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  • Although the amino acid sequences of CUL A and CUL


    Although the amino SGC707 sale sequences of CUL4A and CUL4B share 82% identity, the existing studies have shown that these two proteins do not have significant functional redundancy. Elevated expression of CUL4A has been observed in a variety of cancer cells, such as breast cancer, ovarian cancer, liver cancer, and medulloblastoma. CRL4A-based E3 ubiquitin ligase can recognize a series of substrate proteins, such as DDB2, XPC, UNG2, and SMUG1, as well as histones H2A, H3, and H4, which are widely involved in DNA damage and repair process. In addition, CRL4ACTD2 E3 ligase can recognize a number of cell-cycle regulatory proteins, such as CTD1, p21, and Chk1, as substrates to regulate cancer cell-cycle progression. Interestingly, CRL4A-based E3 ligases are also involved in the p53 and Wnt signaling pathways. However, the function of CUL4B in cancer remains unclear. Previous studies mainly focused on its function in cell development; for instance, CUL4B mutations lead to defects in nervous system and cardiac development.47, 48 In recent years, studies have also shown that CUL4B plays an important role in the process of tumorigenesis, and high expression of CUL4B has been reported in many cancers. For example, in esophageal cancer cells, CRL4B-based E3 ubiquitin ligase can ubiquitinate histone H2A and can coordinate with multi-comb inhibitory complex PRC2 to promote tumor cell proliferation and invasion. Silencing CUL4B in HeLa cells can lead to cyclin E aggregation and can cause cell-cycle arrest in S phase, thereby inhibiting cell proliferation. In prognosis studies of colorectal cancer patients, high expression of CUL4B was associated with a poor prognosis, but the molecular mechanism was unclear. Moreover, our previous results also showed that CUL4B is upregulated in human osteosarcoma cells, and its silencing could effectively inhibit osteosarcoma cell proliferation and induce apoptosis. In the current investigation, we identified the individual components of CRL4BDCAF13 E3 ligase through Y2H screening and LC-MS/MS methods, and we also identified the specific target of this E3 ligase: PTEN. By carefully analyzing the ubiquitination pattern of PTEN, we found that PTEN is modified by polyubiquitination through our in vitro and in vivo studies, indicating that more than one lysine (K) site is modified. Through amino acid sequence analysis, we found that PTEN contains a total of 33 K sites; however, these K sites are distributed everywhere, making it difficult to find the modified sites in our case. Some studies have noted that the C terminus of PTEN plays a crucial role in antagonizing NEDD4-1-mediated ubiquitination, and some K sites, such as K13 and K289, were reportedly modified in 293 cells. However, we cannot conclude which sites were modified in our system. We are currently mapping the modified sites through point mutagenesis. In recent years, many studies have found that a series of miRNAs, such as miR-17-5p, miR-26a, miR-34a, miR-133b, miR-150, and miR-370, exhibit abnormal expression in osteosarcoma cells. In the investigation into the CUL4B overexpression mechanism, we found that 9 out of 20 miRNAs that were predicted to target the 3′ UTR of CUL4B showed downregulation in osteosarcoma cells. However, we focused our studies on the function of miR-300, the most downregulated miRNA. In addition, there were 86 miRNAs, the expression of which we did not detect in osteosarcoma cells. Therefore, we cannot conclude that miR-300 alone regulates the expression of CUL4B. However, there is no doubt that miR-300 plays a very important role in the regulation of miR-300 expression. For the mechanism of miRNA downregulation, DNA methylation and histone modification are the two major causes that inhibit miRNA expression. In this study, we primarily detected the effects of the DNA methylation inhibitor AZA and the acetylation inhibitor TSA on miR-300 expression. AZA treatment, but not TSA treatment, significantly inhibited miR-300 expression, suggesting the major role of DNA methylation is regulating miR-300 expression in osteosarcoma cells. Interestingly, both ectopic expression of miR-300 and AZA treatment showed similar effects on CRL4BDCAF13 E3 ligase stability and PTEN ubiquitination patterns, as well as on cell proliferation. These consistent results suggest that DNA hypermethylation of the CpG island upstream of the miR-300 genomic locus is the primary reason for tumorigenesis in human osteosarcoma cells.