Histidine phosphatase-ferroptosis crosstalk modulation for efficient hepatocellular carcinoma treatment.

Altering the mechanisms of tumor cell death and overcoming the limitations of traditional chemotherapy is pivotal to contemporary tumor treatment. Inducing ferroptosis, while circumventing safety concerns associated with ferrous vectors, through nonferrous ferroptosis is a promising but underexplored frontier in cancer therapy. Histidine phosphatase (LHPP) has emerged as a novel therapeutic target in treating hepatocellular carcinoma (HCC), but the precise mechanism of LHPP against HCC remains unclear. Herein, we explore the effects of upregulating LHPP expression on ferroptosis and tumor immunogenicity induction by simply delivering a miRNA-363-5p inhibitor (miR-363-5pi) via a previously optimized gemcitabine-oleic acid (GOA) prodrug. Efficient miRNA encapsulation was achieved through hydrogen bonding at an optimized GOA/miRNA molar feed ratio of 250:1, affording spherical nanoparticles with a uniform hydrodynamic size of 147.1 nm and a negative potential of -21.5 mV. The mechanism of this LHPP-ferroptosis crosstalk is disclosed to be an inhibited phosphorylation of the PI3K/Akt pathway, leading to a remarkable tumor inhibition rate of 88.2% in nude mice bearing Bel-7402 tumor xenografts via a combination of LHPP-triggered nonferrous ferroptosis and GOA-induced chemotherapy. The biocompatibility of GOA/miR-363-5pi is strongly supported by their non-hematologic toxicity and insignificant organ damage. In addition, the tumor immunogenic activation potential of GOA/miR-363-5pi was finally explored. Overall, this study is the first work that elucidates the precise mechanism of LHPP for treating HCC via ferroptosis induction and achieves the transformation of chemotherapy and gene therapy into ferroptosis activation with tumor cell immunogenicity, which lays a new therapeutic foundation for the clinical treatment of HCC.

Optimized workflow to modify microRNA expression in primary human intravascular cells.

BACKGROUND: A comprehensive dissection of the role of microRNAs (miRNAs) in gene regulation and subsequent cell functions requires a specific and efficient knockdown or overexpression of the miRNA of interest; these are achieved by transfecting the cell of interest with a miRNA inhibitor or a miRNA mimic, respectively. Inhibitors and mimics of miRNAs with a unique chemistry and/or structural modifications are available commercially and require different transfection conditions. Here, we aimed to investigate how various conditions affect the transfection efficacy of two miRNAs with high and low endogenous expression, miR-15a-5p and miR-20b-5p respectively, in human primary cells. RESULTS: MiRNA inhibitors and mimics from two commonly used commercial vendors were employed, i.e., mirVana (Thermo Fisher Scientific) and locked nucleic acid (LNA) miRNA (Qiagen). We systematically examined and optimized the transfection conditions of such miRNA inhibitors and mimics to primary endothelial cells and monocytes using either a lipid-based carrier (lipofectamine) for delivery or an unassisted uptake. Transfection of LNA inhibitors with either phosphodiester (PE)- or phosphorothioate (PS)-modified nucleotide bonds, delivered using a lipid-based carrier, efficiently downregulated the expression levels of miR-15a-5p already 24 h following transfection. MirVana miR-15a-5p inhibitor displayed a less efficient inhibitory effect, which was not improved 48 h following a single transfection or two consecutive transfections. Interestingly, LNA-PS miR-15a-5p inhibitor efficiently reduced the levels of miR-15a-5p when delivered without a lipid-based carrier in both ECs and monocytes. When using a carrier, mirVana and LNA miR-15a-5p and miR-20b-5p mimics showed similar efficiency 48 h following transfection to ECs and monocytes. None of the miRNA mimics effectively induced overexpression of the respective miRNA when given to primary cells without a carrier. CONCLUSION: LNA miRNA inhibitors efficiently downregulated the cellular expression of miRNA, such as miR-15a-5p. Furthermore, our findings suggest that LNA-PS miRNA inhibitors can be delivered in the absence of a lipid-based carrier, whereas miRNA mimics need the aid of a lipid-based carrier to achieve sufficient cellular uptake.

