BackgroundBerberine 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.ResultsWe 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.ConclusionsOur 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.

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 (AMOmiR-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, AMOmiR-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)

Drug insensitivity or resistance is a major obstacle for successful treatment of acute myeloid leukemia. MicroRNAs (miRNAs) are small non-coding RNA molecules. Increasing evidence suggests that miRNAs modulate cellular sensitivity to anticancer drugs. We used a specific anti-miR-21 oligonucleotide (AMO-miR-21) to sensitize leukemic HL60 cells to arabinosylcytosine (Ara-C) by down-regulating miR-21. AMO-miR-21 alone effectively inhibited HL60 cell viability as measured by MTT assays and induced apoptosis as evaluated by flow cytometry, whereas AMO-miR-21 in combination with Ara-C enhanced HL60 cells to Ara-C-sensitivity and promoted Ara-C-induced apoptosis. Levels of miR-21 and its target PDCD4, quantified by real-time PCR, showed that expression of miR-21 was significantly decreased after AMO-miR-21 treatment. PDCD4 as a direct target of miR-21 in leukemic HL60 cells was confirmed by the dual-luciferase reporter assay. Our study suggests that AMO-miR-21 significantly sensitizes HL60 cells to Ara-C by inducing apoptosis and these effects of AMO-miR-21 may be partially due to its up-regulation of PDCD4. Therefore, exploiting synergistic effects between AMO-miR-21 and Ara-C might be an effective clinical strategy for leukemia chemotherapy.

MicroRNAs (miRNAs) are small non-coding RNA molecules that are widely involved in cancer-related processes. The microRNA-21 (miR-21) has been identified as the only miRNA overexpressed in a variety of cancers, including leukemia. However, the function of miR-21 is yet unknown in chronic myelogenous leukemia (CML). Antisense oligonucleotides (ASOs), as inhibitors of miRNAs, have already been applied to therapeutic development and functional identification in miRNA research. In this study, we found that the antisense inhibition of miR-21 in K562 cells suppressed cell migration, promoted cell apoptosis, and inhibited cell growth, and up-regulated the expression of the tumor suppressor gene PDCD4. Meanwhile, pre-miRNA-21 increased migration and decreased cell apoptosis without affecting proliferation. We also validated that PDCD4 is a functional target of miR-21 in K562 cells. These effects of miR-21 might be partially due to its regulation of PDCD4. Our data suggest that miR-21 may play an oncogenic role in the cellular processes of CML, and antisense inhibition of miR-21 may therefore be useful as CML therapy.

MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules involved in modulation of cellular sensitivity to anti-cancer drugs. miRNA-21 (miR-21), one of the most prominent miRNAs in the genesis and progression of many human cancers, has been rarely characterized in myelogenous leukemia. Arsenic trioxide (ATO) was successfully used in the treatment of acute promyelocytic leukemia (APL) etc. However, cytotoxicity or insensitivity is a major concern in the successful treatment of leukemia. Here, we used a specific precursor miRNA-21 (pre-miR-21) or anti-miRNA-21 oligonucleotide (AMO-miR-21) to study sensitivity of HL60 and K562 cells to ATO. Cell viability and cell cycle were evaluated by MTT assay and PI assay using flow cytometry, respectively. Levels of miR-21 and its target PDCD4 were quantified by real-time PCR and/or western blot. AMO-miR-21 or ATO alone led to growth inhibition, apoptosis and G1 phase arrest of cell cycle. Apoptotic cells were confirmed morphologically with Hoechst staining. Moreover, there was somewhat synergistic effect of AMO-miR-21 and ATO in growth inhibition and apoptosis promotion. Meanwhile, enforced pre-miR-21 expression increased resistance to ATO, nevertheless not affecting cell growth alone. Dual-luciferase reporter vector containing two tandem PDCD4 3' UTR validated that PDCD4 was directly up-regulated by miR-21. Therefore, miRNA-21 by targeting PDCD4 may play a functional role in modulating ATO-induced cell death, and strategy using AMO-miR-21 and its combination with ATO may be useful as a myelogenous leukemia therapy.