Luciferase Reporter Vector Research Articles

Regorafenib inhibits tumor progression through suppression of ERK/NF-κB activation in hepatocellular carcinoma bearing mice.

Regorafenib has been demonstrated in our previous study to trigger apoptosis through suppression of extracellular signal-regulated kinase (ERK)/nuclear factor-κB (NF-κB) activation in hepatocellular carcinoma (HCC) SK-Hep1 cells in vitro. However, the effect of regorafenib on NF-κB-modulated tumor progression in HCC in vivo is ambiguous. The aim of the present study is to investigate the effect of regorafenib on NF-κB-modulated tumor progression in HCC bearing mouse model. pGL4.50 luciferase reporter vector transfected SK-Hep1 (SK-Hep1/luc2) and Hep3B 2.1-7 tumor bearing mice were established and used for the present study. Mice were treated with vehicle or regorafenib (20 mg/kg/day by gavage) for 14 days. Effects of regorafenib on tumor growth and protein expression together with toxicity of regorafenib were evaluated with digital caliper and bioluminescence imaging (BLI), ex vivo Western blotting immunohistochemistry (IHC) staining, and measurement of body weight and pathological examination of liver tissue, respectively, in SK-Hep1/luc2 and Hep3B 2.1-7 tumor bearing mice. The results indicated regorafenib significantly reduced tumor growth and expression of phosphorylated ERK, NF-κB p65 (Ser536), phosphorylated AKT, and tumor progression-associated proteins. In addition, we found regorafenib induced both extrinsic and intrinsic apoptotic pathways. Body weight and liver morphology were not affected by regorafenib treatment. Our findings present the mechanism of tumor progression inhibition by regorafenib is linked to suppression of ERK/NF-κB signaling in SK-Hep1/luc2 and Hep3B 2.1-7 tumor bearing mice.

Mutations in the thyroid transcription factor gene NKX2-1 result in decreased expression of SFTPB and SFTPC

Mutations in the NK2 homeobox 1 (NKX2-1) gene are associated with lung disease in infants and children. We hypothesize that disruption of normal surfactant gene expression with these mutations contributes to the respiratory phenotypes observed. To assess transactivational activity, cotransfection of luciferase reporter vectors containing surfactant protein B or C (SFTPB or SFTPC) promoters with NKX2-1 plasmids was performed and luciferase activity was measured. To assess the binding of mutated proteins to target DNA, electrophoretic mobility shift assays (EMSA) were performed using nuclear protein labeled with oligonucleotide probes representing NKX2-1 consensus binding sequences followed by gel electrophoresis. The effect of overexpression of wild-type (WT) and mutant NKX2-1 on SFTPB and SFTPC was evaluated with quantitative real-time PCR. Decreased transactivation of the SFTPB promoter by both mutants and decreased transactivation of the SFTPC promoter by the L197P mutation was observed. EMSA demonstrated decreased DNA binding of both mutations to NKX2-1 consensus binding sequences. Transfection of A549 cells with NKX2-1 expression vectors demonstrated decreased stimulation of SFTPB and SFTPC expression by mutant proteins compared with that of WT. Disruption of transcriptional activation of surfactant protein genes by these DNA-binding domain mutations is a plausible biological mechanism for disruption of surfactant function and subsequent respiratory distress.

MiR-1908 improves cardiac fibrosis after myocardial infarction by targeting TGF-β1.

To investigate the role and mechanism of micro ribonucleic acid (miR)-1908 in myocardial fibrosis after myocardial infarction. In in-vivo experiments, the rat model of myocardial infarction was established, and miR-1908 was up-regulated by lentivirus with miR-1908 overexpression. Cardiac function of rats was detected by echocardiography. Transforming growth factor-β1 (TGF-β1) and Smad2/3 expressions in infarction border zone were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Masson staining was used to detect the fibrosis, thus studying the role of miR-1908 in the myocardial fibrosis model. In in-vitro experiments, myocardial fibroblasts were isolated and cultured. Oxygen glucose deprivation (OGD) model was established to mimicking the ischemic condition; the relationship between miR-1908 and TGF-β1 was verified using luciferase reporter vector, lentivirus and small-interfering RNA (siRNA) in TGF-β1. In-vivo experiments showed that the miR-1908 expression was down-regulated at 4 weeks after myocardial infarction. The up-regulation of miR-1908 significantly improved the cardiac function, reduced the myocardial fibrosis, and inhibited the expressions of TGF-β1 and Smad2/3. In-vitro experiments revealed that TGF-β1 was a target gene of miR-1908 and miR-1908 could inhibit the Smad2/3 expression through TGF-β1. MiR-1908 can improve the myocardial fibrosis through the target gene TGF-β1.