Structural improvement of LidNA: delta-type LidNA is a potent miRNA inhibitor constructed with unmodified DNA.

Many miRNA inhibitors have been developed, including chemically modified oligonucleotides, such as 2'-O-methylated RNA and locked nucleic acid (LNA). Unmodified DNA has not yet been reported as a miRNA inhibitor due to relatively low DNA/miRNA binding affinity. We designed a structured DNA, LidNA, which was constructed with unmodified DNA, consisting of a complementary sequence to the target miRNA flanked by two structured DNA regions, such as double-stranded DNA. LidNA inhibited miRNA activity more potently than 2'-O-methylated RNA or LNA. To optimize LidNA, two double-stranded regions were joined, causing the molecule to assume a delta-like shape, which we termed delta-type LidNA. Delta-type LidNAs were developed to target endogenous and exogenous miRNAs, and exhibited potent miRNA inhibitory effects with a duration of at least 10 days. Delta-type LidNA-21, which targeted miR-21, inhibited the growth of cancer cell lines. This newly developed LidNA could contribute to miRNA studies across multiple fields.Abbreviations: LidNA: DNA that puts a lid on miRNA function; LNA: locked nucleic acid; 3'-UTR: 3'-untranslated regions; RISC: RNA-induced silencing complex; MBL: Molecular beacon-like LidNA; YMBL: Y-type molecular beacon-like LidNA; TDMD: target-directed microRNA degradation.

Improvement of a miRNA inhibitor by intracellular selection.

Sequences surrounding the miRNA binding domain of the miRNA inhibitor LidNA were selected intracellularly. The library was transfected into cells, and then, inhibitors that were associated with argonaute 2 were selected. The potent inhibitors were slowly degraded intracellularly, while the lower-activity inhibitors were rapidly degraded. A combination of the selected sequences surrounding the miRNA binding domain enhanced miRNA inhibitory activity. ABBREVIATIONS: LidNA: DNA that puts a lid on miRNA function; LNA: locked nucleic acid; Ago2: argonaute 2; LNA: locked nucleic acid.

MicroRNAs cooperatively inhibit a network of tumor suppressor genes to promote pancreatic tumor growth and progression.

BACKGROUND & AIMS: There has not been a broad analysis of the combined effects of altered activities of microRNAs (miRNAs) in pancreatic ductal adenocarcinoma (PDAC) cells, and it is unclear how these might affect tumor progression or patient outcomes. METHODS: We combined data from miRNA and messenger RNA (mRNA) expression profiles and bioinformatic analyses to identify an miRNA-mRNA regulatory network in PDAC cell lines (PANC-1 and MIA PaCa-2) and in PDAC samples from patients. We used this information to identify miRNAs that contribute most to tumorigenesis. RESULTS: We identified 3 miRNAs (MIR21, MIR23A, and MIR27A) that acted as cooperative repressors of a network of tumor suppressor genes that included PDCD4, BTG2, and NEDD4L. Inhibition of MIR21, MIR23A, and MIR27A had synergistic effects in reducing proliferation of PDAC cells in culture and growth of xenograft tumors in mice. The level of inhibition was greater than that of inhibition of MIR21 alone. In 91 PDAC samples from patients, high levels of a combination of MIR21, MIR23A, and MIR27A were associated with shorter survival times after surgical resection. CONCLUSIONS: In an integrated data analysis, we identified functional miRNA-mRNA interactions that contribute to growth of PDACs. These findings indicate that miRNAs act together to promote tumor progression; therapeutic strategies might require inhibition of several miRNAs.

