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DOE OSTI2017

BRG1 promotes VEGF-A expression and angiogenesis in human colorectal cancer cells

Angiogenesis plays an important role in tumor growth and progression in solid tumors. Vascular endothelial growth factor (VEGF) is one of the most critical and specific factors that stimulate both physiological and pathological angiogenesis. Here, we report a novel role of BRG1, the core subunit of SWI/SNF family complexes, in angiogenesis. In this study, we demonstrate that BRG1 is overexpressed in colorectal cancer and decreased expression of BRG1 not only blocks cell proliferation but remarkably inhibits the ability of HUVECs to form capillary-like structures. Moreover, our study shows that BRG1 can regulate the expression of VEGF-A by interacting with HIF-1α. Furthermore, we find VEGF-A is overexpressed in colorectal cancer and is positively correlated with BRG1 expression. Taken together, our study demonstrated that BRG1 can promote VEGF-A expression and angiogenesis in colorectal cancer and BRG1 may be a novel drug target for the treatment of colorectal cancer. - Highlights: • BRG1 is highly expressed in colorectal cancer. • The deficiency of BRG1 in colorectal cancer cells inhibits the proliferation and tube formation of HUVECs. • BRG1 can regulate the expression of VEGF-A by interacting with HIF-1α. • BRG1 and VEGF-A expression are significantly correlated in colorectal tissues.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Up-regulation of caveolin-1 by DJ-1 attenuates rat pulmonary arterial hypertension by inhibiting TGFβ/Smad signaling pathway

Highlights: • Knockout of DJ-1-aggravated hypoxic pulmonary arterial hypertension of rats. • Overexpression of DJ-1 alleviated PASMs injury induced by hypoxia. • DJ-1 alleviated hypoxia-induced PASMCs injury via Cav-1 by inhibiting TGFβ/Smad. Pulmonary arterial hypertension (PAH), characterized by excessive proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs), is closely associated with the imbalance in vasoactive mediators and massive remodeling of pulmonary vasculature. DJ-1/park7, a multifunctional protein, plays a critical defense role in several cytobiological activity, such as transcriptional regulation, anti-oxidative stress and tumor formation. In this study, we investigated the effects of DJ-1 on hypoxia-induced PAH model rats and PASMCs, as well as its possible molecular mechanism. First, the low expressions of DJ-1 and caveolin-1 (Cav-1) were synchronously detected in lung tissue of PAH model rats and hypoxia-induced PASMCs by Western blot. Then, the DJ-1 wild type (WT) or Knock out (KO) rats were exposed to chronic hypoxia to mimic a hypoxic PAH condition. The protein level of Cav-1 was markedly decreased in the tissue of DJ-1 KO rats, and additionally lower in tissue of the hypoxia group than that in the normoxia group for DJ-1 WT and KO rats. In vivo, hemodynamic data showed that the pulmonary arterial pressure (mPAP), right ventricle systolic pressure (RVSP) and pulmonary arterial systolic pressure (PASP), as well as the weight of the right ventricle/left ventricle plus septum (RV/LV+S) ratio of PAH model rats were higher in the DJ-1 KO group than those in the DJ-1 WT group. Moreover, knockout of DJ-1 also results in the phenotype switch from contractile to synthetic PASMC, which is reflected by reduced calponin and SM22α expressions. In vitro, DJ-1 overexpression reversed hypoxia-induced elevation of PASMC cell proliferation, migration and Ca{sup 2+} concentration, which were not obviously observed in Cav-1 shRNA (sh-Cav-1) and DJ-1 co-transfected cells. Then the increased levels of calponin and SM22α were detected in the DJ-1 group; similarly those levels were not changed in the DJ-1+sh-Cav-1 group. Finally, the expression of TGFβ1, p-Smad2 and p-Smad3 were obviously decreased in the ad-DJ-1 group, however those were all elevated in the DJ-1 and sh-Cav-1 co-transfected groups. In conclusion, these results indicate that DJ-1 may alleviate hypoxia-induced PASMCs injury by Cav-1 through inhibiting the TGFβ/Smad signaling pathway.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Tsc1-dependent transcriptional programming of dendritic cell homeostasis and function

