Activity Reporter Assay Research Articles

Characterization of GJB2 cis-regulatory elements in the DFNB1 locus.

Although most disease-causing variants are within coding region of genes, it is now well established that cis-acting regulatory sequences, depending on 3D-chromatin organization, are required for temporal and spatial control of gene expression. Disruptions of such regulatory elements and/or chromatin conformation are likely to play a critical role in human genetic disease. Hence, recurrent monoallelic cases, who present the most common hereditary type of nonsyndromic hearing loss (i.e., DFNB1), carry only one identified pathogenic allele. This strongly suggests the presence of uncharacterized distal cis-acting elements in the missing allele. Here within, we study the spatial organization of a large DFNB1 locus encompassing the gap junction protein beta 2 (GJB2) gene, the most frequently mutated gene in this inherited hearing loss phenotype, with the chromosome conformation capture carbon copy technology (5C). By combining this approach with functional activity reporter assays and mapping of CCCTC-binding factor (CTCF) along the DFNB1 locus, we identify a novel set of cooperating GJB2 cis-acting elements and suggest a DFNB1 three-dimensional looping regulation model.

MiR-129-5p improves cardiac function in rats with chronic heart failure through targeting HMGB1.

Increasing evidence shows that miRNAs play pivotal roles in cardiovascular diseases, including heart failure (HF). The aim of this study was to investigate the role of miR-129-5p in chronic heart failure and the underlying mechanisms. The levels of miR-129-5p and HMGB1 in chronic heart failure patients (CHF) and normal controls were examined by RT-qPCR and ELISA. Cardiac function, hemodynamics parameters, oxidative stress, and inflammation factors were analyzed in CHF rat model after transfection of miR-129-5p or HMGB1. Dual-luciferase activity reporter assay was conducted to validate the interaction between miR-129-5p and HMGB1. Downregulation of miR-129-5p and upregulation of HMGB1 were observed in the serum of CHF patients, respectively. Transfection of miR-129-5p improved heart function and hemodynamic parameters, as well as attenuated oxidative stress and inflammation factors in CHF rats. We further confirmed that HMGB1 is a direct target of miR-129-5p. Transfection of miR-129-5p also decreased the mRNA and protein levels of HMGB1 in myocardial tissues of CHF rats. Overexpression of HMGB1 diminished the effects of miR-129-5p on ameliorating oxidative stress and inflammatory response in rats with CHF. Our findings suggest that miR-129-5p protects the heart by targeting HMGB1.

MiR-652-3p promotes bladder cancer migration and invasion by targeting KCNN3.

Increasing evidence indicated that microRNAs (miRNAs) are crucial regulators for cancer development. Bladder cancer (BCa) is a major threat to human health. The aim of this study was to analyze the roles of miR-652-3p in BCa, and to explore the associated mechanisms. MiR-652-3p expression in BCa cell lines was explored using Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) method. MiR-652-3p expression level in BCa tissues was explored at StarBase. Cell Counting Kit-8 (CCK-8) assay, wound-healing assay, and transwell invasion assay were conducted to investigate the biological roles of miR-652-3p. The underlying mechanisms of miR-652-3p in NSCLC were investigated using luciferase activity reporter assay and rescue experiments. We showed that miR-652-3p expression level was upregulated in both BCa tissues and cell lines. The knockdown of miR-652-3p significantly inhibited BCa cell proliferation, migration, and invasion in vitro. Moreover, we showed that potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3 (KCNN3) was a functional target for miR-652-3p. Besides, the expression of KCNN3 in BCa tissues was negatively correlated with miR-652-3p. Collectively, these results showed that miR-652-3p could promote BCa cell proliferation, migration, and invasion via directly regulating KCNN3, which may provide a novel therapeutic target for BCa treatment.