Engineered Cancer-Derived Small Extracellular Vesicle-Liposome Hybrid Delivery System for Targeted Treatment of Breast Cancer.

Cancer-derived small extracellular vesicles (sEVs) may be a promising drug delivery system that targets cancer cells due to their unique features, such as native homing ability, biological barrier crossing capability, and low immune response. However, the oncogenic cargos within them pose safety concerns, hence limiting their application thus far. We proposed using an electroporation-based strategy to extract the endogenous cargos from cancer-derived sEVs and demonstrated that their homing ability was still retained. A membrane fusion technique was used to fuse these sEVs with liposomes to form hybrid particles, which possessed both benefits of sEVs and liposomes. Anti-EGFR monoclonal antibodies were modified on the hybrid particles to improve their targeting ability further. The engineered hybrid particles showed higher drug loading ability that is 33.75 and 43.88% higher than that of liposomes and sEVs, respectively, and improved targeting ability by 52.23% higher than hybrid particles without modification. This delivery system showed >90% cell viability and enhanced treatment efficiency with 91.58 and 79.26% cell migration inhibition rates for the miR-21 inhibitor and gemcitabine, respectively.

LidNA, a miRNA inhibitor constructed with unmodified DNA, requires an xxxA insertion sequence in miRNA binding site for its potent inhibitory activity.

The involvement of miRNAs in the pathogenesis of various diseases, including cancer, poses the need for developing miRNA inhibitors. Previously, using unmodified DNA, we designed LidNA, which inhibited miRNA function more potently than 2'-O-methylated RNA and locked nucleic acid. LidNA consists of a complementary sequence to miRNA flanked by two structured DNAs. Alterations in the connected sequences between the complementary region and structured region modestly affect miRNA inhibition activity. Surprisingly, variations in the mismatched insertion sequence in the center of the complementary sequence significantly affect activity. The central insertion sequence xxxA is required for the potent miRNA inhibitory effects of LidNA. This suggests that both the structure and insertion sequence of LidNA and other miRNA inhibitors should be considered for maximal miRNA inhibitory activity.

Downregulation of miR-9 correlates with poor prognosis in colorectal cancer.

INTRODUCTION: microRNAs (miRNAs) are frequently dysregulated in many human cancers including colorectal cancer (CRC) and are useful candidate biomarkers in liquid biopsy of cancer for their stability in the blood. METHODS: We compared the expression of microRNA-9 (miR-9) in tissues (n = 357) and sera (n = 109) of CRC patients to determine whether miR-9 in serum reflects that in the cancer tissue in parallel. Also, we examined the miR-9 role in CRC by in vitro functional studies in four CRC cell lines. RESULTS: On multivariate analysis of colorectal cancer tissues and sera, miR-9 low expressions were significantly associated pN stage (tissues; p < 0.01, serum; p = 0.013), and clinical stage (tissues; p < 0.01, serum; p = 0.031). Moreover, patients with low miR-9 expression had shorter survival than those with high miR-9 expression (log-rank test, tissue; p = 0.021, serum; p = 0.011). miR-9 level in serum reflects that in the tumor. The CRC cells with low miR-9 expression was significantly increased cell proliferation, migration, invasion and colony formation than cells with high miR-9 expression. CONCLUSION: Serum miR-9 is an useful early detection marker in liquid biopsy of CRC and overexpression of miR-9 in CRC may be a novel prognostic marker as well.

Direct interaction analysis of microRNA-351-5p and nuclear scaffold lamin B1 mRNA by the cell-free in vitro mRNA/miRNA binding evaluation system.

We previously demonstrated that miR-351-5p regulates nuclear scaffold lamin B1 expression and mediates the anticancer floxuridine-induced necrosis shift to apoptosis in mammalian tumor cells. Notably, it is unknown whether lamin B1 mRNA is a direct target of miR-351-5p. Here, we show that miR-351-5p interacts with a lamin B1 mRNA partial sequence by using the cell-free in vitro miRNA and mRNA binding evaluation system. In addition, the interaction of miR-351-5p/lamin B1 mRNA was suppressed by an miR-351-5p inhibitor. Our findings are important in exploring the functions of miRNAs in cellular processes, including cell death.

