MAPKAPK2-centric transcriptome profiling reveals its major role in governing molecular crosstalk of IGFBP2, MUC4, and PRKAR2B during HNSCC pathogenesis.

Transcriptome analysis of head and neck squamous cell carcinoma (HNSCC) has been pivotal to comprehending the convoluted biology of HNSCC tumors. MAPKAPK2 or MK2 is a critical modulator of the mRNA turnover of crucial genes involved in HNSCC progression. However, MK2-centric transcriptome profiles of tumors are not well known. This study delves into HNSCC progression with MK2 at the nexus to delineate the biological relevance and intricate crosstalk of MK2 in the tumor milieu. We performed next-generation sequencing-based transcriptome profiling of HNSCC cells and xenograft tumors to ascertain mRNA expression profiles in MK2-wild type and MK2-knockdown conditions. The findings were validated using gene expression assays, immunohistochemistry, and transcript turnover studies. Here, we identified a pool of crucial MK2-regulated candidate genes by annotation and differential gene expression analyses. Regulatory network and pathway enrichment revealed their significance and involvement in the HNSCC pathogenesis. Additionally, 3'-UTR-based filtering recognized important MK2-regulated downstream target genes and validated them by nCounter gene expression assays. Finally, immunohistochemistry and transcript stability studies revealed the putative role of MK2 in regulating the transcript turnover of IGFBP2, MUC4, and PRKAR2B in HNSCC. Conclusively, MK2-regulated candidate genes were identified in this study, and their plausible involvement in HNSCC pathogenesis was elucidated. These genes possess investigative values as targets for diagnosis and therapeutic interventions for HNSCC.

miR-30a inhibits the osteogenic differentiation of the tibia-derived MSCs in congenital pseudarthrosis via targeting HOXD8.

Background: Congenital pseudarthrosis of the tibia (CPT) is an uncommon congenital deformity and a special subtype of bone nonunion. The lower ability of osteogenic differentiation in CPT-derived mesenchymal stem cells (MSCs) could result in progression of CPT, and miR-30a could inhibit osteogenic differentiation. However, the role of miR-30a in CPT-derived MSCs remains unclear. Methods: The osteogenic differentiation of CPT-derived MSCs treated with the miR-30a inhibitor was tested by Alizarin Red S staining and alkaline phosphatase (ALP) activity. The expression levels of protein and mRNA were assessed by Western blot or quantitative reverse transcription-polymerase chain reaction (RT-qPCR), respectively. The interplay between miR-30a and HOXD8 was investigated by a dual-luciferase reporter assay. Chromatin immunoprecipitation (ChIP) was conducted to assess the binding relationship between HOXD8 and RUNX2 promoter. Results: CPT-derived MSCs showed a lower ability of osteogenic differentiation than normal MSCs. miR-30a increased in CPT-derived MSCs, and miR-30a downregulation promoted the osteogenic differentiation of CPT-derived MSCs. Meanwhile, HOXD8 is a direct target for miR-30a, and HOXD8 could transcriptionally activate RUNX2. In addition, miR-30a could inhibit the osteogenic differentiation of CPT-derived MSCs by negatively regulating HOXD8. Conclusion: miR-30a inhibits the osteogenic differentiation of CPT-derived MSCs by targeting HOXD8. Thus, this study might supply a novel strategy against CPT.

Genistein promotes M1 macrophage apoptosis and reduces inflammatory response by disrupting miR-21/TIPE2 pathway.

Cardiovascular diseases are a major cause of mortality, and vascular injury, a common pathological basis of cardiovascular disease, is deeply correlated with macrophage apoptosis and inflammatory response. Genistein, a type of phytoestrogen, exerts cardiovascular protective activities, but the underlying mechanism has not been fully elucidated. In this study, RAW264.7 cells were treated with genistein, lipopolysaccharide (LPS), nuclear factor-kappa B (NF-κB) inhibitor, and/or protein kinase B (AKT) agonist to determine the role of genistein in apoptosis and inflammation in LPS-stimulated cells. Simultaneously, high fat diet-fed C57BL/6 mice were administered genistein to evaluate the function of genistein on LPS-induced cardiovascular injury mouse model. Here, we demonstrated that LPS obviously increased apoptosis resistance and inflammatory response of macrophages by promoting miR-21 expression, and miR-21 downregulated tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) expression by targeting the coding region. Genistein reduced miR-21 expression by inhibiting NF-κB, then blocked toll-like receptor 4 (TLR4) pathway and AKT phosphorylation dependent on TIPE2, resulting in inhibition of LPS. Our research suggests that miR-21/TIPE2 pathway is involved in M1 macrophage apoptosis and inflammatory response, and genistein inhibits the progression of LPS-induced cardiovascular injury at the epigenetic level via regulating the promoter region of Vmp1 by NF-κB.

