0.001).Conclusion: m6 A RNA methylation regulators are key factors involved in the malignant progression of hepatocellular carcinoma,and have potential value in prediction and treatment decision-making.PART Ⅱ Analysis of expression and prognostic value of YTHDF1 and YTHDF2 in hepatocellular carcinomaObjective: To verify the expression of YTHDF1 and YTHDF2 in hepatocellular carcinoma and its correlation with clinicopathology and prognosis.Methods: Collected surgically resected cancer tissues and adjacent liver tissues from patients with hepatocellular carcinoma in the First Affiliated Hospital of Guangxi Medical University,38 fresh specimens and 30 tissue wax blocks were collected,q RT-PCR and immunohistochemistry(IHC)were performed to detect YTHDF1 and YTHDF2 expression at the m RNA and protein levels.The hepatocellular carcinoma tissue chip provided by Shanghai Xinchao Biotechnology Co.,Ltd.was used to detect YTHDF1 and YTHDF2 protein expression by IHC.Collected the clinical pathological information of the patient,and analyzed the correlation between protein expression and the clinicopathology of the patient by chi-square test.Kaplan-Meier method was used to explore the relationship between protein expression and patient’s OS and recurrence-free survival(RFS);univariate and multivariate Cox regression were used to explore the prognostic value of YTHDF2 protein expression in HCC patients.Results:(1)Compared with the adjacent tissues of liver cancer,q RT-PCR test showed that YTHDF2 mRNA was not significantly different between the two groups;IHC test results showed that the expression of YTHDF2 protein in the former was significantly higher than that in the latter(P < 0.05);its high expression was related to worse pathological grade,higher AFP expression,lower survival rate and higher recurrence rate(P < 0.05);it could be used as an independent prognostic factor for evaluating RFS(P<0.05).(2)Compared with the adjacent tissues of liver cancer,42 samples underwent q RT-PCR and 30 samples underwent IHC detection indicating that there was no significant difference in the expression of YTHDF1 between the two groups;IHC detection on tissue microarray indicated that the expression of YTHDF1 protein in the former was significantly higher than that in the latter and the difference between the two reached statistical significance(P < 0.05);its expression was not significantly correlated with clinicopathology and prognosis,except for agerelated(P<0.05).Conclusion: YTHDF2 may be used as a new biological biomarker for evaluating the prognosis of liver cancer.PART Ⅲ Study on the mechanism of YTHDF2 in promoting hepatocellular carcinomaObjective: To explore the mechanism of YTHDF2 in promoting hepatocellular carcinoma.Methods: Lentiviral infection was used to construct stable cell lines targeting silencing YTHDF2(sh YTHDF2)and non-targeting control(NTC)of human liver cancer Huh7 cells;q RT-PCR and Western blot were used to detect the silencing effect.The CCK8 and the cell clone formation test were used to detect the differences in cell proliferation and clonal formation ability between the sh YTHDF2 and NTC groups;the cell scratch test and the transwell test were used to detect the differences in cell migration ability between the sh YTHDF2 and NTC groups.Secondly,perform gene-set enrichment analysis(GSEA)of YTHDF2 to explore its potential pathways.Finally,the micro RNAs that target and regulate YTHDF2 was predicted through online networks.Results:(1)Successfully constructed a stable human hepatocarcinoma cell line Huh7 to target and silence YTHDF2;(2)The CCK8 experiment showed that compared with the NTC group,the cell proliferation ability of the sh YTHDF2 group was significantly weakened,and the difference between the two reached statistical significance(P < 0.05);(3)The cloning experiment indicated that compared with the NTC group,the cloning ability of the sh YTHDF2 group was significantly weakened,and the difference between the two reached statistical significance(P<0.05);(4)There was no significant difference in cell migration between the sh YTHDF2 group and the NTC group by scratch and transwell test;(5)The pathway enrichment results showed that the expression of YTHDF2 was positively correlated with multiple cancer signaling pathways;(6)According to online predictions,mi R-5195-3p may target YTHDF2.Conclusion: YTHDF2 may be targeted and regulated by mi R-5195-3p,and promote the progression of liver cancer by enhancing cell proliferation.