Feng et alfurther demonstrate that genetic alterations in PolqSL genes could be observed in around 30% of TCGA breast cancer samples, indicating that inhibiting POLQ could be a promising therapeutic strategy for breast cancer treatment [21]. our study revealed that POLQ may participate in the development of HCC, depletion of which may be a promising treatment strategy for HCC. gene in mammalian genomes. POLQ is the defining enzyme for a pathway of DNA double-strand break (DSB) repair termed “alternative end-joining” (altEJ) or “theta-mediated end-joining” [7]. The enzyme GPI-1046 has several unique properties, including low fidelity and the ability to insert and extend past abasic sites and thymine glycollesions. It is important for maintaining genetic stability of cells and protecting cells from DNA breaks formed by ionizing radiation and topoisomerase inhibitors, breaks arising at stalled DNA replication forks, breaks introduced during diversification steps of the mammalian immune system, and DSB induced by CRISPR-Cas9 [8]. Three “insertion” sequence elements present in POLQ cannot be found in any other A-family DNA polymerase, and it has been proposed that they may lend some unique properties to POLQ [9]. Studies concerning the biological functions of POLQ in human diseases is rarely GPI-1046 seen. High POLQ expression was directly associated with defective DNA replication fork progression and chromosomal damage [10]. The upregulation of POLQ in breast cancer was deduced to play a great role in increasing intrinsic radio-sensitivity [11, 12]. Despite, to the best of our knowledge, the relationship between POLQ and HCC has KMT2D not been well documented. In this study, the role of POLQ in HCC was initially investigated by bioinformatics based on The Cancer Genome Atlas (TCGA) database and immunohistochemical staining of HCC tissues and normal tissues. Loss-of-function studies were carried out both in vitro and in vivo to explore the regulatory effects of POLQ on HCC development and progression. The underlying mechanism was explored through the application of an apoptosis-related signaling pathways antibody array analysis. Materials and methods Cell culture and cell infection Hepatocellular carcinoma cell lines Huh7, MHCC-97L, SK-HEP-1, BEL-7404 and HepG2 were purchased from BeNa Technology (Hangzhou, China) and cultured in a cell incubator with a humid condition at 37?C with 5% CO2. Cells were cultured in 90% RPMI-1640 (31800022, GIBCOA) supplemented GPI-1046 with 10% fetal bovine serum (FBS) (10099-141, GIBCO). The shPOLQ SK-HEP-1 and BEL-7404 cells and the control cells were established by knockdown lentiviral vector (1??108 TU/mL) infection with ENI.S and Polybrene. After culturing for 72?h, the infection efficiency was detected by GFP fluorescence. Clinical specimens To assess the protein POLQ (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_199420″,”term_id”:”1519242779″,”term_text”:”NM_199420″NM_199420) expression level, we analyzed 180 pathological sections via IHC assay. Endogenous peroxidase was deactivated by 3% H2O2 and non-specific-binding sites were blocked with 4% skim milk powder for 30?min. Sections were immersed GPI-1046 into antigen-retrieval solution for antigen retrieval at 95?C for 30?min. The sections were then incubated with primary antibody for POLQ protein overnight at 4?C. The corresponding secondary antibody was then added to incubate for 30?min at room temperature. All slides were stained DAB solution and counterstained with 10% Harris hematoxylin. IHC results were judged by positive cell score and staining color intensity score. POLQ expression positive cell scored as follows: negative: no positive signal; positive: GPI-1046 1 point, 0%? ?positive cells accounted for? ?25%; 2 points, 25%??positive cells proportion? ?50%; 3 points, 50%??positive cells? ?75%; 4 points, positive cells proportion??75%. The intensity was scored as: 0 point, no positive signal; 1 point, weak; 2 points, moderate; 3 points intense. Lentiviral vector construction According to the principle of RNA interference sequence design, multiple 19C21 nt were designed based on the POLQ template RNA interferes with the target sequence. After the evaluation and determination of the design.