LncRNA NEAT1 Promotes Inflammatory Response in Sepsis via the miR-31-5p/POU2F1 Axis.

Sepsis is considered to be a systemic inflammatory response, which results in organ dysfunction. LncRNA nuclear-enriched abundant transcript 1 (NEAT1) involved in sepsis progression has been reported. However, the underlying mechanism of NEAT1 in sepsis-induced inflammatory response remains to be revealed. In this study, NEAT1 and POU domain class 2 transcription factor 1 (POU2F1) were highly expressed in LPS-induced septic RAW264.7 cells, opposite to miR-31-5p expression. Furthermore, we found that NEAT1 silencing inhibited LPS-induced inflammatory response and cell proliferation, and promoted cell apoptosis. Subsequently, we found that miR-31-5p interacted with NEAT1 and targeted the 3'UTR of POU2F1, and in LPS-induced RAW264.7 cells, the inhibition of NEAT1 silencing was reversed by miR-31-5p knockdown, while POU2F1 downregulation could cover the functions of miR-31-5p knockdown. In a word, this study indicates that NEAT1 inhibits the LPS-induced progression of sepsis in RAW264.7 cells by modulating miR-31-5p/POU2F1 axis, suggesting that NEAT1 will be the potential therapeutic target for sepsis.

Influence of MRP3 Genetics and Hepatic Expression Ontogeny for Morphine Disposition in Neonatal and Pediatric Patients.

We have previously reported the influences of OCT1 ontogeny and genetic variation on morphine clearance in neonatal and pediatric patients. In the latter study, plasma morphine-glucuronide levels correlated with patient genotype for the rs4793665 single-nucleotide polymorphism (SNP) at the locus of MRP3, an efflux transporter of morphine glucuronides between hepatocytes and circulating blood. The link between MRP3 activity and overall morphine clearance has not been thoroughly investigated however, and the developmental profile of hepatic MRP3 protein expression remains thinly defined between neonates and adults. In the current study, previously determined morphine clearance values for neonatal (24-58 weeks postmenstrual age, N = 57) and pediatric (5-16 years, n = 85) patients were reanalyzed for correlation to the SNP genotype of patient rs4793665. Among OCT1 wild-type patients, pediatric morphine clearance showed a significant decreasing trend by MRP3 genotypes in the order of CC > CT > TT (P = .014), whereas for neonates, an identical but nonsignificant trend was observed. Pharmacogenetic differences in MRP3 and OCT1 ontogeny were evaluated by Western blot of hepatic membrane fractions from 50 subjects aged 1 day postnatal to 33 years old. Hepatic MRP3 protein level did not vary by rs4793665 genotype, and followed an atypical developmental pattern of increase up to 1-2 years of age, thereafter decreasing during preadolescence before increasing again to adult levels at maturity (17-33 years). By comparison, OCT1 expression was significantly decreased in OCT1 *1/*3 genotyped patients older than 1 year and followed a trajectory consistent with prior studies. Our results suggest that consideration of MRP3 pharmacogenetics and ontogeny may aid in identifying pediatric patients having different/atypical morphine requirements.

Metabolic Acidosis Alters Expression of Slc22 Transporters in Mouse Kidney.

INTRODUCTION: The kidneys play a central role in eliminating metabolic waste products and drugs through transporter-mediated excretion along the proximal tubule. This task is mostly achieved through a variety of transporters from the solute carrier family 22 (SLC22) family of organic cation and anion transporters. Metabolic acidosis modulates metabolic and renal functions and also affects the clearance of metabolites and drugs from the body. We had previously shown that induction of metabolic acidosis in mice alters a large set of transcripts, among them also many transporters including transporters from the Slc22 family. OBJECTIVE: Here we further investigated the impact of acidosis on Slc22 family members. METHODS: Metabolic acidosis was induced for 2 or 7 days with NH4Cl, some animals also received the uricase inhibitor oxonic acid for comparison. Expression of transporters was studied by qPCR and immunoblotting. RESULTS: NH4Cl induced no significant changes in plasma or urine uric acid levels but caused downregulation of Slc22a1 (Oct1), Slc22a6 (Oat1), Slc22a19 (Oat5), and -Slc22a12 (Urat1) at mRNA level. In contrast, Slc22a4 mRNA (Octn1) was upregulated. On protein level, NH4Cl increased Octn1 (after 7 days) and Urat1 (after 2 days) abundance and decreased Oat1 (after 2 days) and Urat1 (after 7 days). Oxonic acid had no impact on protein abundance of any of the transporters tested. CONCLUSION: In summary, metabolic acidosis alters expression of several transporters involved in renal excretion of metabolic waste products and drugs. This may have implications for drug kinetics and clearance of waste metabolites.

