Investigation of the role of mitophagic genes and their genetic and epigenetic profile in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common liver cancer with the highest mortality rate and ranks 2nd in cancer-related deaths in the world. Under physiological circumstances, the activation of cell death pathways becomes imperative when the genetic material of a cell sustains irreparable damage. Mitophagy, a specific subtype of autophagy entrusted with the maintenance of mitochondrial quality control, emerges as a pivotal cellular process impeding the genesis of cancer within the cell. During the carcinogenesis process, disruptions in epigenetic mechanisms, which don't involve permanent changes in DNA sequences, are implicated in causing cancer. DNA methylation, a specific epigenetic mechanism, can lead to the development of cancer by hypermethylating tumor suppressor genes, causing their suppression and promoting carcinogenesis. In our investigation we aim to unraveling the roles of AMBRA1, FUNDC1, PGAM5, and LC3 genes in hepatocellular carcinoma (HCC) carcinogenesis through mitophagy and we examined samples from HepG2 and HUVEC cell lines. We utilized qRT-PCR, QMSP, and ELISA methods to determine the mRNA expression level, promoter methylation, and protein levels.When comparing tumor samples to normal ones for AMBRA1, 60% (3/5) of the samples showed an increase in mRNA expression alongside a decrease in protein levels. In 20% (1/5) of the samples showed expression decrease while 20% (1/5) showed no change. Hypermethylation of the promoter region of the AMBRA1 gene was detected in 40% (2/5) of tumor samples and 80% (4/5) of normal samples. While a statistically significant decrease in protein level was observed for FUNDC1 (p=0.024), an 80% (4/5) increase in expression and a 20% (1/5) decrease in mRNA expression was observed. When promoter hypermethylation was examined, FUNDC1 promoter hypermethylation was observed only in 20% (1/5) of the tumor samples. In tumor samples, both the protein level of PGAM5 decreased significantly (p = 0.0005) and an 80% (4/5) increase in PGAM5 expression was observed in its mRNA expression. Promoter methylation was detected in 40% (2/5) and 40% (2/5) of both tumor and normal samples. In 60% (3/5) of the tumor cells, there was an observed decrease in mRNA expression of the LC3 gene, whereas 20% (1/5) of samples showed elevated expression level and 20% (1/5), no expression change is noticed. The protein level in the tumor group increased compared to the normal group. Hypermethylation of the promoter region of the LC3 gene was detected in 40% (2/5) of tumor samples and 80% (4/5) of normal samples. Statistical significance was found between the protein and methylation levels of AMBRA1, FUNDC1, PGAM5 and LC3 (p<0.0001, p=0.001, p=0.002, p<0.0001, respectively). Studying the interactions of these mitophagic genes examined in vitro with other genes in larger patient cohorts can be used to create both therapeutic and biomarker strategies.