Regulation of PLPP3 gene expression by NF-κB family transcription factors

Lipid phosphate phosphatase 3 (LPP3), encoded by the PLPP3 gene, is an integral membrane enzyme that dephosphorylates phosphate esters of glycero- and sphingophospholipids. Cell surface LPP3 can terminate the signaling actions of bioactive lysophosphatidic acid (LPA) and sphingosine 1 phosphate, which likely explains its role in developmental angiogenesis, vascular injury responses, and cell migration. Heritable variants in the final intron PLPP3 associate with interindividual variability in coronary artery disease risk that may result from disruption of enhancer sequences that normally act in cis to increase expression of the gene. However, the mechanisms regulating PLPP3 expression are not well understood. We show that the human PLPP3 promoter contains three functional NF-κB response elements. All of these are required for maximal induction of PLPP3 promoter activity in reporter assays. The identified sequences recruit RelA and RelB components of the NF-κB transcription complex to chromatin, and these transcription factors bind to the identified target sequences in two different cell types. LPA promotes binding of Rel family transcription factors to the PLPP3 promoter and increases PLPP3 gene expression through mechanisms that are attenuated by an NF-κB inhibitor, LPA receptor antagonists, and inhibitors of phosphoinositide 3 kinase. These findings indicate that up-regulation of PLPP3 during inflammation and atherosclerosis results from canonical activation of the NF-κB signaling cascade to increase PLPP3 expression through nuclear import and binding of RelA and RelB transcription factors to the PLPP3 promoter and suggest a mechanism by which the LPP3 substrate, LPA, can regulate PLPP3 expression.

MiR-885-5p promotes gastric cancer proliferation and invasion through regulating YPEL1.

Dysregulation of microRNAs (miRNAs) was found to play crucial roles in the carcinogenesis of multiple human cancers. This study was aimed to investigate the biological function of miR-885-5p and associated mechanisms in gastric cancer (GC). Reverse Transcription-quantitative Polymerase Chain Reaction was used to measure miR-885-5p level in GC cell lines and normal cell line. The effects of miR-885-5p expression on cell proliferation, colony formation, and invasion were investigated by Cell Counting Kit-8 (CCK-8) assay, colony formation assay, and transwell invasion assay, respectively. Furthermore, Luciferase activity reporter assay and Western blot were conducted to validate Yippee-like-1 (YPEL1) as a direct target of miR-885-5p. We found that miR-885-5p expression level was elevated in GC cell lines compared with normal cell line. Additionally, the knockdown of miR-885-5p inhibits GC cell proliferation, colony formation, and cell invasion in vitro. Notably, rescue experiments demonstrated that the knockdown of YPEL1 partially reversed the effects of miR-885-5p on GC cell growth and invasion. The present study suggested that miR-885-5p regulates GC proliferation, colony formation, and invasion via targeting YPEL1.

MiR-1249-3p accelerates the malignancy phenotype of hepatocellular carcinoma by directly targeting HNRNPK.

BackgroundmicroRNAs (miRNAs) have been implicated to play crucial roles in carcinogenesis. miR‐1249‐3p was reported to be abnormally expressed in multiple human cancers. However, its biological role and the associated underlying mechanisms in hepatocellular carcinoma (HCC) remain largely unknown.Methods miR‐1249‐3p expression level in HCC cell lines and normal cell line was measured by quantitative real‐time PCR. Role of miR‐1249‐3p on HCC cell proliferation, colony formation, and invasion was examined by cell counting kit‐8 assay, colony formation assay, and transwell invasion assay, respectively. Luciferase activity reporter assay and western blot were performed to validate whether heterogeneous nuclear ribonucleoprotein K (HNRNPK) was a direct target of miR‐1249‐3p. Effect of miR‐1249‐3p on overall survival of HCC patients was analyzed at KM Plotter website.ResultsWe found miR‐1249‐3p expression level was increased, while HNRNPK expression level was decreased in HCC cell lines compared with normal cell line. Knockdown miR‐1249‐3p expression inhibits HCC cell proliferation, colony formation, and cell invasion through regulating HNRNPK in vitro. We also showed high miR‐1249‐3p expression was a predictor for poor overall survival of HCC patients.ConclusionsThese findings about miR‐1249‐3p/HNRNPK pair provide a novel therapeutic method for HCC patients.