MicroRNA-34c-5p provokes isoprenaline-induced cardiac hypertrophy by modulating autophagy via targeting ATG4B.

Pathological cardiac hypertrophy serves as a significant foundation for cardiac dysfunction and heart failure. Recently, growing evidence has revealed that microRNAs (miRNAs) play multiple roles in biological processes and participate in cardiovascular diseases. In the present research, we investigate the impact of miRNA-34c-5p on cardiac hypertrophy and the mechanism involved. The expression of miR-34c-5p was proved to be elevated in heart tissues from isoprenaline (ISO)-infused mice. ISO also promoted miR-34c-5p level in primary cultures of neonatal rat cardiomyocytes (NRCMs). Transfection with miR-34c-5p mimic enhanced cell surface area and expression levels of foetal-type genes atrial natriuretic factor (Anf) and ß-myosin heavy chain (ß-Mhc) in NRCMs. In contrast, treatment with miR-34c-5p inhibitor attenuated ISO-induced hypertrophic responses. Enforced expression of miR-34c-5p by tail intravenous injection of its agomir led to cardiac dysfunction and hypertrophy in mice, whereas inhibiting miR-34c-5p by specific antagomir could protect the animals against ISO-triggered hypertrophic abnormalities. Mechanistically, miR-34c-5p suppressed autophagic flux in cardiomyocytes, which contributed to the development of hypertrophy. Furthermore, the autophagy-related gene 4B (ATG4B) was identified as a direct target of miR-34c-5p, and miR-34c-5p was certified to interact with 3' untranslated region of Atg4b mRNA by dual-luciferase reporter assay. miR-34c-5p reduced the expression of ATG4B, thereby resulting in decreased autophagy activity and induction of hypertrophy. Inhibition of miR-34c-5p abolished the detrimental effects of ISO by restoring ATG4B and increasing autophagy. In conclusion, our findings illuminate that miR-34c-5p participates in ISO-induced cardiac hypertrophy, at least partly through suppressing ATG4B and autophagy. It suggests that regulation of miR-34c-5p may offer a new way for handling hypertrophy-related cardiac dysfunction.

The generation of PD-L1 and PD-L2 in cancer cells: From nuclear chromatin reorganization to extracellular presentation.

The immune checkpoint blockade (ICB) targeting on PD-1/PD-L1 has shown remarkable promise in treating cancers. However, the low response rate and frequently observed severe side effects limit its broad benefits. It is partially due to less understanding of the biological regulation of PD-L1. Here, we systematically and comprehensively summarized the regulation of PD-L1 from nuclear chromatin reorganization to extracellular presentation. In PD-L1 and PD-L2 highly expressed cancer cells, a new TAD (topologically associating domain) (chr9: 5,400,000-5,600,000) around CD274 and CD273 was discovered, which includes a reported super-enhancer to drive synchronous transcription of PD-L1 and PD-L2. The re-shaped TAD allows transcription factors such as STAT3 and IRF1 recruit to PD-L1 locus in order to guide the expression of PD-L1. After transcription, the PD-L1 is tightly regulated by miRNAs and RNA-binding proteins via the long 3'UTR. At translational level, PD-L1 protein and its membrane presentation are tightly regulated by post-translational modification such as glycosylation and ubiquitination. In addition, PD-L1 can be secreted via exosome to systematically inhibit immune response. Therefore, fully dissecting the regulation of PD-L1/PD-L2 and thoroughly detecting PD-L1/PD-L2 as well as their regulatory networks will bring more insights in ICB and ICB-based combinational therapy.