Highlights: • Transcriptional mechanisms of Tsc1-regulated DC homeostasis and function. • Tsc1 controls DC survival, proliferation, metabolism and antigen presentation. • Tsc1affect DC gene expression partially through inhibition of mTORC1 signal. Dendritic cells (DCs) are pivotal to initiating adaptive immune response. Emerging evidence highlights important roles of tuberous sclerosis complex 1 (Tsc1) in DC development and activation. Our previous study also showed that Tsc1 expression in DCs was required to promote T-cell homeostasis and response partially through inhibiting mammalian target of rapamycin complex1 (mTORC1). However, the molecular mechanism of transcriptional regulation by which Tsc1 control DC homeostasis and function remains largely unknown. Here we globally identified the Tsc1-regulated genes by comparing the transcriptional profiling of Tsc1-deficient DCs with wild-type DCs. It showed that Tsc1 specifically regulated the expression of groups of gene sets critically involved in DC survival, proliferation, metabolism and antigen presentation. The impacts of Tsc1 on DC gene expression were partially dependent on inhibition of mTORC1 signal. Our study thus provides a comprehensive molecular basis for understanding how Tsc1 programs the homeostasis and function of DCs through transcriptional regulation.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Up-regulated lncRNA-MSX2P1 promotes the growth of IL-22-stimulated keratinocytes by inhibiting miR-6731-5p and activating S100A7

Highlights: • High expression of MSX2P1 and S100A7 in psoriatic lesion tissues. • MSX2P1, functions as miR-6731-5p sponge, modulates S100A7 expression. • miR-6731-5p inhibits the progression of IL-22-stimulated keratinocytes by S100A7. Competitive endogenous RNAs (ceRNAs) regulate RNA transcripts by competing for shared miRNAs and play critical roles in disease development. Psoriasis is a long-lasting, recurring chronic inflammatory skin disease characterized by hyperproliferation of keratinocytes. The keratinocyte response is triggered by the activation of inflammatory cytokines, like interleukin-22 (IL-22). We used lncRNA array analysis to detect differentially expressed lncRNAs in skin (HaCaT) cells treated with or without IL-22. We used hematoxylin and eosin (H&E) staining to determine the pathological changes in skin cells and immunohistochemistry to evaluate the expression of S100A7. We used qRT-PCR and Western blotting to detect the expression levels of MSX2P1 and S100A7. We down-regulated the expression of MSX2P1 by infecting with lentiviral-vector shRNA. We measured cell proliferation, cell cycle status, and apoptosis by the CCK-8 assay, flow cytometry, and Annexin Ⅴ-FITC/PI staining, respectively. In addition, we used the luciferase reporter gene assay to determine the relationships between MSX2P1 or miR-6731-5p and S100A7, respectively. LncRNA array analysis revealed that 103 lncRNAs were up-regulated and 51 were down-regulated. Furthermore, qRT-PCR showed that the mRNAs levels of MSX2P1 was significantly altered in HaCaT cells treated with IL-22, compared with control cells; and MSX2P1 was mainly in the cytoplasm. Based on the IL-22-stimulated lncRNA-associated ceRNA network, we selected MSX2P1-miR-6731-5p-S100A7 for further study. H&E staining exhibited characteristic features specific to psoriatic lesions. Immunohistochemistry demonstrated significantly increased expression levels of S100A7 in psoriatic lesions, compared with normal skin tissue. We observed a positive correlation between lncRNA-MSX2P1 expression and S100A7 expression. In addition, miR-6731-5p suppressed proliferation, accelerated apoptosis in IL-22-stimulated keratinocytes, and decreased the expressions of S100A7, IL-12β, IL-23, HLA-C, CCHCR1, TNF-α, and NF-κB proteins. Our data demonstrated that MSX2P1 facilitate the progression and growth of IL-22-stimulated keratinocytes by inhibiting miR-6731-5p and activating S100A7. We speculate that the biological network of MSX2P1-miR-6731-5p-S100A7 is a potential novel therapeutic target for the future treatment of psoriasis.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

ADAMTS-1 disrupts HGF/c-MET signaling and HGF-stimulated cellular processes in fibrosarcoma