The human organic cation transporter OCT1 and its role as a target for drug responses.

The human organic cation uptake transporter OCT1, encoded by the SLC22A1 gene, is highly expressed in the liver and reported to possess a broad substrate specificity. OCT1 operates by facilitated diffusion and allows the entry of nutrients into cells. Recent findings revealed that OCT1 can mediate the uptake of drugs for treating various diseases such as cancers. The levels of OCT1 expression correlate with the responses towards many drugs and functionally defective OCT1 lead to drug resistance. It has been recently proposed that OCT1 should be amongst the crucial drug targets used for pharmacogenomic analyses. Several single nucleotide polymorphisms exist and are distributed across the entire OCT1 gene. While there are differences in the OCT1 gene polymorphisms between populations, there are at least five variants that warrant consideration in any genetic screen. To date, and despite two decades of research into OCT1 functional role, it still remains uncertain what are the define substrates for this uptake transporter, although studies from mice revealed that one of the substrates is vitamin B1. It is also unclear how OCT1 recognizes a broad array of ligands and whether this involves specific modifications and interactions with other proteins. In this review, we highlight the current findings related to OCT1 with the aim of propelling further studies on this key uptake transporter.

Long noncoding RNA NEAT1 promotes the growth of cervical cancer cells via sponging miR-9-5p.

Evidence has accumulated demonstrating that long noncoding RNAs (lncRNAs) participate in the initiation and progression of cancers. In this study, we found that the lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) is significantly increased in both cervical cancer tissues and cell lines. Overexpression of NEAT1 promoted the proliferation and migration of cervical cancer cells. Molecular studies uncovered that NEAT1 functions as competitive endogenous RNA (ceRNA), binding the micro-RNA miR-9-5p and suppressing its expression. However, we consistently found that when NEAT1 was highly expressed, it attenuated the inhibitory effect of miR-9-5p on the expression of PTEN and POU2F1, which are the targets of miR-9-5p. Consistent with the negative regulation of NEAT1 on miR-9-5p, restoration of miR-9-5p inhibited the growth-promoting effects of NEAT1 on cervical cancer cells. Taken together, these results indicated that NEAT1 plays an important role in the regulation cervical cancer cell growth by targeting miR-9-5p. Our findings characterized the possible mechanism of NEAT1 in cervical cancer.

hOCT1 gene expression predict for optimal response to Imatinib in Tunisian patients with chronic myeloid leukemia.

PURPOSE: Imatinib mesylate (IM) is considered as a highly effective therapy for chronic myeloid leukemia (CML) patients. However, a minority of patients fail to achieve optimal response due to impaired bioavailability of IM. The human organic cation transporter 1 (OCT1; SLC22A1) has been reported to be the main influx transporter involved in IM uptake into CML cells. Genetic variants and/or hOCT1 expression changes may influence IM response. In this study, we aimed to investigate the impact of both hOCT1 polymorphisms located in exon 7 and hOCT1 mRNA levels on the clinical outcome in CML patients. METHODS: hOCT1 expression profile was determined using the quantitative real-time polymerase chain reaction in 69 CML patients treated with IM (35 responders to IM patients and 34 IM-resistant patients), while genotyping of 69 cases and 51 controls for hOCT1 polymorphisms was performed by direct sequencing after amplification of exon7. RESULTS: Our results showed that the hOCT1 gene was significantly downregulated in the samples of the IM-resistant group when compared with the IM-responder group (p = 0.0211). Moreover, sequencing data show an association in all cases between the SNP 408V>M (g.1222G>A) and an intronic 8 bp (base pairs) insertion of GTAAGTTG (rs36056065) at the 3' end of exon 7. The genotype and allele distribution of the different SNPs did not differ significantly between the two groups of patients. CONCLUSIONS: hOCT1 mRNA expression may serve as a clinical biomarker of response to imatinib and could be useful to predict IM therapy outcome of CML patients.

Developmental Changes in Hepatic Organic Cation Transporter OCT1 Protein Expression from Neonates to Children.