MicroRNA-34c-5p provokes isoprenaline-induced cardiac hypertrophy by modulating autophagy via targeting ATG4B

Pathological cardiac hypertrophy serves as a significant foundation for cardiac dysfunction and heart failure. Recently, growing evidence has revealed that microRNAs (miRNAs) play multiple roles in biological processes and participate in cardiovascular diseases. In the present research, we investigate the impact of miRNA-34c-5p on cardiac hypertrophy and the mechanism involved. The expression of miR-34c-5p was proved to be elevated in heart tissues from isoprenaline (ISO)-infused mice. ISO also promoted miR-34c-5p level in primary cultures of neonatal rat cardiomyocytes (NRCMs). Transfection with miR-34c-5p mimic enhanced cell surface area and expression levels of foetal-type genes atrial natriuretic factor (Anf) and β-myosin heavy chain (β-Mhc) in NRCMs. In contrast, treatment with miR-34c-5p inhibitor attenuated ISO-induced hypertrophic responses. Enforced expression of miR-34c-5p by tail intravenous injection of its agomir led to cardiac dysfunction and hypertrophy in mice, whereas inhibiting miR-34c-5p by specific antagomir could protect the animals against ISO-triggered hypertrophic abnormalities. Mechanistically, miR-34c-5p suppressed autophagic flux in cardiomyocytes, which contributed to the development of hypertrophy. Furthermore, the autophagy-related gene 4B (ATG4B) was identified as a direct target of miR-34c-5p, and miR-34c-5p was certified to interact with 3' untranslated region of Atg4b mRNA by dual-luciferase reporter assay. miR-34c-5p reduced the expression of ATG4B, thereby resulting in decreased autophagy activity and induction of hypertrophy. Inhibition of miR-34c-5p abolished the detrimental effects of ISO by restoring ATG4B and increasing autophagy. In conclusion, our findings illuminate that miR-34c-5p participates in ISO-induced cardiac hypertrophy, at least partly through suppressing ATG4B and autophagy. It suggests that regulation of miR-34c-5p may offer a new way for handling hypertrophy-related cardiac dysfunction.

The generation of PD-L1 and PD-L2 in cancer cells: From nuclear chromatin reorganization to extracellular presentation

The immune checkpoint blockade (ICB) targeting on PD-1/PD-L1 has shown remarkable promise in treating cancers. However, the low response rate and frequently observed severe side effects limit its broad benefits. It is partially due to less understanding of the biological regulation of PD-L1. Here, we systematically and comprehensively summarized the regulation of PD-L1 from nuclear chromatin reorganization to extracellular presentation. In PD-L1 and PD-L2 highly expressed cancer cells, a new TAD (topologically associating domain) (chr9: 5,400,000-5,600,000) around CD274 and CD273 was discovered, which includes a reported super-enhancer to drive synchronous transcription of PD-L1 and PD-L2. The re-shaped TAD allows transcription factors such as STAT3 and IRF1 recruit to PD-L1 locus in order to guide the expression of PD-L1. After transcription, the PD-L1 is tightly regulated by miRNAs and RNA-binding proteins via the long 3'UTR. At translational level, PD-L1 protein and its membrane presentation are tightly regulated by post-translational modification such as glycosylation and ubiquitination. In addition, PD-L1 can be secreted via exosome to systematically inhibit immune response. Therefore, fully dissecting the regulation of PD-L1/PD-L2 and thoroughly detecting PD-L1/PD-L2 as well as their regulatory networks will bring more insights in ICB and ICB-based combinational therapy.

RUNX3 derived hsa_circ_0005752 accelerates the osteogenic differentiation of adipose-derived stem cells via the miR-496/MDM2-p53 pathway.

