Inherited KDM6AA649T facilitates tumor-immune escape and exacerbates colorectal signet-ring cell carcinoma outcomes.

Childhood onset of colorectal signet-ring cell carcinoma (CR-SRCC) is extremely rare and featured as highly malignant with poor prognosis. Here we reported a CR-SRCC case of 11-year-old boy with a novel inherited X-linked KDM6AA694T mutation. The H3K27me3 demethylase KDM6A was frequently mutated in varieties of tumors and acts as a tumor suppressor. In vivo H3K27me3 demethylation assay demonstrated that KDM6AA694T had dampened H3K27me3 demethylase activity. Overexpression of KDM6AA694T in SRCC cell line KATO3 promoted cell proliferation, invasion and migration, which were further confirmed in vivo by constructing orthotopic tumor growth and lung metastasis model. Besides, expression of KDM6AA694T in immune cells suppresses inflammatory macrophage response and effector T cell response. In conclusion, we characterized a novel inherited KDM6AA694T mutant from a childhood-onset SRCC case and demonstrated that the mutant with impaired H3K27me3 demethylase activity could potentiate tumor malignancy and suppress antitumor immunity.

RUVBL1 ubiquitination by DTL promotes RUVBL1/2-ß-catenin-mediated transcriptional regulation of NHEJ pathway and enhances radiation resistance in breast cancer.

Radiotherapy effectiveness in breast cancer is limited by radioresistance. Nevertheless, the mechanisms behind radioresistance are not yet fully understood. RUVBL1 and RUVBL2, referred to as RUVBL1/2, are crucial AAA+ ATPases that act as co-chaperones and are connected to cancer. Our research revealed that RUVBL1, also known as pontin/TIP49, is excessively expressed in MMTV-PyMT mouse models undergoing radiotherapy, which is considered a murine spontaneous breast-tumor model. Our findings suggest that RUVBL1 enhances DNA damage repair and radioresistance in breast cancer cells both in vitro and in vivo. Mechanistically, we discovered that DTL, also known as CDT2 or DCAF2, which is a substrate adapter protein of CRL4, promotes the ubiquitination of RUVBL1 and facilitates its binding to RUVBL2 and transcription cofactor ß-catenin. This interaction, in turn, attenuates its binding to acetyltransferase Tat-interacting protein 60 (TIP60), a comodulator of nuclear receptors. Subsequently, ubiquitinated RUVBL1 promotes the transcriptional regulation of RUVBL1/2-ß-catenin on genes associated with the non-homologous end-joining (NHEJ) repair pathway. This process also attenuates TIP60-mediated H4K16 acetylation and the homologous recombination (HR) repair process. Expanding upon the prior study's discoveries, we exhibited that the ubiquitination of RUVBL1 by DTL advances the interosculation of RUVBL1/2-ß-catenin. And, it then regulates the transcription of NHEJ repair pathway protein. Resulting in an elevated resistance of breast cancer cells to radiation therapy. From the aforementioned, it is evident that targeting DTL-RUVBL1/2-ß-catenin provides a potential radiosensitization approach when treating breast cancer.

DSE inhibits melanoma progression by regulating tumor immune cell infiltration and VCAN.

Dermatan sulfate epimerase (DSE) is a C5 epiminase that plays a key role in converting chondroitin sulfate into dermal sulfate. DSE is often upregulated during carcinogenesis of some types of cancer and can regulate growth factor signaling in cancer cells. However, the expression and function of DSE in human melanoma have not been reported. In this study, we investigated the influence of tumor-derived DSE in melanoma progression and the potential mechanism of their action. First, proteomic analysis of collected melanoma tissues revealed that DSE was significantly down-regulated in melanoma tissues. DSE silenced or overexpressed melanoma cells were constructed to detect the effect of DSE on melanoma cells, and it was found that the up-regulation of DSE significantly inhibited the proliferation, migration and invasion of melanoma cells. Data analysis and flow cytometry were used to evaluate the immune subpopulations in tumors, and it was found that the high expression of DSE was closely related to the invasion of killer immune cells. Mechanistically, DSE promoted the expression of VCAN, which inhibited the biological activity of melanoma cells. Together, these results suggest that DSE is downregulated in melanoma tissues, and that high expression of DSE can promote melanoma progression by inducing immune cell infiltration and VCAN expression.