Androgen receptor dampens tissue factor expression via nuclear factor‐κB and early growth response protein 1

Essentials Androgen deprivation increases the rate of venous thromboembolism in prostate cancer patients.We characterized androgen receptor‐mediated tissue factor regulation in prostate epithelial cells.Androgen receptor is dampening tissue factor expression in prostate epithelial cells.Androgen deprivation could enhance tissue factor expression and raise venous thromboembolism rates. SummaryBackgroundProstate cancer is one of the leading causes of cancer death in men. Advanced prostate cancer is usually treated by androgen deprivation therapy (ADT), which is aimed at reducing circulating testosterone levels to reduce cancer growth. There is growing evidence that ADT can increase the rate of venous thromboembolism (VTE) in prostate cancer patients. The tissue factor (TF) gene is one of the most important mediators of coagulation and VTE, but, so far, there are limited data on androgen receptor (AR)‐mediated TF gene expression.ObjectivesTo characterize AR‐mediated TF regulation in vitro and in vivo.MethodsWe used the androgen‐dependent prostate cancer cell lines LNCaP and MyC‐CaP to test whether TF expression is regulated by AR. Furthermore, we cloned the TF gene promoter into a luciferase reporter vector to identify the transcription factor‐binding sites that mediate TF regulation downstream of AR. Finally, we used castration experiments in mice to characterize AR‐mediated TF regulation in vivo.Results TF is directly regulated by AR. In LNCaP cells, nuclear factor‐κB signaling and EGR1 mediate TF expression. By using castration experiments in mice, we could detect upregulation of TF and early growth response protein 1 mRNA and protein expression in prostate epithelial cells.Conclusion AR is crucial for dampening TF expression, which could be important for increased TF expression and TF‐positive microvesicle release in androgen‐deprived prostate cancer patients.

PH-mediated upregulation of AQP1 gene expression through the Spi-B transcription factor

BackgroundBicarbonate-based peritoneal dialysis (PD) fluids enhance the migratory capacity and damage-repair ability of human peritoneal mesothelial cells by upregulating AQP1. However, little is known about the underlying molecular mechanisms.ResultsHere we used HEK-293T cells to investigate the effect of pH on AQP1 gene transcription levels. We found that AQP1 mRNA levels increases with pH. Transfection of HEK-293T cells with luciferase reporter vectors containing different regions of the AQP1 promoter identified an upstream region in the AQP1 gene between − 2200 and – 2300 bp as an enhancer required for pH-mediated regulation of AQP1 expression. Site-directed mutagenesis of this specific promoter region revealed a critical region between − 2257 and − 2251 bp, and gene knock-down experiments and ChIP assays suggested that the Spi-B transcription factor SPIB is involved in pH-mediated regulation of AQP1 expression.ConclusionsWe identified an upstream region in the AQP1 gene and the transcription factor SPIB that are critically involved in pH-mediated regulation of AQP1 expression. These findings provide the basis for further studies on the pH- and buffer-dependent effects of PD fluids on peritoneal membrane integrity and function.

Androgen receptor mediated epigenetic regulation of CRISP3 promoter in prostate cancer cells

Cysteine-rich secretory protein 3 (CRISP3) is one of the most upregulated genes in prostate cancer. Androgen receptor (AR) plays an important role not only in initial stages of prostate cancer development but also in the advanced stage of castration-resistant prostate cancer (CRPC). Role of AR in regulation of CRISP3 expression is not yet known. In order to understand the regulation of CRISP3 expression, various overlapping fragments of CRISP3 promoter were cloned in pGL3 luciferase reporter vector. All constructs were transiently and stably transfected in PC3 (CRISP3 negative) and LNCaP (CRISP3 positive) cell lines and promoter activity was measured by luciferase assay. Promoter activity of LNCaP stable clones was significantly higher than PC3 stable clones. Further in CRISP3 negative PC3 and RWPE-1 cells, CRISP3 promoter was shown to be silenced by histone deacetylation. Treatment of LNCaP cells with DHT resulted in increase in levels of CRISP3 transcript and protein. AR dependency of CRISP3 promoter was also evaluated in LNCaP stable clones by luciferase assay. To provide molecular evidence of epigenetic regulation of CRISP3 promoter and its response to DHT, ChIP PCR was performed in PC3 and LNCaP cells. Our results demonstrate that CRISP3 expression in prostate cancer cells is androgen dependent and in AR positive cells, CRISP3 promoter is epigenetically regulated by AR.