BACKGROUND: Circular RNAs (circRNAs) are non-coding RNAs that play a pivotal role in bone diseases. RUNX3 was an essential transcriptional regulator during osteogenesis. However, it is unknown whether RUNX3 regulates hsa_circ_0005752 during osteogenic differentiation. METHODS: The levels of hsa_circ_0005752 and RUNX3 were measured by qRT-PCR after osteogenic differentiation of ADSCs. The osteogenic differentiation was analyzed by Alkaline phosphatase (ALP) staining and Alizarin red staining (ARS). qRT-PCR and western blot were used to assess the expressions of osteogenic differentiation-related molecules. RNA pull-down, RIP, and luciferase reporter assays determine the interactions between miR-496 and hsa_circ_0005752 or MDM2 mRNA. CHIP-PCR analyzed the interaction between RUNX3 and LPAR1. Finally, the potential roles of RUNX3 were investigated during osteogenic differentiation with or without hsa_circ_0005752 knockdown. RESULTS: Hsa_circ_0005752 and RUNX3 were significantly increased, and miR-496 was remarkably decreased in ADSCs after osteogenic differentiation. Hsa_circ_0005752 could promote osteogenic differentiation, as shown by enhancing ALP and ARS staining intensity. Hsa_circ_0005752 enhanced the expressions of Runx2, ALP, Osx, and OCN. Furthermore, hsa_circ_0005752 directly targeted miR-496, which can directly bind to MDM2. RUNX3 bound to the LPAR1 promoter and enhanced hsa_circ_0005752 expressions. Moreover, the enhanced expression of hsa_circ_0005752 by RUNX3 could promote osteogenic differentiation, whereas knockdown of hsa_circ_0005752 partially antagonized the effects of RUNX3. CONCLUSION: Our study demonstrated that RUNX3 promoted osteogenic differentiation via regulating the hsa_circ_0005752/miR-496/MDM2 axis and thus provided a new therapeutic strategy for osteoporosis.

Characteristics of miRNA-SNPs in healthy Japanese subjects and non-small cell lung cancer, colorectal cancer, and soft tissue sarcoma patients.

Single nucleotide polymorphisms in genes encoding microRNAs (miRNA-SNPs) may affect the maturation steps of miRNAs or target mRNA recognition, leading to changes in the expression of target mRNAs to cause gain- or loss-of-function changes. Several miRNA-SNPs are known to be associated with the risk of diseases such as cancer. The purpose of this study was to comprehensively determine the miRNA-SNPs in Japanese individuals to evaluate the differences in allele frequencies between ethnicities by comparing data from the global population in the 1000 Genomes Project and differences between healthy subjects and cancer patients. We performed next-generation sequencing targeting genes encoding 1809 pre-miRNAs. As a result, 403 miRNA-SNPs (146 miRNA-SNPs per subject on average) were identified in 28 healthy Japanese subjects. We observed significant differences in the allele frequencies between ethnicities in 33 of the 403 miRNA-SNPs. The numbers of miRNA-SNPs per subject in 44 non-small cell lung cancer (NSCLC), 33 colorectal cancer (CRC), and 15 soft tissue sarcoma (STS) patients were almost equal to those in healthy subjects. Significant differences in allele frequencies were observed for 14, 11, and 9 miRNA-SNPs in NSCLC, CRC, and STS patients compared with the frequencies in healthy subjects, suggesting that these SNPs can be biomarkers of risk for each type of cancer assessed. In summary, we comprehensively characterized miRNA-SNPs in Japanese individuals and found differences in allele frequencies of several miRNA-SNPs between ethnicities and between healthy subjects and cancer patients. Studies investigating a larger number of subjects should be performed to confirm the potential of miRNA-SNPs as biomarkers of cancer risk.

Hepatic miR-192-3p reactivation alleviates steatosis by targeting glucocorticoid receptor.