Fatty Acids Support the Fitness and Functionality of Tumor-Resident CD8+ T Cells by Maintaining SCML4 Expression.

CD8+ tissue-resident memory T (Trm) cells and tumor-infiltrating lymphocytes (TIL) regulate tumor immunity and immune surveillance. Characterization of Trm cells and TILs could help identify potential strategies to boost antitumor immunity. Here, we found that the transcription factor SCML4 was required for the progression and polyfunctionality of Trm cells and was associated with a better prognosis in patients with cancer. Moreover, SCML4 maintained multiple functions of TILs. Increased expression of SCML4 in CD8+ cells significantly reduced the growth of multiple types of tumors in mice, while deletion of SCML4 reduced antitumor immunity and promoted CD8+ T-cell exhaustion. Mechanistically, SCML4 recruited the HBO1-BRPF2-ING4 complex to reprogram the expression of T cell-specific genes, thereby enhancing the survival and effector functions of Trm cells and TILs. SCML4 expression was promoted by fatty acid metabolism through mTOR-IRF4-PRDM1 signaling, and fatty acid metabolism-induced epigenetic modifications that promoted tissue-resident and multifunctional gene expression in Trm cells and TILs. SCML4 increased the therapeutic effect of anti-PD-1 treatment by elevating the expression of effector molecules in TILs and inhibiting the apoptosis of TILs, which could be further enhanced by adding an inhibitor of H3K14ac deacetylation. These results provide a mechanistic perspective of functional regulation of tumor-localized Trm cells and TILs and identify an important activation target for tumor immunotherapy. SIGNIFICANCE: SCML4 upregulation in CD8+ Trm cells and tumor-infiltrating lymphocytes induced by fatty acid metabolism enhances antitumor immune responses, providing an immunometabolic axis to target for cancer treatment. See related commentary by Chakraborty et al., p. 3321.

From NAFLD to HCC: Advances in noninvasive diagnosis.

Non-alcoholic fatty liver disease (NAFLD) has gradually become one of the major liver health problems in the world. The dynamic course of the disease goes through steatosis, inflammation, fibrosis, and carcinoma. Before progressing to carcinoma, timely and effective intervention will make the condition better, which highlights the importance of early diagnosis. With the further study of the biological mechanism in the pathogenesis and progression of NAFLD, some potential biomarkers have been discovered, and the possibility of their clinical application is gradually being discussed. At the same time, the progress of imaging technology and the emergence of new materials and methods also provide more possibilities for the diagnosis of NAFLD. This article reviews the diagnostic markers and advanced diagnostic methods of NAFLD in recent years.

m6A methylation regulates hypoxia-induced pancreatic cancer glycolytic metabolism through ALKBH5-HDAC4-HIF1α positive feedback loop.

Pancreatic cancer (PC) is the most hypoxic cancer type among solid tumors. The dynamic changes of RNA N6-methyl-adenosine (m6A) contribute to tumor cells adaption to hypoxic microenvironmental. However, the regulatory mechanisms of hypoxia response in PC remains elusive. Here, we reported that the m6A demethylase ALKBH5 mediated a decrease of total mRNA m6A modification during hypoxia. Subsequently, methylated RNA immunoprecipitation sequencing (MeRIP-seq) combined with RNA sequencing (RNA-seq) revealed transcriptome-wide gene expression alteration and identified histone deacetylase type 4 (HDAC4) as a key target gene of m6A modification under hypoxic conditionds. Mechanistically, m6A methylation recognized by m6A reader-YTHDF2 enhanced the stability of HDAC4, and then promoted glycolytic metabolism and migration of PC cells. Our assays also demonstrated that hypoxia-induced HDAC4 enhanced HIF1a protein stability, and overexpressed HIF1a promoted transcription of ALKBH5 in hypoxic pancreatic cancer cells. Together, these results found a ALKBH5/HDAC4/HIF1α positive feedback loop for cellular response to hypoxia in pancreatic cancer. Our studies uncover the crosstalk between histone acetylation and RNA methylation modification on layer of epigenetic regulation.