Sp1 is an important transcriptional regulation factor for forkhead box N1 in pig

The transcription factor forkhead box N1 (Foxn1) plays an important role in the development and function of thymic epithelial cells (TECs) in vertebrates. However, the transcriptional regulation of Foxn1 is still unknown. A series of dual luciferase report vectors were constructed and their relative activities were also detected. The 5'-untranslated regions contains two cis-acting elements, Sp1 and GATA-1, as well as trans-acting elements between positions -332 to -438. Nevertheless, the relative activities of Foxn1 promoter were significantly increased in PK15 cells only when the Sp1 was overexpressed, suggesting that the Sp1 was the most important sequence for Foxn1 transcription activation in the pig. Regrettably, the exact trans-acting elements were not found.

MicroRNA-298 regulates apoptosis of cardiomyocytes after myocardial infarction.

To investigate the role and mechanism of micro ribonucleic acid (miR)-298 in myocardial apoptosis after myocardial infarction. In vivo experiments, the rat model of myocardial infarction was established, and miR-298 was up-regulated via lentivirus with miR-298 overexpression. Cardiac function of rats was detected via echocardiography, Bcl-2 associated X protein (BAX) expressions in infarction border zone were detected via Real-time Quantitative PCR (qT-PCR) and Western blot, and TUNEL assay was used to detect the myocardial apoptosis. In vitro experiments, myocardial cells were isolated and cultured, an oxygen-glucose deprivation (OGD) model was established to mimicking the ischemic condition, the relationship between miR-298 and BAX was verified using luciferase reporter vector, lentivirus and small-interfering RNA (siRNA) in BAX. In vivo experiments showed that the miR-298 expression was down-regulated at 2 and 4 weeks after myocardial infarction. The up-regulation of miR-298 significantly improved the cardiac function, decreased the expressions of BAX, reduced the myocardial apoptosis and inhibit the apoptosis proteins expression including cytochrome-c and cleaved caspase-3. In vitro experiments revealed that BAX was a target gene of miR-298 and further proof that miR-298 could inhibit the cytochrome-c and cleaved caspase-3 expression and myocardial apoptosis through BAX. MiR-298 can improve the myocardial apoptosis through the target gene BAX.

Amentoflavone Inhibits Hepatocellular Carcinoma Progression Through Blockage of ERK/NF-ĸB Activation.

The aim of the present study was to confirm therapeutic efficacy and find probable mechanism of action of amentoflavone in hepatocellular carcinoma (HCC) in vivo. Luciferase reporter vector pGL4.50_transfected SK-Hep1 (SK-Hep1/luc2) tumor-bearing mice were treated with vehicle or amentoflavone (100 mg/kg/day by gavage) for 14 days. Tumor growth, amentoflavone toxicity, and extracellular signal-regulated kinase (ERK)/nuclear factor-kappaB (NF-ĸB) signaling in tumor progression were evaluated with digital caliper, bioluminescence imaging, computed tomography, body weight, pathological examination of liver, and immunohistochemistry staining. Amentoflavone significantly inhibited tumor growth, ERK/NF-ĸB activation, and expression of tumor progression-associated proteins as compared to vehicle-treated group. In addition, body weight and liver morphology of mice were not influenced by amentoflavone treatment. These results suggest that amentoflavone inhibits HCC progression through suppression of ERK/NF-ĸB signaling.