BACKGROUND & AIMS: The paradox of hepatic insulin resistance describes the inability for liver to respond to bioenergetics hormones in suppressing gluconeogenesis whilst maintaining lipid synthesis. Here, we report the deficiency of miR-192-3p in the livers of mice with diabetes and its role in alleviating hepatic steatosis. METHODS: As conventional pre-microRNA (miRNA) stem-loop overexpression only boosts guiding strand (i.e. miR-192-5p) expression, we adopted an artificial AAV(DJ)-directed, RNA Pol III promoter-driven miRNA hairpin construct for star-strand-specific overexpression in the liver. Liver steatosis and insulin resistance markers were evaluated in primary hepatocytes, mice with diabetes, and mice with excessive carbohydrate consumption. RESULTS: Functional loss of miR-192-3p in liver exacerbated hepatic micro-vesicular steatosis and insulin resistance in either mice with diabetes or wild-type mice with excessive fructose consumption. Liver-specific overexpression of miR-192-3p effectively halted hepatic steatosis and ameliorated insulin resistance in these mice models. Likewise, hepatocytes overexpressing miR-192-3p exhibited improved lipid accumulation, accompanied with decreases in lipogenesis and lipid-accumulation-related transcripts. Mechanistically, glucocorticoid receptor (GCR, also known as nuclear receptor subfamily 3, group C, member 1 [NR3C1]) was demonstrated to be negatively regulated by miR-192-3p. The effect of miR-192-3p on mitigating micro-vesicular steatosis was ablated by the reactivation of NR3C1. CONCLUSIONS: The star strand miR-192-3p was an undermined glycerolipid regulator involved in controlling fat accumulation and insulin sensitivity in liver through blockade of hepatic GCR signalling; this miRNA may serve as a potential therapeutic option for the common co-mobility of diabetic mellitus and fatty liver disease. LAY SUMMARY: The potential regulatory activity of star strand microRNA (miRNA) species has been substantially underestimated. In this study, we investigate the role and mechanism of an overlooked star strand miRNA (miR-192-3p) in regulating hepatic steatosis and insulin signalling in the livers of mice with diabetes and mice under excessive carbohydrate consumption.

miR-22 inhibition reduces hepatic steatosis via FGF21 and FGFR1 induction.

BACKGROUND & AIMS: Metabolism supports cell proliferation and growth. Surprisingly, the tumor suppressor miR-22 is induced by metabolic stimulators like bile acids. Thus, this study examines whether miR-22 could be a metabolic silencer. METHODS: The relationship between miR-22 and the expression of fibroblast growth factor 21 (FGF21) and its receptor FGFR1 was studied in cells and fatty livers obtained from patients and mouse models. We evaluated the effect of an miR-22 inhibitor alone and in combination with obeticholic acid (OCA) for the treatment of steatosis. RESULTS: The levels of miR-22 were inversely correlated with those of FGF21, FGFR1, and PGC1α in human and mouse fatty livers, suggesting that hepatic miR-22 acts as a metabolic silencer. Indeed, miR-22 reduced FGFR1 by direct targeting and decreased FGF21 by reducing the recruitment of PPARα and PGC1α to their binding motifs. In contrast, an miR-22 inhibitor increases hepatic FGF21 and FGFR1, leading to AMPK and ERK1/2 activation, which was effective in treating alcoholic steatosis in mouse models. The farnesoid x receptor-agonist OCA induced FGF21 and FGFR1, as well as their inhibitor miR-22. An miR-22 inhibitor and OCA were effective in treating diet-induced steatosis, both alone and in combination. The combined treatment was the most effective at improving insulin sensitivity, releasing glucagon-like peptide 1, and reducing hepatic triglyceride in obese mice. CONCLUSION: The simultaneous induction of miR-22, FGF21 and FGFR1 by metabolic stimulators may maintain FGF21 homeostasis and restrict ERK1/2 activation. Reducing miR-22 enhances hepatic FGF21 and activates AMPK, which could be a novel approach to treat steatosis and insulin resistance. LAY SUMMARY: This study examines the metabolic role of a tumor suppressor, miR-22, that can be induced by metabolic stimulators such as bile acids. Our novel data revealed that the metabolic silencing effect of miR-22 occurs as a result of reductions in metabolic stimulators, which likely contribute to the development of fatty liver. Consistent with this finding, an miR-22 inhibitor effectively reversed both alcohol- and diet-induced fatty liver; miR-22 inhibition is a promising therapeutic option which could be used in combination with obeticholic acid.

The 3'-untranslated region contributes to the pregnane X receptor (PXR) expression down-regulation by PXR ligands and up-regulation by glucocorticoids.

