LncRNA H19-elevated LIN28B promotes lung cancer progression through sequestering miR-196b

<p>LncRNA H19 is involved in the development of multiple cancers. Here, we firstly provide new evidence that H19 can induce LIN28B, a conserved RNA binding protein, to accelerate lung cancer growth through sponging miR-196b. Abundance in LIN28B was observed in clinical lung cancer samples. A positive link was observed between H19 and LIN28B in clinical lung cancer samples. In lung cancer cells, H19 was capable of increasing LIN28B expression. Mechanistically, miR-196b directly targeted LIN28B to inhibit LIN28B expression. H19 was capable of promoting LIN28B expression through sequestering miR-196b. Functionally, H19-increased LIN28B conferred the cell proliferation of lung cancer. Our finding indicates that H19 depresses miR-196b to elevate LIN28B, resulting in accelerating cell proliferation in lung cancer.</p

Two new prenylated phloroglucinol derivatives from <i>Hypericum scabrum</i>

<p>Two new prenylated phloroglucinol derivatives (<b>1</b>–<b>2</b>), and a known compound furohyperforim isomer 2 (<b>3</b>), were isolated from the aerial parts of <i>Hypericum scabrum</i>. Their structures were elucidated by various spectroscopic methods, including MS, IR, UV, and NMR.</p

Prx-I knockdown inhibited the proliferation of T24 cells in vitro.

<p>The growth rates in Prx-I knockdown group was significantly reduced, compared with the parental and con sh groups, measured by CCK8 assay.</p

miR-1254 down-regulates HO-1 transcription via targeting TFAP2A.

<p>(A) qRT-PCR analysis of TFAP2A, USF1 and NF-κB mRNA levels in cells transfected with indicated oligonucleotides. (B) Luciferase activity assays of the PGL-HO1 reporter in HEK293 cells with TFAP2A knockdown (left) or over-expression (right). (C) Western blot analysis of the effect of TFAP2A knockdown on HO-1 protein expression compared with miR-1254 in A549 cells. (D) The mRNA levels of TFAP2A, USF1 and NF-κB in CRISPR/Cas9-modified miR-1254 knockdown A549 cells. (E) Western blot analysis of the protein level of TFAP2A in the CRISPR/Cas9-modified miR-1254 knockdown A549 cells. (F) HO-1 protein levels in cells transfected with the indicated oligonucleotides (miR-1254 and its mutants) for 48 h assayed by immunoblotting. Left: Representative images. Right: Statistical results. (G) Western blot analysis of the TFAP2A over-expression effect on HO-1 protein expression compared with miR-1254 in A549 cells. (H) ChIP analysis of the polⅡ and TFAP2A enrichment in HO-1 promoter in A549 cells using antibodies against polⅡand TFAP2A, with IgG as a negative control. Left: Representative images. Right: Statistical results. Data are presented as the mean ± SEM of three independent experiments. *<i>P</i><0.05, **<i>P</i> and ***<i>P</i> <0.01 vs. nc; #<i>P</i><0.05, ##<i>P</i> and ###<i>P</i> <0.01 vs miR-1254.</p

MiR-1254 suppresses NSCLC tumor growth in vivo.

<p>(A) Representative photographs of the tumors at day 35 after inoculation with either the A549/miR-1254 or A549/miR-Control cells. (B) Comparison of HO-1 mRNA levels in paired NSCLC tumor samples and normal lung tissue specimens (n = 34). (C) Analysis of the TFAP2A expression in paired tumor and normal samples from patients with NSCLC in The Cancer Genome Atlas (TCGA) database (n = 57). (D) Inverse correlation between HO-1 expression and miR-1254 level in NSCLC tumor samples. miR-1254 and HO-1 mRNA levels in tumor specimens (n = 34) were determined by qRT–PCR, with U6 andβ-actin as respective internal normalized references. The correlation between the miR-1254 and HO-1 expression in these tumors was examined using the Spearman’s correlation test. (E) qRT–PCR analysis of the expression of miR-1254 and HO-1 in the lung cancer cell lines (NCI-1395, NCI-H1975, A549, MSTO-211H, NCI-H292, Calu-3). U6 and β-actin served as internal normalized references for miR-1254 and HO-1, respectively. (F) Diagram of working model. Data in all experiments were mean ± SEM of three independent experiments in triplicates. <i>*P</i> < 0.05, <i>***P</i> < 0.01 vs. nc.</p

HO-1 plays a critical role in miR-1254-modified cell growth suppression of NSCLC cells.

<p>(A-C) Inhibition of cell growth by miR-1254 in A549 cells. Cells were transfected with miR-1254 mimics or negative control oligonucleotides (nc), 20μM hemin was used to rescue the expression of HO-1 as a inducer. (A) Trypan blue staining assays. Cells were counted 72h after transfection. (B) MTT analysis of cell viability in A549 cells transfected with miR-1254 mimics or nc. (C) Colony formation in A549 cells transfected with miR-1254 mimics compared with nc. Upper: Representative image of the colony formation. Bottom: Statistical results. (D) Western blot analysis of the protein levels of proliferating cell nuclear antigen (PCNA) and HO-1 in the cells transfected with indicated plasmids and oligonucleotides, β-actin served as internal normalized reference. (E and F) Flow cytometry analysis of cell cycle (E) and apoptosis (F) in A549 cells. Upper: Representative images. Bottom: Statistical results. Data are presented as the mean ± SEM of three independent experiments. *<i>P</i><0.05, **<i>P</i> and ***<i>P</i> <0.01 vs. nc; #<i>P</i><0.05, ##<i>P</i> and ###<i>P</i> <0.01 vs miR-1254.</p