USP48 Stabilizes Gasdermin E to Promote Pyroptosis in Cancer.

Pyroptosis is a type of programmed cell death characterized by the activation of inflammatory caspases and the cleavage of gasdermin proteins. Pyroptosis can suppress tumor development and induce antitumor immunity, and activating pyroptosis is a potential treatment strategy for cancer. To uncover approaches to harness the anticancer effects of pyroptosis, we aimed to identify regulators of pyroptosis in cancer. A CRISPR-Cas9 screen identified that loss of USP48, a deubiquitinating enzyme, significantly inhibited cell pyroptosis. USP48 promoted pyroptosis by stabilizing gasdermin E (GSDME). USP48 bound GSDME and removed K48-linked ubiquitination at positions K120 and K189. Clinical tissue testing confirmed that the expression of USP48 positively correlated with GSDME and pyroptosis-related factors. Single-cell sequencing showed that the functions of T cells and tumor-associated macrophages in the tumor microenvironment were inhibited after USP48 knockout. Finally, overexpression of USP48 enhanced the therapeutic efficacy of programmed cell death protein 1 inhibitors in tumors in mouse models. Together, these findings define a pyroptosis regulation pathway and indicate that pharmacologic activation of USP48 may provide an effective strategy to sensitize cancer cells to pyroptosis and improve response to immunotherapy. SIGNIFICANCE: USP48 promotes pyroptosis by deubiquitinating GSDME and enhances antitumor immunity, indicating that increasing USP48 activity may be a future therapeutic strategy for treating cancer.

Characterisation of a novel transcript LNPPS acting as tumour suppressor in bladder cancer via PDCD5-mediated p53 degradation blockage.

BACKGROUND: Long non-coding RNAs (lncRNAs) play a crucial role in tumour initiation and progression. However, little is known about their contributions to p53-related bladder cancer (BC) inhibition. METHODS: By using high-throughput sequencing, we screened the expression profiles of lncRNAs in BC and adjacent non-tumour tissues. The roles of a novel lncRNA, named LNPPS [a lncRNA for programmed cell death 5 (PDCD5) and p53 stability], were determined by gain- and loss-of-function assays. RNA pull-down followed by mass spectrometry analysis, RNA immunoprecipitation assays and other immunoprecipitation assays were performed to reveal the interactions among LNPPS, PDCD5 and p53, and the regulatory effect of LNPPS on the complex ubiquitination network comprising PDCD5, p53 and mouse double minute 2 homologue (MDM2). RESULTS: LNPPS was downregulated in BC and markedly inhibited the viability of BC cells by inducing PDCD5/p53-related apoptosis in vivo and in vitro. Mechanistically, LNPPS, serving as a scaffold, connected PDCD5 and p53 with nucleotides (nt) located at 121-251 nt and 251-306 nt of LNPPS, respectively. This process allowed LNPPS to protect PDCD5 from proteasomal degradation by blocking its K20 site ubiquitination. On the other hand, the increased interaction between PDCD5 and p53 displaced p53 from the MDM2-p53 ubiquitination complex, resulting in an increase in p53 expression and related apoptosis levels. Moreover, LNPPS could induce the accumulation of PDCD5 and p53 in the nucleus and exert a synergistic effect on the prevention of protein degradation. In addition, we confirmed that the downregulation of LNPPS in BC was mediated by the decreased N6-methyladenosine (m6 A) modification. CONCLUSION: Our findings highlight a novel cross-talk between LNPPS and the PDCD5/p53/MDM2 ubiquitination axis in BC development, indicating its potential as a therapeutic target for BC patients.