Pregnane X receptor (PXR) is the major regulator of xenobiotic metabolism. PXR itself is controlled by various signaling molecules including glucocorticoids. Moreover, negative feed-back regulation has been proposed at the transcriptional level. We examined the involvement of the 3'-untranslated region (3'-UTR) of NR1I2 mRNA and microRNAs in PXR- and glucocorticoid receptor (GR)-mediated regulation of NR1I2 gene expression. PXR ligands were found to significantly downregulate NR1I2 mRNA expression in a set of 14 human hepatocyte cultures. Similarly, PXR was downregulated by PCN in the C57/BL6 mice liver. In mechanistic studies with the full-length 3'-UTR cloned into luciferase reporter or expression vectors, we showed that the 3'-UTR reduces PXR expression. From the miRNAs tested, miR-18a-5p inhibited both NR1I2 expression and CYP3A4 gene induction. Importantly, we observed significant upregulation of miR-18a-5p expression 6 h after treatment with the PXR ligand rifampicin, which indicates a putative mechanism underlying NR1I2 negative feed-back regulation in hepatic cells. Additionally, glucocorticoids upregulated NR1I2 expression not only through the promoter region but also via 3'-UTR regulation, which likely involves downregulation of miR-18a-5p. We conclude that miR-18a-5p is involved in the down-regulation of NR1I2 expression by its ligands and in the upregulation of NR1I2 mRNA expression by glucocorticoids in hepatic cells.

Differential modulation of Ahr and Arid5a: A promising therapeutic strategy for autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease that involves demyelination of axons in the central nervous system (CNS) and affects patients worldwide. It has been demonstrated that ligand-activated aryl hydrocarbon receptor (Ahr) ameliorates experimental autoimmune encephalomyelitis (EAE), a murine model of MS, by increasing CD4+FoxP3+ T cells. Recent evidence indicates that AT-rich interactive domain-containing protein 5a (Arid5a) is required for EAE pathogenesis by stabilizing Il6 and OX40 mRNAs. However, the differential modulation of Ahr and Arid5a in autoimmunity as a therapeutic strategy is unexplored. Herein, an in silico, in vitro and in vivo approach identified Flavipin (3,4,5-trihydroxy-6-methylphthalaldehyde) as an Ahr agonist that induces the expression of Ahr downstream genes in mouse CD4+ T cells and CD11b+ macrophages. Interestingly, Flavipin inhibited the stabilizing function of Arid5a and its counteracting effects on Regnase-1 on the 3' untranslated region (3'UTR) of target mRNAs. Furthermore, it inhibited the stabilizing function of Arid5a on Il23a 3'UTR, a newly identified target mRNA. In EAE, Flavipin ameliorated disease severity, with reduced CD4+IL-17+ T cells, IL-6 and TNF-α and increased CD4+FoxP3+ T cells. Moreover, EAE amelioration was concomitant with reduced CD4+OX40+ and CD4+CD45+ T cells in the CNS. RNA interference showed that the modulatory effects of Flavipin on pro- and anti-inflammatory mediators in CD4+ T cells and macrophages were Ahr- and/or Arid5a-dependent. In conclusion, our findings reveal differential modulation of Ahr and Arid5a as a new therapeutic strategy for MS.

The 3'-untranslated region contributes to the pregnane X receptor (PXR) expression down-regulation by PXR ligands and up-regulation by glucocorticoids

Pregnane X receptor (PXR) is the major regulator of xenobiotic metabolism. PXR itself is controlled by various signaling molecules including glucocorticoids. Moreover, negative feed-back regulation has been proposed at the transcriptional level. We examined the involvement of the 3'-untranslated region (3'-UTR) of mRNA and microRNAs in PXR- and glucocorticoid receptor (GR)-mediated regulation of gene expression. PXR ligands were found to significantly downregulate mRNA expression in a set of 14 human hepatocyte cultures. Similarly, PXR was downregulated by PCN in the C57/BL6 mice liver. In mechanistic studies with the full-length 3'-UTR cloned into luciferase reporter or expression vectors, we showed that the 3'-UTR reduces PXR expression. From the miRNAs tested, miR-18a-5p inhibited both expression and gene induction. Importantly, we observed significant upregulation of miR-18a-5p expression 6 h after treatment with the PXR ligand rifampicin, which indicates a putative mechanism underlying negative feed-back regulation in hepatic cells. Additionally, glucocorticoids upregulated expression not only through the promoter region but also 3'-UTR regulation, which likely involves downregulation of miR-18a-5p. We conclude that miR-18a-5p is involved in the down-regulation of expression by its ligands and in the upregulation of mRNA expression by glucocorticoids in hepatic cells.