A Simplified System to Express Circularized Inhibitors of miRNA for Stable and Potent Suppression of miRNA Functions.

MicroRNAs (miRNAs) are an evolutionarily conserved class of small regulatory noncoding RNAs, binding to complementary target mRNAs and resulting in mRNA translational inhibition or degradation, and they play an important role in regulating many aspects of physiologic and pathologic processes in mammalian cells. Thus, efficient manipulations of miRNA functions may be exploited as promising therapeutics for human diseases. Two commonly used strategies to inhibit miRNA functions include direct transfection of chemically synthesized miRNA inhibitors and delivery of a gene vector that instructs intracellular transcription of miRNA inhibitors. While most miRNA inhibitors are based on antisense molecules to bind and sequester miRNAs from their natural targets, it is challenging to achieve effective and stable miRNA inhibition. Here we develop a user-friendly system to express circular inhibitors of miRNA (CimiRs) by exploiting the noncanonical head-to-tail backsplicing mechanism for generating endogenous circular RNA sponges. In our proof-of-principle experiments, we demonstrate that the circular forms of the hsa-miR223-binding site of human ß-arrestin1 (ARRB1) 3' UTR sponge RNA (BUTR), the bulged anti-miR223 (cirBulg223) and bulged anti-miR21 (cirBulg21), exhibit more potent suppression of miRNA functions than their linear counterparts. Therefore, the engineered CimiR expression system should be a valuable tool to target miRNAs for basic and translational research.

Simple Methods to Investigate MicroRNA Induction in Response to Toll-Like Receptors.

In this chapter, we describe simple methods to investigate microRNA (miRNA) induction in response to lipopolysaccharide, the ligand for Toll-Like Receptor-4 activation. In brief, we demonstrate how to investigate global miRNA induction and/or repression in bone marrow-derived macrophages using TaqMan MicroRNA Arrays, followed by methods to measure individual miRNAs and target mRNA expression. Moreover, we explain step-by-step instructions on how to modulate endogenous miRNA expression through the use of miRNA inhibitors and mimics as well as highlight how miRNA modulation can be used to confirm mRNA targeting via Luciferase reporter assay. Moreover, these methods can be applied to whichever cell type and cellular function under investigation.

Utilization of inherent miRNAs in functional analyses of Toxoplasma gondii genes.

MicroRNAs (miRNAs) are crucial genetic effectors partaking in numerous mechanisms of gene regulation in eukaryotic organisms. Recent discoveries of miRNA in Toxoplasma gondii, an intracellular obligate parasite of the phylum Apicomplexa, suggested possible roles of T. gondii miRNAs (Tg-miRNAs) in the post-transcriptional gene regulation and in the cell biology of the parasite. To gain a better understanding of the involvement of Tg-miRNAs in regulating the parasite gene expression, a dual luciferase reporter system was used in the examination and evaluation of the effects of endogenous Tg-miRNAs, their mimics and inhibitors. A Renilla luciferase (Rnluc) transcript was engineered to carry independent binding sites of two abundant species, namely Tg-miR-60a and Tg-miR-4a, so that the expression of Rnluc was silenced in a sequence specific manner by Tg-miR-60a and Tg-miR-4a. Notably, Tg-miR-60a, but not Tg-miR-4a, caused the levels of Rnluc transcripts to decrease. These findings strongly suggested that T. gondii employs the Tg-miRNA species-specific mode of silencing actions: transcript degradation by Tg-miR-60a, and translational suppression by Tg-miR-4a. Herein we developed a genetic system that exploits and directs the most abundant Tg-miR-60a for loss-of-function analyses in T. gondii. As a proof of principle, we showed that when the binding sites for Tg-miR-60a were introduced into the parasite transcripts via homologous recombination at the locus of (i) DEAD-box RNA helicase (TgHoDI), or (ii) lactate dehydrogenase isoform 1 (TgLDH1), the expression levels of the selected genes can be altered. It was thus proven that inherit Tg-miR-60a could be directed and used to assist in the loss-of-function analyses.