LncRNA XIST regulates proliferation and migration of hepatocellular carcinoma cells by acting as miR-497-5p molecular sponge and targeting PDCD4

BackgroundMicroRNAs (miRNAs) play a pivotal role in hepatocellular carcinoma (HCC) progression and have been confirmed to participate in the carcinogenesis and development of HCC. However, the relationship between miR-497-5p and HCC remains unclear.MethodsKaplan–Meier curve analysis and the log-rank test were used to investigate the efficacy of miR-497-5p on overall survival (OS) and disease-free survival (DFS) in patients with HCC. According to in vitro experiments, programmed cell death 4 (PDCD4) was a target of miR-497-5p by the dual-luciferase activity assay. The efficacy of PDCD4 on cell proliferation and metastasis in HCC was examined by transwell assays, CCK-8 assays and reverse transcription quantitative PCR (RT-qPCR). Additionally, we conducted a luciferase activity reporter assay to confirm the interaction between lncRNA XIST and miR-49-5p. Then, to evaluate the relationship between lncRNA XIST and miR-497-5p, several mechanistic experiments, including qRT-PCR, Western blotting, transwell assays and tumor xenograft assays, were performed.ResultsmiR-497-5p was upregulated in HCC tissues, and high expression of miR-497-5p resulted in increases in tumor size and tumor number and a higher tumor-node-metastasis (TNM) stage and Edmondson grade in patients with HCC. Silencing miR-497-5p inhibited the proliferation and migration of HCC cells. PDCD4, which was downregulated in HCC tissues, was shown to be a target of miR-497-5p and was negatively correlated with the expression of miR-497-5p. lncRNA XIST was found to act as a miR-497-5p sponge and to regulate the level of PDCD4, which is targeted by miR-497-5p. lncRNA XIST was observed to be downregulated in the HCC tissues and positively correlated with the expression of PDCD4.ConclusionsOur findings reveal that the XIST/miR-497-5p/PDCD4 axis participates in HCC development and that XIST could be used as a biomarker of HCC.

Vitamin D Alleviates Rotavirus Infection through a Microrna-155-5p Mediated Regulation of the TBK1/IRF3 Signaling Pathway In Vivo and In Vitro.

(1) Background: Vitamin D (VD) plays a vital role in anti-viral innate immunity. However, the role of VD in anti-rotavirus and its mechanism is still unclear. The present study was performed to investigate whether VD alleviates rotavirus (RV) infection through a microRNA-155-5p (miR-155-5p)-mediated regulation of TANK-binding kinase 1 (TBK1)/interferon regulatory factors 3 (IRF3) signaling pathway in vivo and in vitro. (2) Methods: The efficacy of VD treatment was evaluated in DLY pig and IPEC-J2. Dual-luciferase reporter activity assay was performed to verify the role of miR-155-5p in 1α,25-dihydroxy-VD3 (1,25D3) mediating the regulation of the TBK1/IRF3 signaling pathway. (3) Results: A 5000 IU·kg–1 dietary VD3 supplementation attenuated RV-induced the decrease of the villus height and crypt depth (p < 0.05), and up-regulated TBK1, IRF3, and IFN-β mRNA expressions in the jejunum (p < 0.05). Incubation with 1,25D3 significantly decreased the RV mRNA expression and the RV antigen concentration, and increased the TBK1 mRNA and protein levels, and the phosphoprotein IRF3 (p-IRF3) level (p < 0.05). The expression of miR-155-5p was up-regulated in response to an RV infection in vivo and in vitro (p < 0.05). 1,25D3 significantly repressed the up-regulation of miR-155-5p in vivo and in vitro (p < 0.05). Overexpression of miR-155-5p remarkably suppressed the mRNA and protein levels of TBK1 and p-IRF3 (p < 0.01), while the inhibition of miR-155-5p had an opposite effect. Luciferase activity assays confirmed that miR-155-5p regulated RV replication by directly targeting TBK1, and miR-155-5p suppressed the TBK1 protein level (p < 0.01). (4) Conclusions: These results indicate that miR-155-5p is involved in 1,25D3 mediating the regulation of the TBK1/IRF3 signaling pathway by directly targeting TBK1.