Development of precision medicine approaches based on inter-individual variability of BCRP/ABCG2.

Precision medicine is a rapidly-developing modality of medicine in human healthcare. Based on each patient׳s unique characteristics, more accurate dosages and drug selection can be made to achieve better therapeutic efficacy and less adverse reactions in precision medicine. A patient׳s individual parameters that affect drug transporter action can be used to develop a precision medicine guidance, due to the fact that therapeutic efficacy and adverse reactions of drugs can both be affected by expression and function of drug transporters on the cell membrane surface. The purpose of this review is to summarize unique characteristics of human breast cancer resistant protein (BCRP) and the genetic variability in the BCRP encoded gene ABCG2 in the development of precision medicine. Inter-individual variability of BCRP/ABCG2 can impact choices and outcomes of drug treatment for several diseases, including cancer chemotherapy. Several factors have been implicated in expression and function of BCRP, including genetic, epigenetic, physiologic, pathologic, and environmental factors. Understanding the roles of these factors in controlling expression and function of BCRP is critical for the development of precision medicine based on BCRP-mediated drug transport.

Bioengineered miR-27b-3p and miR-328-3p modulate drug metabolism and disposition via the regulation of target ADME gene expression.

Drug-metabolizing enzymes, transporters, and nuclear receptors are essential for the absorption, distribution, metabolism, and excretion (ADME) of drugs and xenobiotics. MicroRNAs participate in the regulation of ADME gene expression via imperfect complementary Watson-Crick base pairings with target transcripts. We have previously reported that Cytochrome P450 3A4 (CYP3A4) and ATP-binding cassette sub-family G member 2 (ABCG2) are regulated by miR-27b-3p and miR-328-3p, respectively. Here we employed our newly established RNA bioengineering technology to produce bioengineered RNA agents (BERA), namely BERA/miR-27b-3p and BERA/miR-328-3p, via fermentation. When introduced into human cells, BERA/miR-27b-3p and BERA/miR-328-3p were selectively processed to target miRNAs and thus knock down CYP3A4 and ABCG2 mRNA and their protein levels, respectively, as compared to cells treated with vehicle or control RNA. Consequently, BERA/miR-27b-3p led to a lower midazolam 1'-hydroxylase activity, indicating the reduction of CYP3A4 activity. Likewise, BERA/miR-328-3p treatment elevated the intracellular accumulation of anticancer drug mitoxantrone, a classic substrate of ABCG2, hence sensitized the cells to chemotherapy. The results indicate that biologic miRNA agents made by RNA biotechnology may be applied to research on miRNA functions in the regulation of drug metabolism and disposition that could provide insights into the development of more effective therapies.

Bioengineered miR-27b-3p and miR-328-3p modulate drug metabolism and disposition the regulation of target ADME gene expression

Drug-metabolizing enzymes, transporters, and nuclear receptors are essential for the absorption, distribution, metabolism, and excretion (ADME) of drugs and xenobiotics. MicroRNAs participate in the regulation of ADME gene expression imperfect complementary Watson-Crick base pairings with target transcripts. We have previously reported that Cytochrome P450 3A4 (CYP3A4) and ATP-binding cassette sub-family G member 2 (ABCG2) are regulated by miR-27b-3p and miR-328-3p, respectively. Here we employed our newly established RNA bioengineering technology to produce bioengineered RNA agents (BERA), namely BERA/miR-27b-3p and BERA/miR-328-3p, fermentation. When introduced into human cells, BERA/miR-27b-3p and BERA/miR-328-3p were selectively processed to target miRNAs and thus knock down and mRNA and their protein levels, respectively, as compared to cells treated with vehicle or control RNA. Consequently, BERA/miR-27b-3p led to a lower midazolam 1'-hydroxylase activity, indicating the reduction of CYP3A4 activity. Likewise, BERA/miR-328-3p treatment elevated the intracellular accumulation of anticancer drug mitoxantrone, a classic substrate of ABCG2, hence sensitized the cells to chemotherapy. The results indicate that biologic miRNA agents made by RNA biotechnology may be applied to research on miRNA functions in the regulation of drug metabolism and disposition that could provide insights into the development of more effective therapies.

