Abstract:

Background and purpose: Silent information regulator proteins (sirtuins, SIRT) are a class Ⅲ histone deacetylases with nicotinamide adenine dinucleotide (NAD⁺) as coenzyme. YME1 like 1 ATPase (YME1L1) is essential for the maintenance of mitochondrial morphology, function and plasticity. Optic atrophy 1 (OPA1) mainly mediates mitochondrial fusion. The aim of this study was to explore the expression of SIRT3 in the endocrine resistance of breast cancer, the relationship between SIRT3 and YME1L1 and OPA1, and the mechanism of SIRT3 in the endocrine resistance of breast cancer. Methods: 4-hydroxytamoxifen was used to induce tamoxifen-resistant MCF-7/TAM cells. cell counting kit-8 (CCK-8) was used to detect cell proliferation and verify drug resistance. The mitochondrial morphology was observed by transmission electron microscopy (TEM) and immunofluorescence staining. The expressions of SIRT3 and OPA1 were detected by real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) and Western blot. JC-1 staining was used to detect mitochondrial membrane potential, and dihydroethidium (DHE) staining was used to detect reactive oxygen species (ROS) to verify mitochondrial function. SIRT3 was knocked down in drug-resistant cells by RNA interference, and SIRT3 and YME1L1 wild type (WT), simulated acetylation state mutant (MUT K237Q), and simulated deacetylation state mutant (MUT K237R) were overexpressed in parental cells by overexpression plasmid. Immunoprecipitation assay (IP) and immunofluorescence (IF) were used to verify the interaction between SIRT3 and YME1L1. Results: RTFQ-PCR and Western blot results showed that SIRT3 gene expression and protein level was significantly higher in drug-resistant cells than in parental cells. Overexpression of SIRT3 in parental cells decreased the sensitivity of breast cancer cells to tamoxifen. Knockdown of SIRT3 in drug-resistant cells enhanced the sensitivity of drug-resistant cells to tamoxifen. DHE staining showed that the ROS level was lower in tamoxifen resistant cells than in parental cells at the same concentration. Transmission electron microscopy and fluorescence staining showed that the mitochondria of the drug-resistant cells were elongated compared with the parental cells. Western blot results showed that the expression level of L-OPA1 protein was higher in drug-resistant cells than in parental cells. Overexpression of SIRT3 in the parental cells resulted in enhanced mitochondrial function and longer mitochondrial morphology compared with the control cells. Western blot showed that the expression of L-OPA1 was upregulated. When SIRT3 was knocked down in drug-resistant cells, the opposite result was obtained. We further verified how SIRT3 regulated OPA1 protein, affected the morphology and function of mitochondria, and promoted drug resistance of breast cancer. Overexpression of YME1L1 (wild-type and mutant plasmids) in parental cells showed that overexpression of YME1L1 in the simulated deacetylation state resulted in similar results as overexpression of SIRT3, and overexpression of YME1L1 in the acetylated state resulted in similar results as knockdown of SIRT3. IP assay confirmed the interaction between SIRT3 and YME1L1 in breast cancer cells. The acetylation level of YME1L1 was different at different SIRT3 expression levels. IF assay showed that YME1L1 was co-localized with SIRT3 in MCF-7 cells. Conclusion: SIRT3 is highly expressed in tamoxifen-resistant breast cancer cells. SIRT3 upregulates L-OPA1 expression by deacetylating YME1L1, thereby promoting mitochondrial fusion and enhancing mitochondrial function, and promotes tamoxifen resistance in breast cancer.

Key words: Breast cancer, Endocrine resistance, SIRT3, YME1L1, OPA1