Abstract 3670: Transcriptional co-activators TAZ/YAP are novel regulators of PAX3-FOXO1 transcriptional programing and fusion-positive rhabdomyosarcoma cancer cell stemness

Abstract Introduction: Fusion Positive Rhabdomyosarcoma (FP-RMS), a soft tissue sarcoma of adolescents and young adults, is driven by the oncogenic transcription factor PAX3-FOXO1 (P3F). Although most patients with FP-RMS initially respond to therapy, tumors frequently become refractory or relapse, underscoring a need to target resistance mechanisms and cancer stem cells. Because P3F is inherently disordered with no viable drug binding sites, it is currently not amendable to direct inhibition. Our prior work demonstrated that transcriptional co-activators TAZ and YAP are highly abundant in human FP-RMS and mediate several cancer phenotypes including apoptosis, differentiation, proliferation, and xenograft tumor growth. TAZ/YAP are essential co-activators for TEADs and AP1, which are among the top enriched transcription factor motifs at P3F binding sites. Here, we show that TAZ/YAP complex with P3F to positively regulate P3F transcriptional activity and that TAZ/YAP mediate FP-RMS stemness. Methods: Functional interactions of TAZ/YAP and P3F were investigated using P3F reporters, immunoblots, and co-immunoprecipitation (co-IP); while co-IP-coupled mass spectrometry (IP-MS) to identify the TAZ/YAP/P3F interactome is currently underway. For reporter assays, gain- and loss-of-function vectors expressing control, wild-type TAZ/YAP (WT), constitutively active TAZ/YAP (S89A/S127A), or shRNA knockdown (KD) were used. Epitope-tagged TAZ, YAP, and P3F were used for co-IPs and IP-MS. To investigate the role of TAZ/YAP in stemness, we evaluated the expression of stem cell genes in FP-RMS 3D-spheres expressing vector, WT, S89A/S127A, or KD. We are now performing RNA-Seq as an unbiased approach to examine genes and pathways regulating FP-RMS stemness. Results: TAZ/YAP functionally augment P3F transcriptional activity in reporter assays and also regulate expression of P3F targets. Immunoblotting of 293T cells co-expressing epitope-tagged proteins demonstrate an interaction between TAZ/YAP and P3F. A TAZ/YAP/P3F complex is also seen in co-IPs of endogenous proteins in FP-RMS cells. Serial passaging of spheres increases TAZ/YAP expression, suggesting these may be important for stem cell enrichment. Indeed, expression of SOX2, OCT4, and NANOG are TAZ/YAP-dependent, whereby their expression decreases with KD and increases with S89A/S127A. Functionally, TAZ/YAP are required for FP-RMS sphere formation in limiting dilution assays. Conclusions: These studies expand on prior work showing TAZ/YAP are potent oncoproteins in FP-RMS. We identify a novel complex between TAZ/YAP and P3F, show TAZ/YAP are positive regulators of P3F transcriptional activity, and demonstrate that TAZ/YAP regulate FP-RMS cancer cell stemness. Thus, targeting TAZ/YAP could be a vulnerability of P3F and for inhibiting the cancer stem cell population. Citation Format: Breanne A. Burgess, Katherine K. Slemmons, Corinne M. Linardic, Michael D. Deel. Transcriptional co-activators TAZ/YAP are novel regulators of PAX3-FOXO1 transcriptional programing and fusion-positive rhabdomyosarcoma cancer cell stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3670.

Long noncoding RNA DLEU1 aggravates glioma progression via the miR-421/MEF2D axis.

