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

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

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

miR-1254 targets TFAP2A 3`UTR via its non-seed sequence.

<p>(A) Luciferase activity of psi-TFAP2A reporter in HEK293 cells transfected with indicated oligonucleotides. (B) Sequence complementarity between the 3′UTR of TFAP2A mRNA and the different region of miR-1254. (C) Luciferase activity in HEK293 cells transfected with miR-1254 mimics and reporter plasmids containing wt or mt TFAP2A 3′-UTR normalized to that in cells transfected with negative control (nc). (D) TFAP2A and HO-1 mRNA levels in A549 cells transfected with indicated oligonucleotides. (E) Upper: Schematic representation of the CRISPR/Cas9 modified nucleotides knockout in TFAP2A 3`UTR, the PAM sequence is italic. Bottom: DNA sequencing used to confirm the CRISPR/Cas9 modified genome editing.(F) qRT-PCR analysis of the mRNA levels of TFAP2A and HO-1 expression in the CRISPR-sites A549 cells. CRISPR-sites A549 cells: CRISPR/Cas9-modified A549 cells which the binding sites of miR-1254 on TFAP2A 3`UTR were deleted.(G) Western blot analysis of the effects of miR-1254 on TFAP2A and HO-1 expression in wild-type or CRISPR-sites A549 cell lines. Left: Representative images. Right: Statistical results. Data are presented as the mean ± SEM of three independent experiments. **<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 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

miR-1254 represses HO-1 transcription.

<p>(A) Sequence alignment of wild-type/5mt miR-1254 with wild-type/mutant HO-1 3`UTR. (B) Luciferase activity in HEK293 cells transfected with miR-1254 mimics and reporter plasmids containing wt or mt HO-1 3′-UTR normalized to activity in cells transfected with nc. (C and D) HO-1 mRNA (C) and protein (D) levels in A549 cells transfected with the indicated nucleotides (MiR-1254 and its 5`mt) for 48 h were measured by qRT-PCR and immunoblotting respectively. (E) The effect of miR-1254 on HO-1 mRNA stability. A549 and NCI-H1975 cells were transiently transfected with the indicated oligonucleotides, and after 4 hours each culture was treated with 5 μg/ml actinomycin D (ActD) for the indicated times. (F) ChIP analysis of the polⅡ binding to the HO-1 promoter in the A549 cells using antibodies against polⅡ, with IgG as a negative control. Top: Representative images. Bottom: Statistical results. (G) Nucleotides with mutations (underlined and italic) used in all experiments. (H and I) Luciferase activity in HEK293 cells (H) and HO-1 mRNA expression in A549 cells (I), cells were all transfected with indicated oligonuclitides and harvested 48h post-transfection. 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

Selection of sensitive and specific genotoxic stress responsive genes.

<p>(<b>A</b>) Hierarchical clustering of top 50 scored up-regulated genes shown in gene symbol. Red and green indicate up-regulation and down-regulation, respectively. The orange box represents genes whose expression could distinguish GTXs from NGTXs. The blue box represents the gene with the highest score, BC005512. (<b>B</b> and <b>C</b>) Microarray and quantitative PCR (qPCR) data showing BC expression levels in livers of mice dosed with indicated chemicals at 4 h or 20 h after administration. Microarray data represented pooled samples from 4 animals per group. Quantitative PCR data were mean ± s.d. (n = 4).</p

Expression of BC was specifically induced by GTXs in NIH/3T3 cells.

<p>Data from quantitative PCR showing transcriptional expression of BC in NIH/3T3 cells treated with genotoxic or non-genotoxic chemicals for indicated time. Data were mean ± s.d. of three independent experiments.</p

Induced expression level of BC correlated with DNA damage in NIH/3T3 cells.

<p>(<b>A, B, D, E, G and H</b>) Comparison between expression level of BC and DNA damage in NIH/3T3 cells exposed to MMS (A, B) for 8 h, or to colchicine (D, E) or paclitaxel (G, H) for 24 h. DNA damage was measured by olive tail moment (tail length × percentage of DNA in tail) in an alkaline comet assay (representative figures are shown in B, E and H). Data were mean±s.d. of three independent experiments. (<b>C</b>) Linear regression analysis between expression level of BC and DNA damage, reflected by olive tail moment. Each dot represents the mean of data shown in (A), (D) and (G). (<b>F</b> and <b>I</b>) Micronucleus frequency in NIH/3T3 cells exposed to colchicine or paclitaxel for 24 h. Data were mean ± s.d. of three independent experiments. Values shown on top of bars are <i>P</i> values <i>vs</i> control.</p