OBJECTIVES: To investigate the role of PTEN-induced putative kinase 1 (PINK1) in regulating the viability, migration, and apoptosis of colorectal cancer (CRC) cells, and to explore its potential epigenetic mechanisms. METHODS: PINK1 was overex-pressed or knocked down in HCT116 and DLD1 CRC cell lines using lentiviral vectors, with efficiency verified by qRT-PCR and Western blotting. Cell proliferation, colony formation, migration, and apoptosis were assessed using CCK-8, colony formation, wound healing, Transwell, and Hoechst 33258 staining assays, respectively. Protein levels of apoptosis-related and histone modification-related markers were analyzed by Western blotting. Genome-wide chromatin accessibility was profiled using ATAC-seq. RESULTS: PINK1 expression was significantly downregulated at both mRNA and protein levels in CRC tissues compared to normal tissues. PINK1 overexpression inhibited cell prolifera-tion, colony formation, and migration in HCT116 and DLD1 cells (all P<0.05), whereas PINK1 knockdown promoted these malignant phenotypes (all P<0.05). PINK1 overex-pression induced apoptosis, associated with decreased levels of anti-apoptotic proteins (Mcl-1, Bcl-2, Bcl-xl) and increased pro-apoptotic Bax (all P<0.05), without altering p53 expression. Mechanistically, PINK1 overexpression reduced the expression of histone H3 lysine 9 trimethylation (H3K9me3) and histone H3 lysine 27 trimethylation (H3K27me3), and increased the expression of histone H3 lysine 9 acetylation (H3K9ac) and histone H3 lysine 27 acetylation (H3K27ac). It also downregulated key histone-modifying enzymes, including EZH2, EZH1, SUZ12, and histone deacetylase 3 (HDAC3) (all P<0.01). ATAC-seq revealed that PINK1 overexpression increased chromatin accessibility, particularly around transcription start sites. CONCLUSIONS PINK1 acts as a tumor suppressor in colorectal cancer by inhibiting proliferation and migration, promoting apoptosis, and remodeling the epigenetic landscape through altering histone modifications and enhancing chromatin accessibility.

Abstract: To develop novel live attenuated influenza vaccine, we explored the feasibility to attenuate influenza virus by codon deoptimization of NS1. According to the codon usage bias in influenza A virus, we designed and synthesized a condon-deoptimized NS gene by substituting codons of 110 amino acids in the NS1 gene of A/Puerto Rico/8/34(H1N1) with unpreferred synonymous codons. The influenza A virus with the codon deoptimized NS1 gene (deoNS virus) was rescued by reverse genetics. Plaque forming assay and virus growth curve showed that the growth of deoNS virus was reduced about 1000 times in MDCK cells compared to that of the wild-type virus. Intranasal inoculation with deoNS virus did not cause death or evident disease in infected BALB/c mice. Furthermore, the virus titer in the lungs of mice infected with deoNS virus was significantly lower (i.e. 100-1000 times) than that of wild-type virus. Our results indicated that influenza virus could be effectively attenuated by synonymous codon deoptimization of NS1 gene. This strategy will be useful to develop new attenuated candidates for the production of live attenuated influenza vaccines.
Animals ; Base Sequence ; Chick Embryo ; Codon ; genetics ; Influenza A virus ; genetics ; pathogenicity ; Influenza Vaccines ; immunology ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Orthomyxoviridae Infections ; immunology ; prevention & control ; Recombination, Genetic ; Vaccines, Attenuated ; immunology ; Viral Nonstructural Proteins ; genetics ; Virulence ; genetics