BackgroundLong noncoding RNA (lncRNA) deleted in lymphocytic leukemia 1 (DLEU1) was reported to be involved in the development and progression of multiple cancers. However, the accurate expression pattern, biological function and potential molecular mechanism of DLEU1 in glioma are not yet known. The present study investigated the role of DLEU in the development and progression of glioma, as well as the potential mechanism played by DLEU1 in glioma.Materials and methodsThe levels of DLEUI in glioma tissues and cell lines were examined using quantitative real-time PCR. The potential effects of DLEU1 on the proliferation, mobility, invasion and apoptosis of glioma cells were evaluated using corresponding in vitro experiments. The association between DLEU1 and microRNA (miR)-421 was also determined using luciferase reporter activity and RNA immunoprecipitation (RIP) assays.ResultsThe results revealed that DLEU1 was significantly upregulated in glioma tissues and cell lines. Increased DLEU1 was positively associated with the high-grade carcinoma (III–IV). Functional studies revealed that knockdown of DLEU1 expression by siRNA led to decreased proliferation, migration and invasion and increased apoptosis in human glioma cells. Furthermore, luciferase reporter activity and RIP assays confirmed that DLEUI could act as a competing endogenous RNA (ceRNA) for miR-421 that functioned as a tumor suppressor in glioma. Moreover, inhibition miR-421 partially restored the effect of DLEU1 knockdown on the glioma cells. DLEU1 could regulate myocyte enhancer factor 2D (MEF2D) expression, a known target of miR-421 in glioma cells.ConclusionTaken together, these findings suggested that DLEU1 regulated MEF2D expression to promote glioma progression by sponging miR-421 and that DLEU1 might be a potential therapeutic target for glioma.

Abstract 654: Dietary interventions ameliorate infectious colitis through differential regulation of Lgr5

Abstract Background: During inflammatory bowel disease (IBD), the disruption of the epithelial barrier and translocation of bacteria drive inappropriate immune responses and unresolved inflammation. Rapid wound healing responses orchestrated by intestinal stem cells (ISCs) are central to inflammatory resolution and normalization of the mucosal barrier. Commensal microbiota ferment fibers to produce short-chain-fatty-acids (SCFAs) such as butyrate and its decrease has been linked to IBD. However, the mechanism through which SCFAs promote wound healing is poorly understood. Aims: The present study was designed to test the hypothesis that dietary fibers/butyrate ameliorate infectious colitis by differentially regulating Lgr5-dependent crypt regeneration and wound healing. Methods: Lgr5CreERT2/+; Rosa26LacZ reporter (Lgr5-R) mice and wild-type littermates were infected with Citrobacter rodentium (CR; 108 CFUs) and fed with either 6% Pectin (Pec) or 6% Tributyrin (Tbt) diets followed by euthanasia at 12 days post-infection. To partially deplete microbiota, mice were also given a cocktail of vancomycin (500mg/L), metronidazole (1g/L) and Ciprofloxacin (0.2g/L) for 10 days starting 3-days post-CR infection. Fresh feces pre- and post-infection/treatments were taken for 16S rDNA sequencing. Lineage tracing post-tamoxifen and Lgr5 promoter reporter activity assays were performed. Results: Both Pec and Tbt reduced the severity of CR-induced colitis as was evidenced by increased body weight and colon length, reduced immune cell infiltration and increased mucus production compared with CR-infected but untreated mice. 16s rDNA sequencing revealed significant dysbiosis during CR infection with the dominance of Proteobacteria and loss of Firmicutes and Bacteroidetes. Both Pec and Tbt diets reduced the levels of Proteobacteria and restored Firmicutes and Bacteroidetes phyla to pre-infection levels. X-gal staining revealed that there was an expansion of LacZ-labeled Lgr5(+) stem cells in the colons of CR infected Lgr5-R mice when subjected to dietary intervention via Pectin and Tributyrin as sources of butyrate compared with controls. Interestingly, Pec-induced Lgr5 regulation was dependent upon the presence of gut microbiota as antibiotics treatment reduced Pec-induced Lgr5 expansion and the extent of crypt regeneration. Tbt-treatment, on the other hand, regulated Lgr5 independently of the microbiota. Butyrate, in a dose-dependent (1-10mM) manner, increased Lgr5 promoter reporter activity. Docking studies further revealed butyrate’s ability to efficiently bind Lgr5 with a -4.0 Kcal/mol binding energy. The cellular thermal shift assay showed that butyrate was indeed able to bind Lgr5. Conclusions: Thus, dietary interventions, by altering the gut microbiota, can differentially regulate Lgr5’s ability to orchestrate crypt regeneration and wound healing to ameliorate colitis. Citation Format: Ishfaq Ahmed, Badal C. Roy, Rita-Marie T. Raach, Sharon J. Manley, Pugazhendhi Srinivasan, Prasad Dandawate, Afreen Sayed, Danny R. Welch, Shrikant Anant, Venkatesh Sampath, Shahid Umar. Dietary interventions ameliorate infectious colitis through differential regulation of Lgr5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 654.