Novel Mechanism of Post-Transcriptional Regulation of PD-L1 Expression By 3'-UTR Binding Proteins

Programmed cell death ligand 1 (PD-L1) plays a key role in tumor immune escape by negatively regulating cytotoxic T-cells (CTLs) via PD-1 receptors. Thus, those tumors with high PD-L1 expression are thought to be sensitive to PD-1/PD-L1 blockade, reactivating CTL reactions to tumors. However, the mechanism that regulates PD-L1 expression in tumor cells has not been fully elucidated, understanding of which would help to develop effective anti-tumor immunotherapy.Recently, we reported that disruption of 3'-UTR of PD-L1 led to a remarkably enhanced expression of PD-L1 in a wide variety of human cancers, particularly adult T-cell leukemia/lymphoma (ATL) and diffuse large B-cell lymphoma. In these cancers, stability of PD-L1 transcripts is negatively regulated via their 3'-UTR sequence, whose disruption thus, results in markedly elevated PD-L1 expression and immune evasion (Kataoka et al., Nature, 2016). It has been well established that non-coding regions (i.e., 5'- and 3'-untranslated regions (UTRs)) play important roles in the regulation of mRNA expression, which is accomplished by several transacting factors that bind to cis-regulatory elements within the UTR. Based on this knowledge, we investigated the mechanism that controls PD-L1 expression through 3'-UTR sequence, primarily focusing on those transacting RNA-binding proteins (RNA-BPs).To determine the relevant regions within the 3'-UTR which are capable of repressing PD-L1, several cell lines were transfected with luciferase reporter vectors, in which a luciferase coding sequence was concatenated to various deletion mutants of the PD-L1 3'-UTR (Panel A). We identified two critical sequences, segment 5 and 2 within the PD-L1 3'-UTR, whose existence significantly decreased luciferase expression (Panel B; 293T data, mean ± SD, * denotes t-test p < .05). Importantly, the deletion of these sequences showed a similar effect on gene expression in various cell lines derived from different tissues. To confirm this finding, we used a CRISPR-mediated tiled 3'-UTR editing in situ technique. We designed all possible single-guide (sg) RNAs in the PD-L1 3'-UTR. The library was virally infected into cells and those with high expression of PD-L1 were concentrated by FACS, and the enrichment of each sgRNA was evaluated by high-throughput sequencer. The enriched sgRNAs localized to positions compatible with the luciferase assay, confirming that the two regions are actually responsible for the regulation of PD-L1 expression.Next, we searched RNA-BPs that bind to these regions in mass spectrometry, utilizing flag-peptide-tagged RNA pull-down method (Panel C). In brief, we synthesized PD-L1 3'-UTR RNA segments along with β-actin mRNA as control in vitro, and conjugated a flag-peptide to their 3'-ends. Using these RNA-baits, an immunoprecipitation experiment was performed in 293T cells and the co-immunoprecipitated proteins were analyzed by mass spectrometry. The data obtained from different segments were compared to each other. In addition to those proteins binding to multiple regions within the PD-L1 3'-UTR, we found proteins that specifically interacted with the repressive segments (segment 5 and 2) identified through luciferase assays.Finally, to confirm that the proteins detected by mass spectrometry actually suppress PD-L1 expression, we performed knock-down experiments using siRNA designed for the RNA-BPs that are presumed to interact with the segment 5 and 2. Three siRNA constructs per gene were transfected to 293T cells and their effect on PD-L1 expression was evaluated by RQ-PCR. Panel D shows relative PD-L1 expressions for each siRNA targets with median line, which are grouped according to relevant PD-L1 3'-UTR segments. The negative regulatory effect of these RNA-PBs on PD-L1 expression was largely confirmed. PD-L1 protein level was also increased, when these genes were knocked out by CRISPR/Cas9 system. Expression of these genes in ATL and other lymphomas was also evaluated.In summary, we identified critical sequences within the PD-L1 3'-UTR and RNA-BPs that bind to these sequences and negatively regulate PD-L1 expression. Our findings should not only provide novel insight into the molecular mechanisms by which PD-L1 expression in tumor cell is regulated but also help to identify potential targets for immune therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Intracellular galectin-7 expression in cancer cells results from an autocrine transcriptional mechanism and endocytosis of extracellular galectin-7.

The β-galactoside binding protein galectin-7 (gal-7) is constitutively expressed at abnormally high levels in the outside milieu and intracellular compartments of many types of epithelial cancer cells, most notably in aggressive forms of ovarian and breast cancer. It is thus of utmost importance to understand how gal-7 traffics between both intracellular and extracellular compartments to develop novel drugs that target the protumorigenic functions of galectin-7. In the present work, we report that extracellular gal-7 plays a central role in controlling intracellular gal-7 in cells. It does so via two distinct yet complementary mechanisms: firstly by increasing the transcriptional activation of lgals7 gene transcription, and secondly via re-entry into the cells. Increased mRNA levels were dose- and time-dependent and occur in all cell lines tested, including ovarian and breast cancer cell lines. Addition of recombinant gal-7 to MDA-MB-231 transfected with a luciferase reporter vector containing response elements of the lgals7 promoter indicated that increased mRNA level of lgals7 occurs via de novo gene transcription. Re-entry of extracellular gal-7 inside cells was rapid, and reached cytosolic and mitochondrial compartments. Taken together, these findings reveal the existence of a positive self-amplification pathway that regulates intracellular gal-7 expression in breast and ovarian cancer cells.