Sirtuin 4 activates autophagy and inhibits tumorigenesis by upregulating the p53 signaling pathway.

The role of autophagy in cancer is context-dependent. In the present study, we aimed to investigate the regulator and underlying mechanism of autophagy. We found that a sirtuin (SIRT) family member, SIRT4, was significantly associated autophagy pathway in pancreatic ductal adenocarcinoma (PDAC). Specifically, in vitro cell culture experiments and in vivo transgenic and xenografted animal models revealed that SIRT4 could inhibit tumor growth and promote autophagy in PDAC. In terms of the mechanism, we demonstrated that SIRT4 activated the phosphorylation of p53 protein by suppressing glutamine metabolism, which was crucial in SIRT4-induced autophagy. AMPKα was implicated in the regulation of autophagy and phosphorylation of p53 mediated by SIRT4, contributing to the suppression of pancreatic tumorigenesis. Notably, the clinical significance of the SIRT4/AMPKα/p53/autophagy axis was demonstrated in human PDAC specimens. Collectively, these findings suggested that SIRT4-induced autophagy further inhibited tumorigenesis and progression of PDAC, highlighting the potential of SIRT4 as a therapeutic target for cancer.

Metabolic reprogramming due to hypoxia in pancreatic cancer: Implications for tumor formation, immunity, and more.

Hypoxia is a common phenomenon in most malignant tumors, especially in pancreatic cancer (PC). Hypoxia is the result of unlimited tumor growth and plays an active role in promoting tumor survival, progression, and invasion. As the part of the hypoxia microenvironment in PC is gradually clarified, hypoxia is becoming a key determinant and an important therapeutic target of pancreatic cancer. To adapt to the severe hypoxia environment, cells have changed their metabolic phenotypes to maintain their survival and proliferation. Enhanced glycolysis is the most prominent feature of cancer cells' metabolic reprogramming in response to hypoxia. It provides the energy source for hypoxic cancer cells (although it provides less than oxidative phosphorylation) and produces metabolites that can be absorbed and utilized by normoxic cancer cells. In addition, the uptake of glutamine and fatty acids by hypoxic cancer cells is also increased, which is also conducive to tumor progression. Their metabolites are pooled in the hexosamine biosynthesis pathway (HBP). As a nutrition sensor, HBP, in turn, can coordinate glucose and glutamine metabolism. Its end product, UDP-GlcNAc, is the substrate of protein post-translational modification (PTM) involved in various signaling pathways supporting tumor progression. Adaptive metabolic changes of cancer cells promote their survival and affect tumor immune cells in the tumor microenvironment (TME), which contributes to tumor immunosuppressive microenvironment and induces tumor immunotherapy resistance. Here, we summarize the hypoxic microenvironment, its effect on metabolic reprogramming, and its contribution to immunotherapy resistance in pancreatic cancer.

USP48 Is Upregulated by Mettl14 to Attenuate Hepatocellular Carcinoma via Regulating SIRT6 Stabilization.

Exploiting cancer metabolism for the clinical benefit of patients with hepatocellular carcinoma (HCC) is a topic under active investigation. Ubiquitin-specific peptidase 48 (USP48), a member of the ubiquitin-specific protease family, is involved in tumor growth, inflammation, and genome stability. However, the role of USP48 in HCC tumorigenesis remains unknown. In this study, we report that expression of USP48 is downregulated in diethylnitrosamine-induced liver tumorigenesis in mice as well as in human HCC. USP48 physically bound and stabilized SIRT6 by K48-linked deubiquitination at the K33 and K128 sites of SIRT6, which impeded metabolic reprogramming to hamper HCC tumorigenesis. Moreover, methyltransferase-like 14 (Mettl14)-induced m6A modification participated in the regulation of USP48 in HCC by maintaining USP48 mRNA stability. Our work uncovers the tumor-suppressive function of the Mettl14-USP48-SIRT6 axis via modulation of glycolysis, providing new insights into the critical roles of metabolic activities in HCC and identifying an attractive target for future treatment studies. SIGNIFICANCE: These findings demonstrate that USP48 is regulated by Mettl14-induced m6A modification and stabilizes SIRT6 to attenuate HCC glycolysis and malignancy.