LncRNA DGCR5 regulates the non-small cell lung cancer cell growth, migration, and invasion through regulating miR-211-5p/EPHB6 axis.

Non-small cell lung cancer (NSCLC) accounts for about 80% of lung cancers worldwide. In recent years, importance of noncoding RNAs including long noncoding RNA and microRNA in regulating tumor progression has been appreciated. Abnormally expression of DiGeorge syndrome critical region gene 5 (DGCR5) was found in multiple human cancers but its function in NSCLC is largely unknown. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was conducted to explore DGCR5 expression level in NSCLC. Bioinformatic analyses were conducted to explore the targets of DGCR5. Cell counting kit-8 assay, wound-healing assay, and transwell invasion assay were performed to analyze functions of DGCR5. RT-qPCR revealed that DGCR5 expression in NSCLC cells was significantly lower than in normal cell. DGCR5 overexpression suppresses NSCLC cell growth, migration, and invasion. Online algorithms found EPH receptor B6 (EPHB6) and DGCR5 contains same miR-211-5p binding region. The predicted connections were further validated by luciferase activity reporter assay. Recue experiments showed DGCR5 regulates NSCLC cell behaviors via targeting miR-211-5p/EPHB6. These findings collectively identified DGCR5/miR-211-5p/EPHB6 triple axis in NSCLC, which may novel understanding regarding the tumorigenesis of NSCLC.

MiR-506-3p acts as a novel tumor suppressor in prostate cancer through targeting GALNT4.

Researches have indicated that microRNA-506-3p (miR-506-3p) was downregulated and functioned as tumor suppressor in cancers. However, the biological role of miR-506-3p in prostate cancer (PCa) remains to be elucidated. Expression of miR-506-3p in PCa cell lines was measured by qRT-PCR. Effects of miR-506-3p expression on PCa cell behaviors were investigated with MTT assay, colony formation assay, and transwell invasion assay. Connection of miR-506-3p and N-Acetylgalactosaminyltransferase-4 (GALNT4) was analyzed with luciferase activity reporter assay and Western blot assay. miR-506-3p expression was downregulated in PCa cell lines. Function studies demonstrated that overexpression of miR-506-3p inhibits PCa tumor progression in vitro. Mechanistic investigations found GALNT4 was a direct target of miR-506-3p. Overexpression of GALNT4 reversed the tumor-suppressive effects of miR-506-3p on PCa cell. Our results elucidated genetic silencing of miR-506-3p enhances GALNT4 oncogene expression to accelerate PCa progression.

MicroRNA-181b-5p modulates tumor necrosis factor-α-induced inflammatory responses by targeting interleukin-6 in cementoblasts.

Tooth cementum is a bone-like mineralized tissue and serves as a microbial barrier against invasion and destruction. Cementum is also responsible for tooth stability and defending pulp from outside stimuli, which is formed by cementoblasts. Although it is crucial for periodontal and periapical diseases, the mechanisms underlying the pathophysiological changes of cementoblasts and their inflammatory responses remain unclear. MiR-181b is found to modulate vascular inflammation and endotoxin tolerance. In this study, miR-181b-5p was downregulated in tumor necrosis factor-α (TNF-α)-stimulated cementoblasts, whereas proinflammatory molecules increased. The mouse periapical lesions have similar results, which imitate an inflammatory environment for cementoblasts in vivo. The bioinformatics analysis and dual luciferase reporter assay suggested that miR-181b-5p targeted interleukin-6 (IL-6). Overexpressing miR-181b-5p negatively regulated IL-6 and proinflammatory chemokine. Western blot analysis and luciferase activity reporter assay verified that miR-181b-5p weakened the NF-κB activity. Hence, miR-181b-5p moderated proinflammatory chemokine production by targeting IL-6 in cementoblasts and NF-κB signaling pathway was involved. Furthermore, miR-181b-5p promoted cementoblast apoptosis, which may enhance the resolution of inflammation. Overall, our data revealed that miR-181b-5p was a negative regulator of TNF-α-induced inflammatory responses in cementoblasts.

