RalA, a GTPase targeted by miR-181a, promotes transformation and progression by activating the Ras-related signaling pathway in chronic myelogenous leukemia.

BCR/ABL is a well-known activator of multiple signaling pathways. RalA, a Ras downstream signaling molecule and a small GTPase, plays an important role in Bcr-Abl-induced leukemogenesis but the exact mechanism remains elusive. Here, we show that RalA GTPase activity is commonly high in chronic myelogenous leukemia (CML) cell lines and patient samples. Overexpression of RalA results in malignant transformation and progression, and induces resistance to imatinib (IM) in BaF3 and K562 cell lines. RalA reduced survival and led to IM resistance in a xenografted mouse model. Ablation of RalA by either siRNA or miR-181a, a RalA targeting microRNA, attenuated the malignant phenotypes in K562 cells. RBC8, a selective Ral inhibitor, enhanced the inhibitory effects of IM in K562, KCL22 and BaF3-P210 cells. Interestingly, the phospho-specific protein microarray assay revealed that multiple phosphorylation signal proteins were decreased by RalA inhibition, including SAPK, JNK, SRC, VEGFR2, P38 MAPK, c-Kit, JunB, and Keratin18. Among them, P38 MAPK and SAPK/JNK are Ras downstream signaling kinases. Taken together, RalA GTPase might be an important oncogene activating the Ras-related signaling pathway in CML.

Systematic analysis of berberine-induced signaling pathway between miRNA clusters and mRNAs and identification of mir-99a ∼ 125b cluster function by seed-targeting inhibitors in multiple myeloma cells.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Seed targeting with tiny anti-miR-155 inhibits malignant progression of multiple myeloma cells.

BACKGROUND: miR-155 acts as a ubiquitous oncogene in major classes of human cancers and is a potential target for therapeutic intervention. However, the role of miR-155 in multiple myeloma is poorly understood. METHODS: To explore the role of miR-155 in multiple myeloma, we assessed the influence of tiny seed-targeting anti-miR-155 (t-anti-miR-155) on multiple myeloma cell line (RPMI-8266) viability and apoptosis in vitro. RESULTS: t-anti-miR-155 significantly inhibited multiple myeloma cell proliferation, migration, and colony formation. Additionally, t-anti-miR-155 significantly increased CD19 positive cell numbers, which are novel biomarkers for multiple myeloma and suppressor of cytokine signaling 1(SOCS1) was shown to be a target gene for miR-155 in multiple myeloma. Finally, the miR-155 signaling pathway was investigated by KEGG assay. CONCLUSION: miR-155 in RPMI-8266 cells is a critical oncomiR in multiple myeloma and seed-targeting t-anti-miR-155 might be a novel strategy for miR-155-based therapeutics.

Integrative analysis of differential miRNA and functional study of miR-21 by seed-targeting inhibition in multiple myeloma cells in response to berberine.

BACKGROUND: 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. RESULTS: We confirmed that berberine down-regulated miRNA-21 expression and significantly up-regulated the expression of programmed cell death 4 (PDCD4), a predicted miR-21 target. Luciferase reporter assays confirmed that PDCD4 was directly regulated by miR-21. Bioinformatic analysis revealed that the miR-21 promoter can be targeted by signal transducer and activator of transcription 3 (STAT3). Down-regulation of interleukin 6 (IL6) by berberine might lead to inhibition of miR-21 transcription through STAT3 down-regulation in multiple myeloma. Furthermore, both berberine and seed-targeting anti-miR-21 oligonucleotide induced apoptosis, G2-phase cell cycle arrest and colony inhibition in multiple myeloma cell lines. Depletion of PDCD4 by short interfering RNA could rescue berberine-induced cytotoxicity in multiple myeloma cells. CONCLUSIONS: Our results suggest that berberine suppresses multiple myeloma cell growth, at least in part, by down-regulating miR-21 levels possibly through IL6/STAT3. This led to increased PDCD4 expression, which is likely to result in suppression of the p53 signaling pathway. These findings may also provide new mechanistic insight into the anti-cancer effects of certain compounds in traditional Chinese herbal medicines.

VEGF depletion enhances bcr-abl-specific sensitivity of arsenic trioxide in chronic myelogenous leukemia.

The development of resistance to imatinib mesylate may partly depend on high bcr-abl expression levels or point mutation(s). Arsenic trioxide (ATO) has bcr-abl suppressing activity in vitro, without cross-resistance to imatinib. Meanwhile, bcr-abl also induces expression of vascular endothelial growth factor (VEGF), which is associated with tumor-related angiogenesis and is involved in chronic myelogenous leukemia (CML) pathogenesis. Here, we investigated ways to improve ATO activity in CML by modulating cellular VEGF levels. K562 and primary CML cells were transfected with a VEGF antisense sequence. Cell viability and survival were assessed using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and trypan blue exclusion assays. Apoptotic cells were detected by flow cytometry following annexin V and propidium iodide staining. The results showed that VEGF depletion effectively promotes enhanced ATO antileukemic activity by repressing bcr-abl protein levels. These data provide a rationale for the clinical development of optimized ATO-based regimens that incorporate VEGF modulator for CML treatment.

miR-181a post-transcriptionally downregulates oncogenic RalA and contributes to growth inhibition and apoptosis in chronic myelogenous leukemia (CML).

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.

Inhibition of small GTPase RalA regulates growth and arsenic-induced apoptosis in chronic myeloid leukemia (CML) cells.

Chronic myelogenous leukemia (CML) results from the transformation of a primitive hematopoietic cell by the bcr-abl gene. RalA, one of the Ras superfamily of small GTPases, is a downstream molecule of bcr-abl fusion protein in ras signaling pathway, but its role in CML is poorly understood. Here, we first detected RalA level in CML cells, which is highly expressed and distributed mainly in the cytoplasm and/or partially in endomembrane. Next, siRNA was used to deplete RalA expression for elucidating its function. The results showed that siRNA RalA effectively inhibited cell viability, induced apoptosis and enhanced sensitivity of arsenic trioxide (ATO), and there are some synergistic effects of anti-CML between RalA siRNA and ATO. Finally, we found that ATO also could downregulate protein level of bcr-abl in K562 and KCL-22. Our research provides evidence that RalA might also serve as linchpin modulators in leukemia, and combinatorial therapies of dual inhibition of bcr-abl and ras signaling pathways have a great potential in treatment of CML.