Knockdown of 5-methylcytosine RNA methyltransferase NOP2/sun RNA methyltransferase 5 in hepatocellular carcinoma cells affects their biological functions

Abstract

BACKGROUND

Hepatocellular carcinoma (HCC) is among the most prevalent cancers worldwide. Recent studies have indicated that 5-methylcytosine (m5c) RNA modifications play crucial roles in various biological processes through interactions with specific regulatory factors, including their involvement in the malignant progression of multiple tumors.

AIM

To examine the impact of NOP2/sun RNA methyltransferase 5 (NSUN5), an m5c methyltransferase, on the functional behavior of HCC cells.

METHODS

NSUN5 expression in HCC and normal liver tissues was analyzed using the Gene Expression Profiling Interactive Analysis bioinformatics tool. Human normal hepatocytes (LO2) and HCC cells were examined. The m5c levels in cellular extracts and culture supernatants were quantified using enzyme-linked immunosorbent assay, while NSUN5 levels were quantified using quantitative reverse transcription polymerase chain reaction (for mRNA levels) and Western blotting (for protein levels). Changes in NSUN5 expression following silencing of NSUN5 using transfection with short hairpin RNA were determined. Functional assays were used to evaluate HCC cell growth (Cell Counting Kit-8 and Colony Formation Assays), migration and invasion (Transwell and Wound-Healing Assays), and cell cycle and apoptosis (flow cytometry).

RESULTS

NSUN5 mRNA was significantly upregulated in HCC compared with normal liver tissues, with expression levels varying across different HCC stages (P < 0.05). HCC cells – HepG2, HEP3B, Huh-7, SMMC7721, and SK-HEP-1 – showed significantly higher NSUN5 mRNA expression levels than the normal human hepatocyte line LO2 (P < 0.05). Following short hairpin RNA-mediated NSUN5 silencing, HepG2 and SMMC7721 cells exhibited reduced proliferation and growth, decreased migration and invasion, and enhanced apoptotic levels (P < 0.05); however, they showed no significant changes in cell cycles (P > 0.05).

CONCLUSION

Both HCC tissues and cell models consistently demonstrated elevated NSNU5 mRNA and protein expression. Genetic inhibition of the m5c methyltransferase NSUN5 suppresses HCC cell growth, reduces invasiveness and migration, and induces apoptosis.

Keywords: RNA 5-methylcytosine; Methyltransferase; NOP2/sun RNA methyltransferase 5; Hepatocellular carcinoma; Biological behavior

Core Tip: Epigenetic modifications are dynamic, reversible, and heritable chemical changes to biomolecules that occur without altering the sequence of nuclear DNA. Among these, the RNA modification 5-methylcytosine has garnered increasing attention. However, the roles of RNA modification 5-methylcytosine modifications and their modifying enzymes in gene regulation and chromatin organization remain largely unclear. While previous studies have reported the involvement of NOP2/sun RNA methyltransferase 5 in human cancers, its comprehensive role in hepatocellular carcinoma remains unexplored. Therefore, this study evaluates the effects of NOP2/sun RNA methyltransferase 5 on hepatocellular carcinoma cell growth, migration, and invasion at the cellular level.