CRL4ADTL degrades DNA-PKcs to modulate NHEJ repair and induce genomic instability and subsequent malignant transformation.

Genomic instability induced by DNA damage and improper DNA damage repair is one of the main causes of malignant transformation and tumorigenesis. DNA double strand breaks (DSBs) are the most detrimental form of DNA damage, and nonhomologous end-joining (NHEJ) mechanisms play dominant and priority roles in initiating DSB repair. A well-studied oncogene, the ubiquitin ligase Cullin 4A (CUL4A), is reported to be recruited to DSB sites in genomic DNA, but whether it regulates NHEJ mechanisms of DSB repair is unclear. Here, we discovered that the CUL4A-DTL ligase complex targeted the DNA-PKcs protein in the NHEJ repair pathway for nuclear degradation. Overexpression of either CUL4A or DTL reduced NHEJ repair efficiency and subsequently increased the accumulation of DSBs. Moreover, we demonstrated that overexpression of either CUL4A or DTL in normal cells led to genomic instability and malignant proliferation. Consistent with the in vitro findings, in human precancerous lesions, CUL4A expression gradually increased with increasing malignant tendency and was negatively correlated with DNA-PKcs and positively correlated with γ-H2AX expression. Collectively, this study provided strong evidence that the CUL4A-DTL axis increases genomic instability and enhances the subsequent malignant transformation of normal cells by inhibiting NHEJ repair. These results also suggested that CUL4A may be a prognostic marker of precancerous lesions and a potential therapeutic target in cancer.

HDAC11 Regulates Glycolysis through the LKB1/AMPK Signaling Pathway to Maintain Hepatocellular Carcinoma Stemness.

Hepatocellular carcinoma (HCC) contains a subset of cancer stem cells (CSC) that cause tumor recurrence, metastasis, and chemical resistance. Histone deacetylase 11 (HDAC11) mediates diverse immune functions and metabolism, yet little is known about its role in HCC CSCs. In this study, we report that HDAC11 is highly expressed in HCC and is closely related to disease prognosis. Depletion of HDAC11 in a conditional knockout mouse model reduced hepatocellular tumorigenesis and prolonged survival. Loss of HDAC11 increased transcription of LKB1 by promoting histone acetylation in its promoter region, thereby activating the AMPK signaling pathway and inhibiting the glycolysis pathway, which in turn leads to the suppression of cancer stemness and HCC progression. Furthermore, HDAC11 overexpression reduced HCC sensitivity to sorafenib. Collectively, these data propose HDAC11 as a new target for combination therapy in patients with kinase-resistant HCC. SIGNIFICANCE: This study finds that HDAC11 suppresses LKB1 expression in HCC to promote cancer stemness, progression, and sorafenib resistance, suggesting the potential of targeting HDAC11 to treat HCC and overcome kinase inhibitor resistance.

Carnosic Acid Reverses the Inhibition of ApoE4 on Cell Surface Level of ApoER2 and Reelin Signaling Pathway.