Drug screening to target nuclear orphan receptor NR4A2 for cancer therapeutics.

Our previous study suggested NR4A2, a subfamily member of orphan nuclear receptors, is essential for survival of human cancer cells such as mucoepidermoid carcinoma (MEC). We conducted high throughput drug screening for NR4A2 inhibitors as a novel therapeutic modality. Positive screening was performed using a luciferase reporter vector containing NR4A2 binding sequence, and a CRE-reporter control vector was used to eliminate false positives. In vitro assays for positive hits were conducted. A total of 23 Food and Drug Administration (FDA) and 43 Life Science Library compounds were identified, including several epidermal growth factor inhibitors and Src inhibitors. Subsequent in vitro assays confirmed that identified compounds were preferentially active in NR4A2+ cancer cells. Several candidate compounds appeared to suppress NR4A2 via inhibition of p-ERK, whereas a novel compound KU0171309 may act as a more direct inhibitor. Further research should focus on homologue selectivity, in vivo activity, and definitively deciphering the mechanism of action of KU0171309.

Prediction of miRNA regulating the potential cancer-promoting gene CCL18 in cutaneous malig-nant melanoma and correlation analysis between CCL18 mRNA and miRNA expression

Objective To explore the miRNA regulating the potential cancer-promoting gene CCL18 in cutaneous malignant melanoma. Methods Bioinformatics analysis was conducted by using online software miRanda and TargetScan, so as to predict the miRNA targeting CCL18 gene. Three kinds of CCL18 3′UTR dual-luciferase reporter vectors, including mutant 3′UTR vector (mutant 3′UTR group) , wild-type 3′UTR vector (wild-type 3′UTR group) and empty vector (blank control group) , as well as miRNA vectors carring selected miRNAs were constructed according to human gene sequence analysis, and then were used to co-transfect 293T cells. After 48-hour treatment, the cells were lysed for detection of luciferase activity. Real-time fluorescence-based quantitative PCR was performed to measure the expression of CCL18 and selected miRNA in 14 fresh malignant melanoma tissue specimens and 14 paracancerous normal skin tissue specimens (control tissues) , and their correlations were analyzed. Results Online software analysis showed that some miRNAs were identified to target the 3′UTR of CCL18 gene, including miR-183, miR-128 and miR-33a. Luciferase reporter vectors and miRNA vectors were constructed successfully. As luciferase activity assay showed, when miR-183 and miR-128 were bound to the CCL18 3′UTR, the luciferase activities were significantly higher in their mutant 3′UTR groups (11.63 ± 0.42; 8.80 ± 0.49) than in their wild-type 3′UTR groups (4.86 ± 0.39; 5.01 ± 0.54; both P 0.05) . Real-time fluorescence-based quantitative PCR revealed higher mRNA expression of the CCL18 gene (3.52 ± 1.68) , but lower expression of miR-183 (0.49 ± 0.32) , miR-128 (0.30 ± 0.20) and miR-33a (0.46 ± 0.40) in the malignant melanoma tissues compared with the control tissues. The mRNA expression of the CCL18 gene was negatively correlated with the expression of miR-128 (rs=-0.548, P 0.05) . Conclusion miR-128 may play a role in regulating the potential malignant melanoma-promoting gene CCL18. Key words: Melanoma; MicroRNAs; RNA, small interfering; Chemokine CCL18

Abstract P162: In vivo miR-431 Inhibition Protects Against Vascular Damage and Hypertension

