Uvm graphpad prism 8
Then, the genetic ablation of AMBRA1 was confirmed by Sanger sequencing. Briefly, the 92.1 cells were transfected with CRISPR-CAS9-based sgRNA (PX330-AMBRA1-sgRNA), and monoclonal were chosen and detected by immunoblotting analysis. Sequences of the primers used in RT-qPCR Target geneĪMBRA1 knockout cell line was generated using the CRISPR-CAS9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) technique as previously described.
The plasmids of pET28a-His6-UB and pET28a-UBA1/UBCH5A/CUL4B/CCND1/USP2cc-His6 were kindly provided by Professor Weiwei Yang (Chinese Academy of Sciences, Shanghai, China). The plasmids containing AMBRA1 were amplified from the complementary DNA (cDNA) of human HEK293T cells and inserted into pCDH, pCADNA3.0-Flag, or pET28a vector. The single guide RNA (sgRNA) targeting AMBRA1 was designed using an online tool ( ), and the designed sgRNAs ( Table 2) were synthesized as oligos (Biosune), annealed, and inserted into a PX330 vector that was digested with BbsI, generating PX330-AMBRA1-sgRNA. The short hairpin RNA (shRNAs) targeting AMBRA1 or CCND1 were synthesized as oligos (Biosune, Shanghai, China), annealed, and inserted into the pLKO.1 vector that was digested with EcoRI and AgeI (NEB, USA), the specific sequences for shRNAs as shown in Table 1. When needed, cells were treated with cycloheximide (CHX 100 μg/mL, Selleck, China) for 6 h before harvesting and then subjected to other analyses. Plasmids were transiently transfected into cells using Lipofectamine 2000 (Life Technologies, USA) according to the manufacturer’s instructions, and the stably transfected cell lines were screened by puromycin (5 μg/mL) or hygromycin B (100 µg/mL). All these cells were cultured in Dulbecco's Modified Eagle Medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Gibco, USA) and 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco), in a 37☌ humidified atmosphere of 5% CO 2. The normal human pigment epithelial cell line ARPE-19 and three human UVM cell lines 92.1, MuM2B, and OMM1 were kindly gifted from Professor Shengfang Ge (Shanghai Jiao Tong University, China). 2 Materials and methods 2.1 Cell culture and transfection In this study, the function of AMBRA1 in UVM was extensively studied. The role of AMBRA1 in UVM has not been reported. The autophagy and beclin 1 regulator 1 (AMBRA1) was identified as the main regulator of cyclin D in several earlier studies, and it mediates ubiquitination and degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. ĭ-type cyclins are central regulators of the cell division cycle and are among the most frequently deregulated therapeutic targets in human cancer. BAP1 is a deubiquitinating enzyme, and its somatic and germline mutations have been found in UVM, malignant mesothelioma, and clear-cell renal cell carcinoma tumors. Ubiquitination includes a sequence of three enzymatic steps, and aberrations in the pathway can lead to tumor development and progression as observed in many cancer types, including UVM. Protein ubiquitination is a dynamic multifaceted posttranslational modification involved in nearly all aspects of eukaryotic biology, including cancer development and progression, cell survival and differentiation, innate and adaptive immunity, and individual development and sexual maturity.
Thus, new targets for melanoma treatment are urgently needed. Different inhibitors specifically target particular mechanisms of UVM occurrence and progression. Novel therapeutics targeting mutant guanine nucleotide-binding protein G(q) subunit alpha/guanine nucleotide-binding protein subunit alpha-11, regulators of G-protein signaling, inactivated BAP1, and immune-checkpoint blockade is emerging. Targeted therapy is widely used in cancer treatment due to its lesser side effects compared to other strategies. So far, no standard treatment exists to reduce the risk of metastasis and improve the overall survival of patients. The treatment of primary UVM includes phototherapy, radiotherapy, and local resection and reservation of enucleation in advanced cases. The ocular treatment usually aims to conserve the eyeball, preserving the visual performance. The causes of somatic mutations in UVM, including sun exposure, radiation, cigarette smoking, and certain chemicals, are known to increase the risk of mutations in genes that control cancers. The majority of UVM is thought to occur as sporadic, meaning everyone is at risk of developing UVM. Approximately, 50% of patients diagnosed with UVM will develop metastasis within 10 years from the primary intraocular tumor.
Uveal melanoma (UVM) is a melanoma of the eyes, which arises from melanocytes in the uvea, 90% of which involve the choroid, 6% of which are confined to the ciliary body, and 4% of which involve the iris.