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  • br Previous reports have highlighted

    2022-04-29


    Previous reports have highlighted that the hepatocellular AKR7A could act as a potent chemo-preventative effector for acetaminophen-induced hepatotoxicity (Ahmed et al., 2011). Therefore, it is necessary to investigate AKR7A-mediated detoxification of AFB1 in liver cancer Ruxolitinib and so to uncover the biological effect of AKR7A on AFB1-associated lipid disorders in the HCC. In the present study, a hospital dossier of HCC patients was collected for further data assay. The plasma samples from the HCC patients were isolated for biochemical analysis. HCC sections and excision samples were harvested for the respective immunoassays and real-time PCR analyses. n cell culture, a human HCC strain of HepG2 was used to focus on the biological effects of AKR7A on AFB1-induced lipid dysmetabolism.
    2. Materials and methods 2.1 Clinical designs
    Twenty HCC patients were subjected to clinical diagnoses through medical biopsy and biochemical analyses, and the hospitalised records of patients were collected for the data analysis. All these patients were not prescribed any medicine before sample collection. Liver cancer and para-cancer (non-cancer) samples were surgically excised from the HCC patients who gave their informed consent between 2013 and 2016 at the Department of Hepatobiliary Surgery of The Eighth Affiliated Hospital of Guangxi Medical University (Guigang, China). In addition, the plasma samples were prepared from the blood in HCC patients for further biochemical tests. Other 10 non-cancer adults were recruited as controls. In a statement, the ethical guidelines in this study were followed by the Declaration of Helsinki (Zhou et al., 2016).
    2.2 Determination of the serological parameters and AFB1 contents
    All the HCC patients’ serological parameters were determined using an automatic biochemical analyser (Hitachi, Japan) from the clinical laboratory of Guigang City People's Hospital. The AFB1 contents and other lipids-associated parameters in sera and liver tissues of the cancer patients and adult controls were calculated using the respective commercial ELISA kits (Hualian Technology, Wuhan, China; Shanghai Elisa Biotech, Shanghai, China), and the final data were produced from a standard curve.
    The human liver cancer cells (HepG2) were obtained from the Chinese Academy of Sciences Cell Resource Centre (Shanghai, China). The cells were cultured in DMEM-high glucose medium (Solarbio, Beijing, China), supplemented with 10% fetal bovine serum (Tianhang Biotechnology, Hangzhou, China) and 1% penicillin/streptomycin (Solarbio, Beijing, China), in a 5% CO2 incubator at 37˚C. After treatment of 8.1, 16.2 ng/ml AFB1 and co-treatment of apolipoprotein B (ApoB)-associated activator of perfluorooctane sulfonate (PFOS, 10 μM) for 72 hours, the supernatants and cells were harvested to conduct the respective biochemical tests and immunoassays (Su et al., 2016).
    2.4 Immunohistochemical and immunofluorescence staining
    The immunohistochemical steps were conducted as described before (Li et al., 2017a). In brief, 5 μm sections from the HCC and non-HCC tissues were subjected to rehydration and permeabilisation through the different concentrations of dimethylbenzene and ethyl alcohol. After being blocked with 5% BSA buffer (Beyotime Biotechnology, China), the hepatic sections and HepG2-coated plate were treated with diluted primary antibodies of AFP, p53, CD36, ApoB, S6K1, AKR7A (1:50, Fuzhou Maixin, China; 1:100, Cell Signaling Technology, USA; 1:100; Santa
    Cruz, USA) at 4℃ overnight, followed by the incubation with related secondary antibodies (1:200; Boster Technology, China) for 1 hour at 37℃. Chemical 3, 3’-diaminobenzidine (DAB) was applied as a developer, followed by the counterstaining with haematoxylin in the cell nucleus. All the sections were mounted and imaged under an optical microscope prior to computing positive cells and assaying data.
    As reported previously (Guo et al., 2017; Wu et al., 2017a), the paraffin-embedded hepatic samples were dewaxed, and HepG2-coated plates were fixed with 4% paraformaldehyde, before being blocked with 5% BSA buffer for 1 hour. And all the samples were incubated with primary antibodies of PCNA, CD36, S6K1, ApoB, AKR7A (1:100; Cell Signaling Technology, USA; 1:100; Santa ruz, USA) at 4°C overnight, and re-incubated with IgG H&L (Alexa Fluor 488 and 647) (1:200; Abcam, UK) for 1 hour. And then, DAPI (Abcam, UK) was used for the nuclear staining before imaging by use of an inverted fluorescence microscope.
    2.4 Real-time PCR assay
    The freshly-isolated HCC tissues were lysed in TRIzol reagent (Thermo Fisher Scientific, USA) to extract the total RNAs. The purified RNA was reverse transcribed to single-strand cDNA with the FastQuant real-time Kit (Tiangen Biotech, Beijing, China) following the manufacturer’s instructions. Fatty acid translocase (CD36) and S6 kinase beta-1 (S6K1) mRNA levels were determined using the quantitative reverse transcription-polymerase chain reaction with specific primers for CD36 and S6K1 (Takara, Japan) using SYBR Green qPCR Master Mix (Applied Biosystems, UK).