Background: Vascular injury is an early manifestation of hypertension. microRNAs (miRNAs) play an important role in cardiovascular disease, but their implication in vascular injury remains unclear. Using small and total RNA sequencing, we identified in murine mesenteric arteries (MAs) a conserved angiotensin (Ang) II-upregulated Dlk1-Dio3 miRNA miR-431 that correlated with blood pressure (BP), and an Ang II-downregulated BP-correlated conserved putative miR-431 target, the transcriptional factor ETS homologous factor ( Ehf ). miR-431 might be involved in vascular remodeling as Ehf regulates expression of extracellular matrix genes including alpha-1 type I collagen ( Col1a1 ) and of other Dlk1-Dio3 miRNAs. In this study, we proposed to validate the miR-431- Ehf - Col1a1 interaction in vitro and in vivo , and determine whether miR-431 inhibition antagonizes angiotensin (Ang) II-induced hypertension and vascular injury. Methods and Results: Transfection of miR-431 mimics into human aortic smooth muscle cells decreased Ehf expression (0.13±0.05 fold, P &lt;0.001) and increased Col1a1 (1.7±0.5 fold, P &lt;0.01), whereas miR-431 inhibitors increased Ehf (1.5±0.2 fold, P &lt;0.001) and decreased Col1a1 (0.89±0.11 fold, P &lt;0.05). Ehf siRNA transfection increased 1.2±0.1 fold Col1a1 ( P &lt;0.01). Co-transfection of miR-431 mimics with luciferase reporter vectors that contain the wild-type but not mutated miR-431 human EHF 3’ UTR binding site decreased 0.51±0.01 fold ( P &lt;0.05) luciferase expression compared to scrambled mimics. miR-431 inhibitor IV injection in mice at day 0 and 7 of Ang II infusion decreased miR-431 (0.16±0.05 fold, P &lt;0.01), Col1a1 (0.58±0.11 fold, P &lt;0.05), increased Ehf (2.9±0.8 fold, P &lt;0.05) in MAs, delayed BP elevation ( P &lt;0.01), improved endothelium-dependent relaxation (33±8 vs 64±7%, P &lt;0.05) and reduced vascular stiffness (strain at 140mmHg: 0.68±0.02 vs 0.58±0.02 ΔD/D, P &lt;0.01) compared to scrambled mimics-injected Ang II-infused mice. Conclusion and Perspectives: miR-431 and its target Ehf may act as master regulators in the pathophysiology of vascular damage in hypertension. miR-431 inhibition has the potential to serve as a novel therapeutic approach for treatment of vascular damage and hypertension.

20(S)-25-methoxyl-dammarane-3β, 12β, 20-triol negatively regulates activation of STAT3 and ERK pathways and exhibits anti-cancer effects in HepG2 cells.

The pro-inflammatory cytokine interleukin 6 (IL-6), via activating its downstream JAK/STAT3 and Ras/ERK signaling pathways, is involved in cell growth, proliferation and anti-apoptotic activities in various malignancies. To screen inhibitors of IL-6 signaling, we constructed a STAT3 and ERK dual-pathway responsive luciferase reporter vector (Co.RE). Among several candidates, the natural compound 20(S)-25-methoxyl-dammarane-3β, 12β, 20-triol (25-OCH3-PPD, GS25) was identified to clearly inhibit the luciferase activity of Co.RE. GS25 was confirmed to indeed inhibit activation of both STAT3 and ERK pathways and expression of downstream target genes of IL-6, and to predominantly decrease the viability of HepG2 cells via induction of cell cycle arrest and apoptosis. Interestingly, GS25 showed preferential inhibition of HepG2 cell viability relative to normal liver L02 cells. Further investigation showed that GS25 could not induce apoptosis and block activation of STAT3 and ERK pathways in L02 cells as efficiently as in HepG2 cells, which may result in differential effects of GS25 on malignant and normal liver cells. In addition, GS25 was found to potently suppress the expression of endogenous STAT3 at a higher concentration and dramatically induce p38 phosphorylation in HepG2 cells, which could mediate its anti-cancer effects. Finally, we demonstrated that GS25 also inhibited tumor growth in HepG2 xenograft mice. Taken together, these findings indicate that GS25 elicits its anti-cancer effects on HepG2 cells through multiple mechanisms and has the potential to be used as an inhibitor of IL-6 signaling. Thus, GS25 may be developed as a treatment for hepatocarcinoma with low toxicity on normal liver tissues as well as other inflammation-associated diseases.