The cell surface level of apolipoprotein E receptor 2 (ApoER2) increases by cyclic transport of ApoER2 and then activates Reelin signaling pathway to exert neuroprotective function in AD. ApoER2 ligand Apolipoprotein E4 (ApoE4) inhibits the recycling of ApoER2 to the cell surface rendering neurons unresponsive to Reelin. Carnosic acid (CA) is proven to possess neuroprotective and neurotrophic functions in Alzheimer's disease (AD) mouse model. However, there are few reports about how ApoE4 impairs the recycling of ApoER2 and if CA can affect the cyclic transport of ApoER2. In this study, we demonstrated that ApoE4 attenuates the binding of sorting nexin 17 (SNX17) to ApoER2 and inhibits the recycling of ApoER2, resulting in decreased cell surface level of ApoER2. Further, we found that CA enhances the binding of SNX17 to ApoER2, counteracts the negative effects of ApoE4 on the cell surface level of ApoER2 to reverse the ApoE4-induced reduction in Reelin signaling activation by increasing the phosphorylation of the N-methyl-D-aspartate receptor (NMDAR) and cAMP-response element-binding protein (CREB) and the expression of Gria2. Thus, CA promotes neurite growth inhibited by ApoE4. Our work suggests that CA may be a potential approach to attenuate the risk of ApoE4-associated AD.

DTL promotes cancer progression by PDCD4 ubiquitin-dependent degradation.

BACKGROUND: Ubiquitin E3 ligase CUL4A plays important oncogenic roles in the development of cancers. DTL, one of the CUL4-DDB1 associated factors (DCAFs), may involve in the process of cancer development. Programmed cell death 4 (PDCD4) is a tumor suppressor gene involved in cell apoptosis, transformation, invasion and tumor progression. METHODS: Affinity-purification mass spectrometry was used to identify potential DTL interaction proteins. Co-immunoprecipitation (Co-IP) was performed to verify protein interaction between DTL and PDCD4. mRNA levels in cancer cells and tissues were detected by Quantitative real-time PCR. Lentivirus was used to establish stable overexpression and knocking down cell lines for DTL and PDCD4. Transwell and wound healing assays were used to determine migration ability of cancer cells. Matrigel assay was used to determine invasion ability of cancer cells. MTT and colony formation assays were used to evaluate proliferation of cancer cells. RESULTS: In this study, programmed cell death 4 (PDCD4) was identified as a potential substrate of DTL. Co-IP and immunofluorescence assays further confirmed the interaction between DTL and PDCD4. Moreover, DTL overexpression decreased the protein level and accelerated the degradation rate of PDCD4. Through in vitro ubiquitination experiment, we proved that PDCD4 was degraded by DTL through ubiquitination. Clinically DTL was significantly up-regulated in cancer tissues than that in normal tissues. The survival curves showed that cancer patients with higher DTL expression owned lower survival rate. Functional experiments showed that DTL not only enhanced the proliferation and migration abilities of cancer cells, but also promoted the tumorigenesis in nude mice. Rescued experiment results demonstrated that silencing PDCD4 simultaneous with DTL recovered the phenotypes defect caused by DTL knocking down. CONCLUSIONS: Our results elucidated that DTL enhanced the motility and proliferation of cancer cells through degrading PDCD4 to promote the development of cancers.

Signal pathways, diseases, and functions associated with the miR-19a/92a cluster and the use of berberine to modulate the expression of this cluster in multiple myeloma cells.

BACKGROUND: Berberine downregulated miR-19a/92a cluster expression in multiple myeloma (MM) cells. METHODS: The cell viability of MM cells after berberine treatment was measured by CCK8 assay. qRT-PCR assay validated miR-19a/92a expression in multiple myeloma cells. TAM database analyzed miR-19a/92a-associated disease. miREnvironment database revealed that effects of environmental factors on the miR-19a/92a cluster. By targeting the seed region in the miRNA, the role of t-anti-miR-19a/92a cluster was evaluated by cell proliferation, migration, and colony formation. RESULTS: Berberine inhibited the cell viability of MM cells and downregulated the expression of miR-19a/92a. Seven kinds of hematological malignancies are closely associated with miR-19a/92a expression. By targeting the seed region of the miRNA, t-anti-miR-19a/92a significantly inhibits multiple myeloma cell proliferation, migration, and colony formation. CONCLUSION: Our findings may exhibit that miR-19a/92a cluster is a therapeutic target for MM and provide new mechanistic insight into the anti-MM effects of certain compounds in traditional Chinese herbal medicines.

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.