PDLIM2 Is a Marker of Adhesion and β-Catenin Activity in Triple-Negative Breast Cancer.

The PDLIM2 protein regulates stability of transcription factors including NF-κB and STATs in epithelial and hemopoietic cells. PDLIM2 is strongly expressed in certain cancer cell lines that exhibit an epithelial-to-mesenchymal phenotype, and its suppression is sufficient to reverse this phenotype. PDLIM2 supports the epithelial polarity of nontransformed breast cells, suggesting distinct roles in tumor suppression and oncogenesis. To better understand its overall function, we investigated PDLIM2 expression and activity in breast cancer. PDLIM2 protein was present in 60% of tumors diagnosed as triple-negative breast cancer (TNBC), and only 20% of other breast cancer subtypes. High PDLIM2 expression in TNBC was positively correlated with adhesion signaling and β-catenin activity. Interestingly, PDLIM2 was restricted to the cytoplasm/membrane of TNBC cells and excluded from the nucleus. In breast cell lines, PDLIM2 retention in the cytoplasm was controlled by cell adhesion, and translocation to the nucleus was stimulated by insulin-like growth factor-1 or TGFβ. Cytoplasmic PDLIM2 was associated with active β-catenin and ectopic expression of PDLIM2 was sufficient to increase β-catenin levels and its transcriptional activity in reporter assays. Suppression of PDLIM2 inhibited tumor growth in vivo, whereas overexpression of PDLIM2 disrupted growth in 3D cultures. These results suggest that PDLIM2 may serve as a predictive biomarker for a large subset of TNBC whose phenotype depends on adhesion-regulated β-catenin activity and which may be amenable to therapies that target these pathways. SIGNIFICANCE: This study shows that PDLIM2 expression defines a subset of triple-negative breast cancer that may benefit from targeting the β-catenin and adhesion signaling pathways. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/10/2619/F1.large.jpg.

MiR-376a inhibits the proliferation and invasion of osteosarcoma by targeting FBXO11.

Dysregulation of microRNAs (miRNAs) is frequently found in the tumorigenesis of osteosarcoma (OS). miR-376a was found to play tumor suppressive roles in human cancers. However, the role of miR-376a in OS remains unclear. The expression of miR-376a was analyzed by quantitative real-time PCR (qRT-PCR) in OS cell lines. Cell proliferation assay, cell invasion assay, and cell apoptosis assay were performed to detect the biological function of miR-376a after synthetic miRNA transfection. The target of miR-376a was predicted by TargetScan and miRDB, and further validated by luciferase activity reporter assay and western blot. miR-376a expression was revealed to be decreased in OS cell lines. In vitro experiments showed that overexpression of miR-376a inhibits OS cell proliferation and invasion but promotes apoptosis. Luciferase activity reporter assay and western blot assay showed F-box protein 11 (FBXO11) was a direct target of miR-376a. Furthermore, FBXO11 mediated the role of miR-376a on the proliferation, invasion, and apoptosis of OS cells. Collectively, these results revealed miR-376a functions as a tumor suppressor by targeting FBXO11 in OS. It may be developed as a therapeutic target for OS patients.

MiR-1294 acts as a tumor suppressor in clear cell renal cell carcinoma through targeting HOXA6.