MiR-30a inhibits the biological function of breast cancer cells by targeting Notch1.

miR-30a is situated on chromosome6q.13 and is produced by an intronic transcriptional unit. However, its role in regulating the apoptosis, invasion and metastasis of breast cancer cells is not yet fully understood. The aim of this study was to research the biological function of miR‑30a and its direct target gene in breast cancer. The biological function of miR‑30a was determined by examining breast cancer cell growth, apoptosis, metastasis and invasion. In addition, Notch1 expression was measured by western blot analysis, and a luciferase reporter vector was constructed to identify the miR‑30a target gene. miR‑30a was found to be significantly downregulated in breast cancer cells. We also found that miR‑30a inhibited breast cancer cell viability, migration and invasion, and induced cell apoptosis. On the whole, our data indicate that miR‑30a attenuates the development of breast cancer by regulating the expression of the downstream target gene, Notch1.

Abstract 1443: Identification of enhancer elements at kidney cancer susceptibility loci using MPRA

Abstract GWAS is an important tool for discovering regions in the genome associated with cancer susceptibility. For renal cell carcinoma (RCC), only two loci have had their functional basis explained (11q13 and 12p12). Here, we used a massively parallel reporter assay (MPRA) to investigate enhancer activity in 20 GWAS regions that had an RCC association p-value of at least 10-7. We selected 784 SNPs with an r2&amp;gt;0.4 or D’&amp;gt;0.5 and MAF&amp;lt;0.05 that had evidence for enhancer activity based on available ENCODE transcription factor or histone ChIP-seq, FAIRE, and DNase I hypersensitive data. The MPRA library was composed of 47461 oligonucleotides 201 bp in length, compelled of 145 bps contained the SNP in the forward or reverse orientation followed by 10 bp of barcode sequence to allow for identification. Each of the 784 SNPs tested were tagged 10 separate times for both orientations. Controls were constructed by randomizing the 10 nucleotides centered on the SNP. The MPRA library was the cloned into a luciferase reporter vector that was transfected into HEK293T and ACHN cell lines. NGS was performed on 5 replicates using the the Illumina HiSeq 2500. After correcting for multiple comparisons, 50 SNPs from 16 regions showed significant p-values. From these 50 SNPs, 29 showed enhancer activity that was independent of the forward/reverse orientation and had the same effect in both cell lines. This analysis has identified possible functional variants at RCC risk loci and opens opportunities to discover new molecular mechanisms of genetic susceptibility of RCC. Citation Format: Leandro Machado Colli, Lea Jessop, Mitchell Machiela, Jiyeon Choi, Timothy Myers, Stephen Chanock. Identification of enhancer elements at kidney cancer susceptibility loci using MPRA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1443. doi:10.1158/1538-7445.AM2017-1443

Abstract 2220: Repurposing statins for prevention of triple-negative breast cancer

Abstract Introduction: Although triple negative breast cancer (TNBC) accounts for 15% of the 250,000 annual cases of breast cancer, its more aggressive nature, coupled with lack of targeted therapy, results in a disproportionate rate of mortality in women with this disease underscoring the critical need for prevention-based approaches. We hypothesized that identifying miRNA-mRNA functional pairs and their driver pathway(s) deregulated during preneoplastic progression would provide the most informative insights into the molecular drivers of TNBC carcinogenesis. Methods: We performed next generation RNA sequencing of TNBC like MCF10A based model system and focused on detailing the molecular changes preceding the development of in situ carcinoma (DCIS). We used ki67, MTT and colony formation assays to study the growth inhibitory effect of miRNA and pathway targeting. To identify direct gene targets of a miRNA, we cloned the 3’untranslated region containing miRNA-binding sites from its predicted gene targets in a luciferase reporter vector, and studied the effect of miRNA overexpression on the repression of luciferase reporter activity. Results: In-silico analyses of small RNA seq data showed loss of miRNA-140-3p as one of the top deregulated miRNA relevant to preneoplastic transition from MCF10A parental to MCF 10AT1 preneoplastic cell line. Staining of breast tissue samples also confirmed loss of miRNA-140-3p in tumors compared to normal breast epithelial cells, suggesting a tumor suppressive role. To investigate the functional role of miRNA-140 during TNBC development, we ectopically expressed miRNA-140 in MCF10.AT1 and MCF10.DCIS cells. Interestingly, these studies revealed miRNA-140 replacement to preferentially inhibit colonizing ability and cell proliferation of preneoplastic MCF10.AT1 cells in contrast to a much weaker cell inhibitory effects in DCIS cells. Next, we identified the functional gene target/s of miRNA-140-3p by integromics studies involving next-generation RNA-seq on the MCF10A model employing bioinformatics pipeline, SigTerms. These analyses identified HMGCR and HMGCS1 as top 2 gene targets of miRNA-140-3p that map in cholesterol biosynthesis pathway. Consistently, we found miRNA-140-3p to directly bind in 3’UTR of HMGCR, HMGCS1 and repress their expression as shown by luciferase assays. miRNA-140 binding of these gene targets is also functionally relevant as inhibition of HMGCR activity by an FDA approved statin inhibitor, fluvastatin, preferentially inhibited the cell proliferation and colonizing ability of preneoplastic MCF10AT1 cells. Further studies focused on testing the potential of fluvastatin to prevent progression of breast atypia cells in mouse xenografts are underway. Conclusions: Our results suggest miRNA-140 and its downstream functional cholesterol biosynthesis pathway targeting, especially by FDA approved Fluvastatin, may be a novel strategy for targeted prevention of TNBC. Citation Format: Anjana Bhardwaj, Harpreet Singh, Kimal Rajapakshe, Cristian Coarfa, Isabelle Bedrosian. Repurposing statins for prevention of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2220. doi:10.1158/1538-7445.AM2017-2220