Simultaneous inhibition of multiple oncogenic miRNAs by a multi-potent microRNA sponge.

The roles of oncogenic miRNAs are widely recognized in many cancers. Inhibition of single miRNA using antagomiR can efficiently knock-down a specific miRNA. However, the effect is transient and often results in subtle phenotype, as there are other miRNAs contribute to tumorigenesis. Here we report a multi-potent miRNA sponge inhibiting multiple miRNAs simultaneously. As a model system, we targeted miR-21, miR-155 and miR-221/222, known as oncogenic miRNAs in multiple tumors including breast and pancreatic cancers. To achieve efficient knockdown, we generated perfect and bulged-matched miRNA binding sites (MBS) and introduced multiple copies of MBS, ranging from one to five, in the multi-potent miRNA sponge. Luciferase reporter assay showed the multi-potent miRNA sponge efficiently inhibited 4 miRNAs in breast and pancreatic cancer cells. Furthermore, a stable and inducible version of the multi-potent miRNA sponge cell line showed the miRNA sponge efficiently reduces the level of 4 target miRNAs and increase target protein level of these oncogenic miRNAs. Finally, we showed the miRNA sponge sensitize cells to cancer drug and attenuate cell migratory activity. Altogether, our study demonstrates the multi-potent miRNA sponge is a useful tool to examine the functional impact of simultaneous inhibition of multiple miRNAs and proposes a therapeutic potential.

Failure of Tooth Formation Mediated by miR-135a Overexpression via BMP Signaling.

MicroRNAs (miRNAs) are known to regulate a variety of gene functions in many tissues and organs, but their expression and function in tooth development are not well-understood. A specific miRNA, miR-135a, was determined to be highly expressed at the bud stage. Interestingly, after the cap stage, miR-135a was expressed in the epithelium and mesenchyme but not in the inner enamel epithelium. To identify the relationship between miR-135a and its putative target genes, Bmpr-Ia and Bmpr-Ib, in early tooth development, miR-135a was ectopically overexpressed with a lentivirus. This overexpression resulted in the repression of Bmpr-Ia and Bmpr-Ib. Furthermore, miR-135a inhibited both Bmpr-Ia and Bmpr-Ib transcription. BMP2 proteins were expressed ectopically in tooth germs during the cap stage to determine the relationship between miR-135a and BMP signaling in early tooth development. When miR-135a was ectopically expressed, no tooth formation was observed after 4 wk of incubation in the kidney capsule. This study suggested that Bmp signaling, specifically Bmpr-Ia and Bmpr-Ib, regulates tooth formation via miR-135a.

Approaches to manipulating microRNAs in neurogenesis.

Neurogenesis in the nervous system is regulated by both protein coding genes and non-coding RNA molecules. microRNAs (miRNAs) are endogenous small non-coding RNAs and usually negatively regulate gene expression by binding to the 3' untranslated region (3'UTR) of target messenger RNAs (mRNAs). miRNAs have been shown to play an essential role in neurogenesis, regulating neuronal proliferation, differentiation, maturation, and migration. An important strategy used to reveal miRNA function is the manipulation of their expression levels and patterns in specific regions and cell types in the nervous system. In this review we will systemically highlight established and new approaches used to achieve gain-of-function and loss-of-function of miRNAs in vitro and in vivo, and will also summarize miRNA delivery techniques. As the development of these leading edge techniques come online, more exciting discoveries of the roles miRNAs play in neural development and function will be uncovered.