Organic cation transporter 1 (OCT1) plays an important role in the disposition of clinically important drugs, and the capacity of OCT1 activity is presumed to be proportional to the protein expression level in organ tissues. Knowledge of OCT1 protein expression in children, especially neonates and small infants, is currently very limited. Here, we report on the characterization of OCT1 protein expression in neonatal, infant, and pediatric liver samples performed using immunoblot analysis. OCT1 protein expression was detected in liver samples from neonates as early as postnatal days 1 and 2. This youngest group showed significantly lower OCT1 expression normalized by glyceraldehyde-6-phosphate dehydrogenase (values given as means ± S.D. in arbitrary units; 0.03 ± 0.02, n = 7) compared with samples from patients aged 3 to 4 weeks (0.08 ± 0.03, n = 5, P < 0.01), 3 to 6 months (0.23 ± 0.15, n = 7, P < 0.01), 11 months to 1 year (0.42 ± 0.32, n = 6, P < 0.01), and 8 to 12 years (1.00 ± 0.44, n = 7, P < 0.01). These data demonstrate an age-dependent increase in OCT1 expression from birth up to 8 to 12 years of age, and the findings of this study contribute to the understanding of OCT1 functional capacity and its effect upon the disposition of OCT1 substrates in neonates and small infants.

Octamer transcription factor 1 mediates epithelial-mesenchymal transition in colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancer types worldwide. Octamer transcription factor 1 (OCT1) is associated with tumor progression and a poor patient survival rate. However, little is known regarding the effect of OCT1 in CRC. Moreover, because the epithelial-to-mesenchymal transition (EMT) is a key player in metastasis, whether OCT1 induces EMT in CRC remains unclear. In the present study, we investigate the role of OCT1 in CRC and its expression pattern and clinical significance. The expression of OCT1 in CRC tissues and the adjacent noncancerous tissues was detected using quantitative real-time PCR (QRT-PCR), Western blot, and immunohistochemistry analyses. In addition, silencing of OCT1 with small interfering RNA (siRNA) was performed in CRC cell lines, and the impact on proliferation, migration, and the EMT marker of CRC was analyzed. Our results found that OCT1 levels were significant higher in CRC tissues compared with the adjacent noncancerous tissues. Furthermore, OCT1 siRNA significantly reduced the proliferation rate of SW620 and LoVo cells, inhibited the migration and invasion, and could reverse EMT in these two CRC cells, indicating that OCT1 plays a critical role in CRC progression.

Differential regulation of hepatic organic cation transporter 1, organic anion-transporting polypeptide 1a4, bile-salt export pump, and multidrug resistance-associated protein 2 transporter expression in lymphocyte-deficient mice associates with interleukin-6 production.

Cholestasis results from interrupted bile flow and is associated with immune-mediated liver diseases. It is unclear how inflammation contributes to cholestasis. The aim of this study was to determine whether T and B cells contribute to hepatic transporter expression under basal and inflammatory conditions. C57BL/6J wild-type mice or strains lacking T, B, or both T and B cells were exposed to lipopolysaccharide (LPS) or saline, and livers were collected 16 hours later. Branched DNA signal amplification was used to assess mRNA levels of organic anion-transporting polypeptides (Oatp) 1a1, 1a4, and 1b2; organic cation transporter (Oct) 1; canalicular bile-salt export pump (Bsep); multidrug resistance-associated proteins (Mrp) 2 and 3; and sodium-taurocholate cotransporting polypeptide (Ntcp). Real-time polymerase chain reaction analysis was used to correlate changes of transporter expression with interleukin-1b (IL-1b), IL-6, IL-17A, IL-17F, tumor necrosis factor-α (TNF-α), and interferon-γ expression in the liver. LPS treatment inhibited Bsep and Oct1 mRNA expression, and this was abrogated with a loss of T cells, but not B cells. In addition, the absence of T cells increased Mrp2 mRNA expression, whereas B cell deficiency attenuated Oatp1a4 mRNA in LPS-treated mice. Oatp1a1, Oatp1b2, Ntcp, and Mrp3 were largely unaffected by T or B cell deficiency. Lymphocyte deficiency altered basal and inflammatory IL-6, but not TNF-α or IL-1b, mRNA expression. Taken together, these data implicate lymphocytes as regulators of basal and inflammatory hepatic transporter expression and suggest that IL-6 signaling may play a critical role.

Exaggerated myocardial oxLDL amount and LOX-1 receptor over-expression associated with coronary microvessel inflammation in unstable angina.