Kaempherol and Luteolin Decrease Claudin-2 Expression Mediated by Inhibition of STAT3 in Lung Adenocarcinoma A549 Cells.

Claudin-2 is highly expressed in human lung adenocarcinoma tissues and may be a novel target for cancer chemotherapy because knockdown of claudin-2 decreases cell proliferation. We found that flavonoids including kaempferol, chrysin, and luteolin concentration-dependently decrease claudin-2 expression in lung adenocarcinoma A549 cells. Claudin-2 expression is up-regulated by mitogen-activated protein kinase kinase (MEK)/ extracellular signal-regulated kinase (ERK)/c-Fos and phosphoinositide 3-kinase (PI3K)/Akt/nuclear factor-κB (NF-κB) pathways, but these activities were not inhibited by kaempferol, chrysin, and luteolin. Promoter deletion assay using luciferase reporter vector showed that kaempferol and luteolin inhibit the function of transcriptional factor that binds to the region between −395 and −144 of claudin-2 promoter. The decrease in promoter activity was suppressed by mutation in signal transducers and activators of transcription (STAT)-binding site, which is located between −395 and −144. The phosphorylation level of STAT3 was not decreased, but the binding of STAT3 on the promoter region is suppressed by kaempferol and luteolin in chromatin immunoprecipitation assay. The inhibition of cell proliferation caused by kaempferol and luteolin was partially recovered by ectopic claudin-2 expression. Taken together, kaempferol and luteolin decreased claudin-2 expression and proliferation in A549 cells mediated by the inhibition of binding of STAT3 on the promoter region of claudin-2. The intake of foods and nutrients rich in these flavonoids may prevent lung adenocarcinoma development.

The interaction of the RNA binding protein with microRNA regulates cytokine production in Th17 cells

Abstract GM-CSF produced by Th17 cells play an essential and non-redundant role in chronic inflammatory and autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis. Although the transcriptional regulation of Th17 cells is well understood, the posttranscriptional regulation of GM-CSF gene expression remains unknown. The RNA-binding protein HuR positively regulates the stability of many target mRNAs via binding the AU-rich elements present in the 3′ untranslated region of many inflammatory cytokines including IL-4, IL-13, IL-17 and TNF-α. However, the regulation of GM-CSF expression by HuR in Th17 cells has not been fully characterized. CD4+ Th17 cells from HuR knockout mice had decreased GM-CSF steady-state mRNA compared with wild-type Th17 cells. Moreover, we demonstrated that HuR directly binds to GM-CSF mRNA 3′ untranslated region (3′UTR) by using RNA immunoprecipitation and biotin pulldown assays. In addition, knockout of HuR increases certain levels of microRNAs in Th17 cells, among them, miR-466i has multiple potential binding sites in the 3′ UTRs of GM-CSF and IL-17 mRNAs. In vitro luciferase assay showed that overexpression of miR-466i reduces activity of luciferase reporter vector containing GM-CSF or IL-17 mRNA 3 UTR, suggesting that miR-466i has the potential to mediate GM-CSF and IL-17 mRNA decay. Taken together, these findings indicate that interaction of HuR with miR-466i orchestrates GM-CSF expression by Th17 cells.