Abstract
RING finger protein 135 has an important role in the occurrence of many cancers; however its regulation and function of RNF135 in hepatocellular carcinoma remains unknown. The promoter methylation status and mRNA expression of RNF135 was evaluated by methylation-specific PCR, semi-quantitative RT-PCR, and real-time quantitative PCR in HCC tissues and cell lines, and further analyzed from The Cancer Genome Atlas database. Wound healing assay, transwell migration, cell viability, and colony formation assay were performed to investigate the function of RNF135. GSEA analysis, TIMER database, and ESTIMATE algorithm were used to decipher the associated pathway and immune infiltration. The survival analysis was applied to assess the prognostic value of RNF135. RNF135 expression was downregulated in HCC tissues and 5 of 8 HCC cell lines, and was negatively correlated with its promoter hypermethylation. Demethylating regent decitabine restored RNF135 expression on the cellular level. Knockdown of RNF135 expression enhanced the migration of HCC cells, while RNF135 overexpression and decitabine treatment repressed cell migration. Bioinformatics analysis and immunohistochemistry revealed a positive relationship between RNF135 expression and six immune cell infiltrates (B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells). Survival analysis disclosed that RNF135 hypermethylation is independently associated with poor clinical outcomes in HCC. Decreased RNF135 expression driven by promoter hypermethylation frequently occurred in HCC and associated with prognosis of HCC. RNF135 functions as a tumor suppressor and is involved in tumor immune microenvironment in HCC.
Highlights
Liver cancer is the sixth most common cancer worldwide with the fourth highest mortality [1]
To validate these results from the The Cancer Genome Atlas (TCGA) data, we detected the mRNA expression level of RING finger protein 135 (RNF135) in 23 pairs of Hepatocellular carcinoma (HCC) tissue samples, and 8 randomly selected samples were analyzed by methylation-specific PCR (MS-PCR)
Decitabine treatment restored RNF135 expression in 4 hypermethylated HCC cell lines (SNU449, Huh7, MHCC-97H, MHCC-97L) (Figure 2C). These results showed RNF135 expression was downregulated partially due to promoter hypermethylation in HCC cell lines, and demethylation agent could rescue its expression
Summary
Liver cancer is the sixth most common cancer worldwide with the fourth highest mortality [1]. Hepatocellular carcinoma (HCC) accounts for up to 90% of primary liver cancer [2]. The pathogenesis of HCC is complex and multifactorial. Risk factors, such as chronic hepatitis virus infection, alcohol, and aflatoxin, may lead to long-term inflammatory infiltration, metabolic imbalance, and cellular and molecular changes, which cause liver cirrhosis and promote HCC formation and development [6]. More questions about tumor heterogeneity, proliferation, invasion and metastasis, and the development of drug resistance remain unanswered by the heritable variation of genes. Epigenetic modifications are different from classical genetic changes, which regulate gene expression without DNA sequence alteration, could be implicated in cancer [8, 9]. DNA methylation is the most widely studied epigenetic modifications compared to histone modification, chromosome remodeling, and non-coding RNA [10]
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