N6-methyladenosine (m6A) RNA methylation regulators are associated with clinical prognosis in hepatocellular carcinoma

Gao Li, Yue Zhang, Xiaowei Du, Wanjun Li, Yanming Zhang, Tao Han, Zhendong Zheng


Background: N -methyladenosine (m A) methylation is a common class of RNA modification. Similar to DNA methylation, m6A methylation regulates most mRNA expressions. At present, most research has found that m6A methylation is related to tumorigenesis and development; however, there are few studies about hepatocellular carcinoma (HCC). This study aimed to analyze the expression level of m6A methylation regulators and their correlation on the clinical features in HCC.
Methods: A total of 13 m6A methylation regulators were evaluated. mRNA data and clinical information were obtained from the Cancer Genome Atlas (TCGA). The Wilcoxon test was utilized to analyze the differences between m6A RNA methylation regulators, and Pearson’s test was used to test the correlation between them. We constructed a tumor subgroup model based on the 13 molecules used for the analysis of the correlations with the clinical features. Two genes (ZC3H13 and YTHDF2) screened by Cox and LASSO regression were used to construct a tumor risk model for analyzing the correlations with clinical features. Finally, we verified the expression of the two molecules in liver cancer and adjacent tissues by Western blot and real-time polymerase chain reaction (PCR) (n=6). P<0.05 was considered statistically significant.
Results: Eleven of the 13 molecules were higher in the liver cancer tissues than the adjacent tissues (P<0.05), and most were significantly positively related. Two subgroup models were constructed. Subgroup 2 patients had higher levels of alpha-fetoprotein (AFP), while grade and the three-year survival were lower than subgroup 1 (49% vs. 77%) with significant differences (P<0.05). The risk model suggested that patients in the high-risk group showed high AFP levels, and the 3- and 5-year survival rates were lower than the low-risk group (3-year survival rate: 19% vs. 31%, 5-year survival rate: 12% vs. 17%). The Western blot test showed that the expression of YTHDF2 in the liver cancer tissues was greater than that in the precancerous tissues (P<0.05), while the expression of ZC3H13 was not significant. Real-time PCR showed that the expression of YTHDF2 mRNA in liver cancer tissues were higher than that in adjacent tissues (7.64±0.44 vs. 4.99±0.61, P=0.006), while the expression of ZC3H13 mRNA had no statistical difference (5.56±0.18 vs. 5.42±0.33, P>0.05). The results of the in vitro experiment were consistent with bioinformatic analysis.
Conclusions: The abnormal expression of m6A methylation regulators in the liver tissues suggest that m6A may play an important role in the development of HCC. Tumor models we constructed that could effectively predict the prognosis of patients, and the clinical correlation results were consistent with clinical practices. Our research is expected to provide a reference for the prognostic stratification and treatment strategy development of HCC.