Critical Role of Organic Anion Transporters 1 and 3 in Kidney Accumulation and Toxicity of Aristolochic Acid I

Ingestion of aristolochic acid (AA), especially its major constituent aristolochic acid I (AAI), results in severe kidney injury known as aristolochic acid nephropathy (AAN). Although hepatic cytochrome P450s metabolize AAI to reduce its kidney toxicity in mice, the mechanism by which AAI is uptaken by renal cells to induce renal toxicity is largely unknown. In this study, we found that organic anion transporters (OATs) 1 and 3, proteins known to transport drugs from the blood into the tubular epithelium, are responsible for the transportation of AAI into renal tubular cells and the subsequent nephrotoxicity. AAI uptake in HEK 293 cells stably transfected with human OAT1 or OAT3 was greatly increased compared to that in the control cells, and this uptake was dependent on the AAI concentration. Administration of probenecid, a well-known OAT inhibitor, to the mice reduced AAI renal accumulation and its urinary excretion and protected mice from AAI-induced acute tubular necrosis. Further, AAI renal accumulation and severe kidney lesions induced by AAl in <i>Oat1</i> and <i>Oat3</i> gene knockout mice all were markedly suppressed compared to those in the wild-type mice. Together, our results suggest that OAT1 and OAT3 have a critical role in AAl renal accumulation and toxicity. These transporters may serve as a potential therapeutic target against AAN

Proteomic Analysis of Bladder Cancer Indicates Prx-I as a Key Molecule in BI-TK/GCV Treatment System

<div><p>In order to understand the molecular mechanisms of Bifidobacterium infantis thymidine kinase/nucleoside analogue ganciclovir (BI-TK/GCV) treatment system which was proven to exhibit sustainable anti-tumor growth activity and induce apoptosis in bladder cancer, a proteomic approach of isobaric tags for relative and absolute quantification (iTRAQ), followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used. 192 down-regulated and 210 up-regulated proteins were identified after treatment with BI-TK/GCV system in Sprague-Dawley (SD) rats. Western blot analysis and immunohistochemistry analysis confirmed that Peroxiredoxin-I (Prx-I) was significantly down-regulated in bladder cancer after treatment. Prx-I silencing by transfection of Prx-I shRNA significantly suppressed growth, promoted apoptosis and regulated the cell cycle in T24 cells and reduced the phospho-NF-κB p50 and p65 protein expression which revealed the links between Prx-I and NF-κB pathway implied by Ingenuity pathway analysis (IPA). These findings yield new insights into the therapy of bladder cancer, revealing Prx-I as a new therapeutic target and indicating BI-TK/GCV system as a prospective therapy by down-regulation of Prx-I through NF-κB signaling pathway.</p></div

miR-1254 inhibits HO-1 expression.

<p>(A) MiR-1254 and HO-1 mRNA expression in A549 and NCI-H1975 cells transfected with miR-1254 mimics or negative control oligonucleotides (nc). (B and C) Western blot analysis of the HO-1 protein levels in the miR-1254 transfected NSCLC cell lines A549 or NCI-H1975. 20μM hemin was used to rescue the expression of HO-1 as a inducer. Upper: Representative blots. Bottom: Quantification of HO-1 protein levels normalized to β-actin protein levels and plotted as fold changes relative to the levels in cells transfected with nc. (D) HO-1 protein (left and middle) and mRNA (right) levels in NSCLC cell lines A549 or NCI-H1975 transfected with the indicated miRNA antisense oligonucleotides (Anti-1254), normalized to β-actin mRNA or protein levels and expressed relative to values in cells transfected with negative control antisense oligonucleotides (Anti-nc). Left: Representative immunoblots. 25nM of miRNA mimics or antisense oligonucleotides used for all experiments unless stated, cells harvested 48 h post-transfection. β-actin served as a loading control. Middle: Quantification of HO-1 protein levels normalized to β-actin. Right: HO-1 mRNA levels in A549 and NCI-H1975 cells. (E) Schematic representation of the CRISPR/Cas9 modified deletion of pri-miR-1254-1. The PAM sequence is italic. (F) Left: Genotyping of CRISPR/Cas9 modified miR-1254 knockdown A549 cells. Right: Taqman RT-PCR measurement of the expression level of miR-1254 in miR-1254 knockdown A549 cells. (G) HO-1 mRNA (upper) or protein (bottom) levels in CRISPR/Cas9 modified miR-1254 knockdown A549 cells. Data are presented as the mean ± SEM of three independent experiments. *<i>P</i><0.05,**<i>P</i> and ***<i>P</i> <0.01 vs. nc; #<i>P</i><0.05, ###<i>P</i><0.01 vs. miR-1254.</p

Bioinformatics and experimental screening identify miR-1254 as a negative regulator of HO-1.

<p>(A) Venn diagrams showing the number of microRNAs potentially bind to the 3’UTR of HO-1 using three predicting search programs of TargetScan, miRanda and PITA. The inhibition effects of predicted miRNAs on HO-1 3`UTR were verified by luciferase assay. (B) Upper: Immunoblots of the extracts from A549 cells transfected with indicated miRNA mimics. Bottom: Quantification of HO-1 protein levels in A549 cells normalized to β-actin. (C) HO-1 protein or mRNA levels in A549 cells transfected with the indicated doses of miR-1254 for 48h. Upper: Representative blots; nc, negative oligonucleotides. Bottom: Quantification of HO-1 mRNA and protein levels normalized to β-actin (mRNA or protein) levels and plotted as fold changes relative to the values in cells transfected with nc. Data are presented as the mean ± SEM of three independent experiments. *<i>P</i><0.05, **<i>P</i> and ***<i>P</i> <0.01 vs. nc.</p