Development of precision medicine approaches based on inter-individual variability of BCRP/

Precision medicine is a rapidly-developing modality of medicine in human healthcare. Based on each patient׳s unique characteristics, more accurate dosages and drug selection can be made to achieve better therapeutic efficacy and less adverse reactions in precision medicine. A patient׳s individual parameters that affect drug transporter action can be used to develop a precision medicine guidance, due to the fact that therapeutic efficacy and adverse reactions of drugs can both be affected by expression and function of drug transporters on the cell membrane surface. The purpose of this review is to summarize unique characteristics of human breast cancer resistant protein (BCRP) and the genetic variability in the BCRP encoded gene in the development of precision medicine. Inter-individual variability of BCRP/ can impact choices and outcomes of drug treatment for several diseases, including cancer chemotherapy. Several factors have been implicated in expression and function of BCRP, including genetic, epigenetic, physiologic, pathologic, and environmental factors. Understanding the roles of these factors in controlling expression and function of BCRP is critical for the development of precision medicine based on BCRP-mediated drug transport.

Brain-derived neurotrophic factor is required for axonal growth of selective groups of neurons in the arcuate nucleus.

OBJECTIVE: Brain-derived neurotrophic factor (BDNF) is a potent regulator of neuronal development, and the Bdnf gene produces two populations of transcripts with either a short or long 3' untranslated region (3' UTR). Deficiencies in BDNF signaling have been shown to cause severe obesity in humans; however, it remains unknown how BDNF signaling impacts the organization of neuronal circuits that control energy balance. METHODS: We examined the role of BDNF on survival, axonal projections, and synaptic inputs of neurons in the arcuate nucleus (ARH), a structure critical for the control of energy balance, using Bdnf (klox/klox) mice, which lack long 3' UTR Bdnf mRNA and develop severe hyperphagic obesity. RESULTS: We found that a small fraction of neurons that express the receptor for BDNF, TrkB, also expressed proopiomelanocortin (POMC) or neuropeptide Y (NPY)/agouti-related protein (AgRP) in the ARH. Bdnf(klox/klox) mice had normal numbers of POMC, NPY, and TrkB neurons in the ARH; however, retrograde labeling revealed a drastic reduction in the number of ARH axons that project to the paraventricular hypothalamus (PVH) in these mice. In addition, fewer POMC and AgRP axons were found in the dorsomedial hypothalamic nucleus (DMH) and the lateral part of PVH, respectively, in Bdnf (klox/klox) mice. Using immunohistochemistry, we examined the impact of BDNF deficiency on inputs to ARH neurons. We found that excitatory inputs onto POMC and NPY neurons were increased and decreased, respectively, in Bdnf (klox/klox) mice, likely due to a compensatory response to marked hyperphagia displayed by the mutant mice. CONCLUSION: This study shows that the majority of TrkB neurons in the ARH are distinct from known neuronal populations and that BDNF plays a critical role in directing projections from these neurons to the DMH and PVH. We propose that hyperphagic obesity due to BDNF deficiency is in part attributable to impaired axonal growth of TrkB-expressing ARH neurons.

Global insights into alternative polyadenylation regulation.

Alternative pre-mRNA processing greatly increases the coding capacity of the human genome and regulatory factors involved in RNA processing play critical roles in tissue development and maintenance. Indeed, abnormal functions of RNA processing factors have been associated with a wide range of human diseases from cancer to neurodegenerative disorders. While many studies have emphasized the importance of alternative splicing (AS), recent high-throughput sequencing efforts have also allowed global surveys of alternative polyadenylation (APA). For the majority of pre-mRNAs, as well as some non-coding transcripts such as lncRNAs, APA selects different 3'-ends and thus modulates the availability of regulatory sites recognized by trans-acting regulatory effectors, including miRs and RNA binding proteins (RBPs). Here, we compare the available technologies for assessing global polyadenylation patterns, summarize the roles of auxiliary factors on APA, and discuss the impact of differential polyA site (pA) selection in the determination of cell fate, transformation and disease.