The pathophysiological relationship between coronary atherosclerosis and coronary microvessels remains undefined and the specific causative role of oxidatively modified low density lipoprotein (oxLDL) in human atherosclerosis is debated. The purposes of this study are to investigate whether coronary microvessels are involved in coronary atherosclerosis and whether increased myocardial oxLDL amount can be associated with coronary microvessel inflammation. A combination of immunohistochemical, RT-PCR and real-time PCR studies performed on myocardial biopsy specimens from patients with mitral stenosis (control hearts, CHs) and from unstable and stable angina patients (UAP and SAP), demonstrated that myocardial oxLDL was associated with a chronic low-grade inflammation in SAP and with a severe high grade inflammation in UAP. oxLDL amount was notably higher in UAP than in SAP and in UAP the high grade of inflammation was correlated with the increased amount of oxLDL in endothelial cells and macrophages. The exaggerated amount of oxLDL in UAP and the interaction of oxLDL with lectin-like oxLDL (LOX-1) receptor are amplified by the activation of transcriptional factor octamere 1 (OCT-1) with consequent activation of a series of inflammatory endothelial feed-back mechanisms resulting in LOX-1 gene over-expression, endothelial inflammation as well as uncontrolled nuclear factor kappa B (NFkB) activation. Moreover, in UAP genes for signal transducer and activator transcriptional factor 1α (STAT1α), angiotensin converting enzyme (ACE) and numerous pro-inflammatory cytokines were over-expressed. The present results may have clinical relevance because they show that coronary atherosclerosis is a disease not confined to the large arteries but involving the whole coronary tree. In UAP the exaggerated amount of myocardial oxLDL is associated with widespread high grade microvessel inflammation.

A far-upstream Oct-1 motif regulates cytokine-induced transcription of the human inducible nitric oxide synthase gene.

Transcriptional regulation of the human inducible nitric oxide synthase (hiNOS) gene is highly complex and requires an orchestrated flow of positive and negative transcription factors that bind to specific cis-acting upstream response elements. Very little specific information exists about the far-upstream region of the hiNOS gene. Oct-1 protein belongs to the Pit-Oct-Unc domain transcription factor family and is constitutively expressed in all dividing cells. It is essential for proliferation, differentiation, and other key cell processes. However, the role of Oct-1 in regulating hiNOS gene expression has not been reported. In this work, the octamer sequence 5'-ATGCAAAT-3' at -10.2 kb in the hiNOS promoter was identified as high-affinity Oct-1 binding by electrophoretic mobility shift assay in vitro and chromatin immunoprecipitation assay in vivo. Mutation of Oct-1 motif at -10.2 kb in the hiNOS promoter decreased cytokine-induced hiNOS promoter activity by 40%. Cytokine-induced hiNOS promoter activity was also significantly reduced by Oct-1 small interfering RNA targeting. Overexpression of Oct-1 increased cytokine-induced hiNOS protein expression in primary human hepatocytes. Furthermore, the Oct-1 motif at -10.2 kb of the hiNOS promoter conferred increased transcriptional activity to the heterologous thymidine kinase promoter irrespective of cytokine induction. Taken together, this work identifies a far-upstream functional Oct-1 enhancer motif at -10.2 kb in the hiNOS promoter that regulates cytokine-induced hiNOS gene transcription and further underscores tight control mechanisms regulating the expression of the hiNOS gene.

[Alternative promoters and oct-1 gene tissue-specific regulation].

Murine oct-1 gene contains exons IU and 1L at 5'-end, which are alternatively presented in oct-1 mRNA. 1U exon is presented in oct-1 mRNA expressed in all tissues whereas lymphoid cells of mouse and human express mRNA containing alternatively IU or 1L exon. Upstream of these exons there are U and L promoters, respectively, which are separated by 67 kb in mice otf-1 locus. Nucleotide sequences located upstream 1U and 1L exons are very different: IU region contains a number of Sp1 sites and 1L region contains many homeo-specific sites and two octamers ATGCAAAT recognized by transcription factors Oct-1, Oct-2 and other POU-domain containing proteins. Oct-1 sites may participate in oct-1 gene autoregulation. The role of U- and L-promoter fragments in oct-1 gene regulation was studied by transfection of luc-containing constructs in lymphoid (NS/0) and non-lymphoid cells. It was shown that L-promoter efficiency is much higher in lymphoid cells than in fibroblast cell line 10/1. U-promoter is active in both types of the cells. Upstream of IL-exon there is the inhibitory nucleotide sequence which elimination increases the expression level by many times. This silencing element is located between initiation starts of transcription and translation. The obtained data suggest that oct-1 gene contains at least two alternative promoters. It seems that U-promoter promises the constitutive synthesis of Oct-1 protein whereas L-promoter is tissue-specific and putatively is inducible and autoregulated.