HiCoP, a simple and robust method for detecting interactions of regulatory regions.

BACKGROUND: Chromatin physical interactions provide essential information for understanding the regulation of cis-elements like enhancers, promoters, and insulators in cell development and differentiation. The Hi-C assay is a technique detecting chromatin structures of the whole genome, but not sensitive to interactions of regulatory elements. Several methods, like HiChIP, DNase-C, and OCEAN-C, have been developed for enriching interactions of regulatory regions, but all of them have some shortcomings. New simple, efficient, and robust methods are still in need for detecting interactions of regulatory regions. RESULTS: We developed a new, simple, and robust assay called CoP (Column Purified chromatin) for profiling of open chromatin regions by directly purifying fragmentized crosslinked chromatin with a DNA purification column. The accessible chromatin regions, including active enhancers, promoters, and insulators, were significantly enriched in CoP chromatin. The CoP-seq assay can efficiently detect open chromatin regions, especially active promoters, with a high signal-to-noise ratio. We integrated the CoP-seq and Hi-C technique (HiCoP) to detect interactions of accessible chromatin regions, which represent active cis-regulatory elements in cells. We observed that the HiCoP captured the peaks in the promoters-associated enhancer regions. HiCoP detected more promoter-enhancer (P-E), promoter-promoter (P-P), and enhancer-enhancer (E-E) interactions within 20 kb-5 Mb than Hi-C. Most of the loops identified by HiCoP are associated with the expressed genes. CONCLUSION: CoP assay can efficiently enrich open chromatin regions. When CoP assay was integrated with Hi-C assay, it provides a simple, robust, alternative technique for profiling accessible chromatin regions and chromatin conformation simultaneously.

STAG2 loss-of-function mutation induces PD-L1 expression in U2OS cells.

BACKGROUND: A tumor suppressor protein, stromal antigen 2 (STAG2), has recurrent mutations or loss of expression in many tumors including in bladder cancer, osteosarcoma (OS), and leukemia. However, the mechanism of STAG2 mutations promoting tumorigenesis is still unclear. METHODS: The distribution of STAG2 mutations in cancer was determined through the COSMIC database; we also generated a STAG2 truncating mutation in OS cell line U2OS cells to mimic a common mutation in OS. CCK-8 assay was employed to evaluate the effect of STAG2 on proliferation and chemo-resistance in OS cells. Cell apoptosis and cell cycle assays were used to assess the effect of STAG2 on apoptosis and the cycle of OS cells. A high throughput RNA sequencing (RNA-Seq) strategy using the Illumina Hiseq 2500 platform was applied to characterize the transcriptome profile from STAG2 knockout and STAG2 WT OS cell lines. RESULTS: We found that STAG2 deficient-cells exhibited reduced cell proliferation and growth; however, they enhanced cell metastasis and invasion, and increased tolerance to chemotherapeutic drugs. We also found that PD-L1, a molecule involved in tumor immune evasion, was up-regulated in the SATG2-lost cells. Expression profile analysis by RNA-seq revealed that there were changes in the expression of many immune-related genes. CONCLUSIONS: Our findings indicated that STAG2 contributes to cell survival and chemo-resistance to cisplatin of OS, suggesting that deletion of STAG2 may promote tumorigenesis by enhancing the immune evasion capacity of cancer cells.

Casticin inhibits nasopharyngeal carcinoma growth by targeting phosphoinositide 3-kinase.

BACKGROUND: Casticin, an isoflavone compound extracted from the herb Fructus Viticis, has demonstrated anti-inflammatory and anticancer activities and properties. The aim of this study was to investigate the effects and mechanisms of casticin in nasopharyngeal carcinoma (NPC) cells and to determine its potential for targeted use as a medicine. METHODS: NPC cells were used to perform the experiments. The CCK­8 assay and colony formation assays were used to assess cell viability. Flow cytometry was used to measure the cell cycle and apoptosis analysis (annexin V/PI assay). A three-dimensional (3D) tumour sphere culture system was used to characterize the effect of casticin on NPC stem cells. In silico molecular docking prediction and high-throughput KINOME scan assays were used to evaluate the binding of casticin to phosphoinositide 3-kinase (PI3K), including wild-type and most of mutants variants. We also used the SelectScreen assay to detect the IC50 of ATP activity in the active site of the target kinase. Western blotting was used to evaluate the changes in key proteins involved cell cycle, apoptosis, stemness, and PI3K/protein kinase B (AKT) signalling. The effect of casticin treatment in vivo was determined by using a xenograft mouse model. RESULTS: Our results indicate that casticin is a new and novel selective PI3K inhibitor that can significantly inhibit NPC proliferation and that it induces G2/GM arrest and apoptosis by upregulating Bax/BCL2 expression. Moreover, casticin was observed to affect the self-renewal ability of the nasopharyngeal carcinoma cell lines, and a combination of casticin with BYL719 was observed to induce a decrease in the level of the phosphorylation of mTORC1 downstream targets in BYL719-insensitive NPC cell lines. CONCLUSION: Casticin is a newly emerging selective PI3K inhibitor with potential for use as a targeted therapeutic treatment for nasopharyngeal carcinoma. Accordingly, casticin might represent a novel and effective agent against NPC and likely has high potential for combined use with pharmacological agents targeting PI3K/AKT.