Arsenic trioxide (ATO), an ancient traditional Chinese medicine, has been successfully used as a therapeutic agent for leukemia. Drug resistance and toxicity are major concerns with the treatment. MicroRNAs (miRNAs) are endogenous small non‐coding RNA molecules that might modulate cellular sensitivity to anticancer drugs. miRNA‐21 (miR‐21) is one of the most prominent miRNAs involved in various aspects of human cancers. However, miR‐21 has been rarely characterized in chronic myelogenous leukemia (CML). Here, we used a specific anti‐miR‐21 oligonucleotide (AMO‐miR‐21) to sensitize K562 cells to ATO by degradation of miR‐21. The results showed that both AMO‐miR‐21 and ATO caused growth inhibition, apoptosis, and G1‐phase arrest in K562 cells. Meanwhile, AMO‐miR‐21 significantly promoted ATO‐mediated growth inhibition and apotosis without affecting the G1 phase. Apoptotic cells were confirmed morphologically with Giemsa’s staining. Furthermore, dual‐luciferase reporter vector, containing two tandem miR‐21 binding sites from PDCD4 3′UTR, validated that PDCD4 was directly regulated by miR‐21. Therefore, AMO‐miR‐21 sensitized leukemic K562 cells to ATO by inducing apoptosis partially due to its up‐regulation of PDCD4 protein level. The combination of ATO and AMO‐miR‐21 present therapeutic potential for CML.(Cancer Sci 2010; 101: 948–954)

MicroRNAs (miRNAs) are a class of short RNAs that regulate gene expression through either translational repression or mRNA cleavage. miRNA-181a (miR-181a), one of the many miRNAs conserved among vertebrates, is differentially expressed in a variety of leukemia. However, its function in leukemia, particularly chronic myelogenous leukemia (CML), is poorly understood. Here we have reported the identification of miR-181a targets by combining TargetScan software prediction and expression profiling through overexpression of miR-181a mimic in leukemic K562 cells. Four overlapping genes were found to be the likely targets of miR-181a. Among the four genes, RalA is a downstream molecule of bcr-abl fusion protein in ras signaling pathway. However, its role in CML remains elusive. Luciferase reporter and Western blot assays confirmed that RalA is a direct target of miR-181a. overexpression of miR-181a effectively suppresses cell growth and induces G2-phase arrest and apoptosis partially by targeting RalA in leukemic K562 cells. Using the KEGG database combined with recent publications, downstream signaling pathway of RalA was graphed by cytoscape software. Therefore, our study is the first to report that RalA is directly regulated by miR-181a and plays an important role in CML. The approach of computational prediction combined with expression profiling might be valuable for the identification of miRNA targets in animal.

Background: Current therapies for multiple myeloma (MM) are associated with toxicity and resistance, highlighting the need for novel effective therapeutics. Berberine (BBR), a botanical alkaloid derived from several Berberis medicinal plants, has exhibited anti-tumor effects, including against multiple myeloma (MM); however, the molecular mechanism underlying the anti-MM effect has not been previously described. This study aimed to identify the target of berberine and related mechanisms involved in its therapeutic activity against MM. Results: Here, we demonstrated that BBR treatment killed MM cells in vitro and prolonged the survival of mice bearing MM xenografts in vivo. A screening approach integrating surface plasmon resonance (SPR) with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified UHRF1 (ubiquitin-like with PHD and RING Finger domains 1) as a potential target of BBR. Combining molecular docking and SPR analysis, we confirmed UHRF1 as a BBR-binding protein and discovered that BBR binds UHRF1 in the tandem tudor domain and plant homeodomain (TTD-PHD domain). BBR treatment induced UHRF1 degradation via the ubiquitin-dependent proteasome system and reactivated p16^INK4A and p73 in MM cells. Overexpression of UHRF1 promoted the MM cell proliferation and rendered MM cells more resistant to BBR, while silencing of UHRF1 with siRNA attenuated BBR-induced cytotoxicity. Conclusions: In summary, our study has identified UHRF1 as a direct target of BBR and uncovered molecular mechanisms involved in the anti-MM activity of BBR. Targeting UHRF1 through BBR may be a novel therapeutic strategy against MM.

