EHHADH is a key gene in fatty acid metabolism pathways in hepatocellular carcinoma: a transcriptomic analysis / 南方医科大学学报

OBJECTIVE@#To explore the driving gene of hepatocellular carcinoma (HCC) occurrence and progression and its potential as new therapeutic target of HCC.@*METHODS@#The transcriptome and genomic data of 858 HCC tissues and 493 adjacent tissues were obtained from TCGA, GEO, and ICGC databases. Gene Set Enrichment Analysis (GSEA) identified EHHADH (encoding enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase) as the hub gene in the significantly enriched differential pathways in HCC. The downregulation of EHHADH expression at the transcriptome level was found to correlate with TP53 mutation based on analysis of the TCGA- HCC dataset, and the mechanism by which TP53 mutation caused EHHADH downregulation was explored through correlation analysis. Analysis of the data from the Metascape database suggested that EHHADH was strongly correlated with the ferroptosis signaling pathway in HCC progression, and to verify this result, immunohistochemical staining was used to examine EHHADH expression in 30 HCC tissues and paired adjacent tissues.@*RESULTS@#All the 3 HCC datasets showed signficnatly lowered EHHADH expression in HCC tissues as compared with the adjacent tissues (P < 0.05) with a close correlation with the degree of hepatocyte de-differentiation (P < 0.01). The somatic landscape of HCC cohort in TCGA dataset showed that HCC patients had the highest genomic TP53 mutation rate. The transcriptomic level of PPARGC1A, the upstream gene of EHHADH, was significantly downregulated in HCC patients with TP53 mutation as compared with those without the mutation (P < 0.05), and was significantly correlated with EHHADH expression level. GO and KEGG enrichment analyses showed that EHHADH expression was significantly correlated with abnormal fatty acid metabolism in HCC. The immunohistochemical results showd that the expression level of EHHADH in HCC tissues was down-regulated, and its expression level was related to the degree of hepatocytes de-differentiation and the process of ferroptosis.@*CONCLUSION@#TP53 mutations may induce abnormal expression of PPARGC1A to cause downregulation of EHHADH expression in HCC. The low expression of EHHADH is closely associated with aggravation of de-differentiation and ferroptosis escape in HCC tissues, suggesting the potential of EHHADH as a therapeutic target for HCC.

[Construction of human mucosa oral epithelial cell lines overexpressing telomerase reverse transcriptase gene mediated by lentivirus].

OBJECTIVE: To construct a cell line of oral mucosa epithelial cells that stably express human telomerase reverse transcriptase (hTERT) by lentiviral vectors, approaches for the establishment of stable and efficient immortalized oral mucosa epithelial cell lines were explored. METHODS: Whole RNA was extracted from 293T cells. The hTERT gene was amplified by polymerase chain reaction (PCR) and cloned into the lentiviral vector as pLVX-puro-hTERT. The lentivirus particles were successfully packaged and used to infect primary oral epithelial cells. The positive cell clones were selected by puromycin. Finally, the expression of hTERT was examined by real-time fluorescent quantitative PCR (qRT-PCR) and Western blot analysis. RESULTS: The sequencing results confirmed the construction of the recombinant lentivirus pLVX-puro-hTERT. The morphology of infected cells was similar to that of normal oral mucosal epithelial cells, with a cobble stone-like appearance. The qRT-PCR and Western blot results showed that hTERT was overexpressed in infected cells compared with the normal group (P<0.05). CONCLUSIONS: The oral epithelial cell line with stable expression of hTERT was successfully established by the lentivirus, which provides an experimental basis for the establishment of a highly efficient and stable oral epithelial immortalized cell line.