Renal cancer represents about 3% of all human cancers. Clear cell renal cell carcinoma (ccRCC) is the main type of renal cancer. MicroRNAs (miRNAs) have been reported to play crucial roles in the carcinogenesis of human cancers. This study was aimed to investigate the expression of miR-1294 and the mechanisms underlying miR-1294-mediated ccRCC progression. The miR-1294 expression levels in ccRCC cell lines were analyzed by quantified real time-PCR (qRT-PCR). The effect of the miR-1294 expression on the overall survival of ccRCC patients was analyzed by the Kaplan-Meier Plotter. Cell proliferation, colony growth, and cell invasion were examined by cell counting kit-8 assay, colony formation assay, and transwell invasion assay, respectively. The luciferase activity reporter assay and Western blot assay were conducted to validate the connection between miR-1294 and homeobox A6 (HOXA6). MiR-1294 was downregulated in ccRCC cell lines and correlated with the poor overall survival of ccRCC patients. The overexpression of miR-1294 inhibits ccRCC cell proliferation, colony growth, and cell invasion. HOXA6 was validated as a target of miR-1294 and negatively regulated by miR-1294. The overexpression of HOXA6 attenuated the miR-1294-mediated effects on ccRCC cellular functions. Our results indicated that miR-1294 functions as a tumor suppressor in ccRCC. MiR-1294 suppressed cell proliferation, colony formation, and invasion in ccRCC partially via targeting HOXA6.

Angiogenin regulates epithelial-mesenchymal transition of hepatocellular carcinoma through upregulation of HMGA2.

Angiogenin (ANG) is known to alter multiple cell behaviors by directly targeting downstream targets, but its role in hepatocellular carcinoma (HCC) remains to be elucidated. The expression of ANG in HCC cell lines was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The effects of ANG expression on cell proliferation, cell migration, and hallmarks of epithelial-mesenchymal transition (EMT) process were also investigated. The relationship between ANG and high mobility group AT-hook 2 (HMGA2) was evaluated. ANG expression was increased in HCC cell lines. Downregulating of ANG inhibits proliferation, migration, and EMT of HCC cells. The direct regulation of ANG on HMGA2 was verified by luciferase activity reporter assay and western blot assay. Furthermore, overexpression of HMGA2 reversed the inhibitory effects of ANG downregulation on HCC cell behaviors. Our results illustrated the mechanism that ANG promote the EMT of HCC through targeting HMGA2.

MiR-376a functions as tumor suppressor by targeting SGK3 in renal cell carcinoma.

Emerging evidence showed that microRNA-376a (miR-376a) functions as a crucial role in human cancers. However, its role in renal cell carcinoma (RCC) remains unclear. MiR-376a expression in RCC cell lines was analyzed by quantitative real time-polymerase chain reaction (qRT-PCR). The target of miR-376a was validated using the luciferase activity reporter assay, and the effects of miR-376a expression on RCC cell behaviors were investigated in vitro. MiR-376a expression was downregulated in RCC cell lines in comparison with HK-2 cell line. Low miR-376a expression was correlated with poor overall survival of RCC patients. Serum/glucocorticoid regulated kinase family member 3 (SGK3) was validated as a direct target of miR-376a. The overexpression of miR-376a inhibits RCC cell proliferation, migration, and invasion through regulating SGK3. Taken together, these results demonstrated the tumor suppressive role of miR-376a via targeting SGK3 in RCC and indicated that miR-376a might represent a novel therapeutic target for RCC treatment.

MiR-584-5p regulates hepatocellular carcinoma cell migration and invasion through targeting KCNE2.

BackgroundHepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancer type. This study was aimed to investigate the role of microRNA‐584‐5p (miR‐584‐5p) in regulating HCC progression.MethodsThe expression of miR‐584‐5p in HCC cell lines was analyzed by quantitative real‐time polymerase chain reaction. Effects of miR‐584‐5p depletion on HCC cell proliferation, migration, and invasion in vitro were analyzed by cell counting kit‐8 assay, wound‐healing assay, and transwell invasion assay. miR‐584‐5p targeting potassium voltage‐gated channel subfamily E regulatory subunit 2 (KCNE2) was identified using bioinformatics algorithm and dual‐luciferase activity reporter assay. Kaplan–Meier Plotter website was used to investigate the effect of miR‐584‐5p or KCNE2 expression on the overall survival of HCC patients.ResultsIn vitro functional assays showed miR‐584‐5p depletion decreased HCC cell proliferation, cell migration, and cell invasion. Moreover, miR‐584‐5p functions by directly targeting KCNE2, and it in turn, mediates the effects of miR‐584‐5p on HCC cell behaviors.ConclusionsThese results demonstrated that miR‐584‐5p functions as an oncogenic miRNA in HCC.