Berberine is a natural alkaloid derived from a traditional Chinese herbal medicine. It is known to modulate microRNA (miRNA) levels, although the mechanism for this action is unknown. Here, we previously demonstrate that the expression of 87 miRNAs is differentially affected by berberine in multiple myeloma cells. Among 49 miRNAs that are down-regulated, nine act as oncomirs, including miR-21. Integrative analysis showed that 28 of the down-regulated miRNAs participate in tumor protein p53 (TP53) signaling and other cancer pathways. miR-21 is involved in all these pathways, and is one of the most important oncomirs to be affected by berberine in multiple myeloma cells.

Berberine (BBR) is a natural alkaloid derived from a traditional Chinese herbal medicine. However, the exact mechanisms underlying the different effects of berberine on MM cells have not been fully elucidated. A systematic analysis assay integrated common signaling pathways modulated by the 3 miRNA clusters and mRNAs in MM cells after BBR treatment. The role of the mir-99a ∼ 125b cluster, an important oncomir in MM, was identified by comparing the effects of t-anti-mirs with complete complementary antisense locked nucleic acids (LNAs) against mature mir-125b (anti-mir-125b). Three miRNAs clusters (miR-99a ∼ 125b, miR-17 ∼ 92 and miR-106 ∼ 25) were significantly down-regulated in BBR-treated MM cells and are involved in multiple cancer-related signaling pathways. Furthermore, the top 5 differentially regulated genes, RAC1, NFκB1, MYC, JUN and CCND1 might play key roles in the progression of MM. Systematic integration revealed that 3 common signaling pathways (TP53, Erb and MAPK) link the 3 miRNA clusters and the 5 key mRNAs. Meanwhile, both BBR and seed-targeting t-anti-mir-99a ∼ 125b cluster LNAs significantly induced apoptosis, G2-phase cell cycle arrest and colony inhibition. our results suggest that BBR suppresses multiple myeloma cells, partly by down-regulating the 3 miRNA clusters and many mRNAs, possibly through TP53, Erb and MAPK signaling pathways. The mir-99a ∼ 125b cluster might be a novel target for MM treatment. These findings provide new mechanistic insight into the anticancer effects of certain traditional Chinese herbal medicine compounds.

Purpose: Malignant pleural effusion (MPE) is an adverse prognostic factor in patients with osteoblastic osteosarcoma; however, the cellular contexts of MPE are largely unknown.Experimental design: We performed single‐cell RNA‐sequencing (scRNA‐seq) on 27 260 cells from seven MPE samples and 91 186 cells from eight osteosarcoma tissues, including one recurrent, one lung metastasis and six primary tumour (PT) samples, to characterize their tumour microenvironment.Results: Thirteen main cell groups were identified in osteosarcoma tumour and MPE samples. Immune cells dominate the cellular contexts in MPE with more T/NK cells and less osteoclasts compared to PT samples. Of T/NK cells, CD8⁺GNLY⁺, CD8⁺KLRC2⁺ T cells and FCGR3A⁺NK cells were enriched in MPE but CD4⁺FOXP3⁺ Tregs were enriched in PT samples. Naïve IGHD⁺ B and immune regulatory IGHA1⁺ B cells were largely identified in MPE, whereas bone metabolism‐related CLEC11A⁺ B cells were significantly enriched in osteosarcoma PT. M2‐type TAMs, including CLEC11A_TAM, C1QC_TAM and Prolif_TAMs, among myeloid cells were enriched in PT, which may suppress cytotoxicity activities of T cells through multiple ligand–receptor interactions. Mature LAMP3⁺ DCs were transformed from CD1C⁺ DC and CLEC9A⁺ DC sub‐clusters when exposure to tumour alloantigens, which may improve T cell cytotoxicity activities on tumour cells under anti‐PD‐L1 treatments. In further, immune cells from MPE usually present up‐regulated glycolysis and down‐regulated oxidative phosphorylation and riboflavin metabolism activities compared to those in PT samples.Conclusions: Our study provided a novel cellular atlas of MPE and PT in patients with advanced osteosarcoma, which may provide potential therapeutic targets in the future.