Extracellular matrix (ECM) serves as a reservoir for biologically active factors, such as growth factors and proteases that influence the tumor cell behavior. ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs) is a secreted protease that has the ability to modify the ECM during physiological and pathological processes. Here, we analyzed the role played by ADAMTS-1 regulating HGF and TGF-β1 activities in the high-grade fibrosarcoma cell line (HT1080). We generated HT1080 and HEK293T cells overexpressing ADAMTS-1. HT1080 cells overexpressing ADAMTS-1 (HT1080-MPA) exhibited a significant decrease in cell proliferation and migration velocity, both in presence of HGF. We obtained similar results with ADAMTS-1-enriched conditioned medium from other cell type. However, ADAMTS-1 overexpression failed to affect TGF-β1 activity associated with HT1080 cell proliferation and migration velocity. Immunoblotting showed that ADAMTS-1 overexpression disturbs c-Met activation upon HGF stimulation. Downstream ERK1/2 and FAK signaling pathways are also influenced by this protease. Additionally, ADAMTS-1 decreased the size of the fibrosarcospheres, both under normal conditions and in the presence of HGF. Likewise, in presence of HGF, ADAMTS-1 overexpression in HT1080 disrupted microtumors formation in vivo. These microtumors, including individual cells, presented characteristics of non-invasive lesions (rounded morphology). Our results suggest that ADAMTS-1 is involved in regulating HGF-related functions on fibrosarcoma cells. This protease may then represent an endogenous mechanism in controlling the bioavailability of different growth factors that have a direct influence on tumor cell behavior.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Overexpression of COPS3 promotes clear cell renal cell carcinoma progression via regulation of Phospho-AKT(Thr308), Cyclin D1 and Caspase-3

Highlights: • COPS3 was overexpressed in ccRCC tissues and cell lines. • Knockdown of COPS3 impaired the viability of ACHN and 786-O cells. • si-RNA mediated downregulation of COPS3 induced G0/G1 phase and promoted apoptosis in ACHN and 786-O cells. • Downregulation of COPS3 repressed Phospho-AKT(Thr308) and Cyclin-D1 expression and upregulated Caspase-3 expression. The third subunit of the COP9 signalosome (COPS3) is associated with cell proliferation and tumorigenesis process in cancer. The present study showed that the expression level of COPS3 was upregulated in malignant cell lines and COPS3 overexpression was related with clinical stage, T stage, historical grade. Kaplan-Meier survival curves showed that COPS3 may function as a prognostic factor for overall survival. CCK-8 and colony formation assays revealed that knockdown of COPS3 in ACHN and 786-O significantly impacted proliferation in vitro. In addition, flow cytometry showed that inhibition of COPS3 induced G0/G1 arrest and promoted apoptosis. COPS3 may promote kidney cancer progression by altering Phospho-AKT(Thr308), Cyclin D1 and Caspase-3 expression. Collectively, Our findings suggest that COPS3 may be a new potential target of ccRCC.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

The RNA binding protein tristetraprolin down-regulates autophagy in lung adenocarcinoma cells

Highlights: • TTP down-regulates autophagy to induce cell death in lung adenocarcinoma cells. • Induction of TTP did not result in cell apoptosis, but result in down-regulation of autophagy in lung adenocarcinoma cells. • A novel mechanism of tumor suppressor by TTP in lung adenocarcinoma is suggested. Tristetraprolin (TTP) is the most well-known member of RNA-binding zinc-finger protein that play a significant role in accelerating mRNA decay. Increasingly studies have reported that TTP was functioned as a tumor suppressor gene in several types of carcinomas, while its underlying mechanism is not clear yet. In the current study, we found that TTP overexpression decreased cell proliferation and increased cell death in lung adenocarcinoma cells, with the cell cycle arrest at the S phase. Remarkably, instead of inducing cell apoptosis directly, TTP overexpression alters cell autophagy. Our studies demonstrate that TTP overexpression has no effect on apoptosis related genes, but decreases the expression of autophagy-related genes, including Beclin 1 and LC3II. The level of autophagy flux assessed by infection with the mGFP–RFP–LC3 adenovirus construction has been blocked by TTP overexpression. Moreover, the autophagic vacuoles number detected by transmission electron microscopy decreased with TTP expression up-regulation. Our results indicate, for the first time, that TTP suppresses cell proliferation and increases cell death through cell autophagy pathway in lung cancer cells. Our study provides a new angle of view for TTP function as a tumor suppressor which could be targeted in tumor treatment.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Diacylglycerol lipase alpha promotes tumorigenesis in oral cancer by cell-cycle progression

Highlights: • DAGLA was up-regulated in human oral squamous cell carcinoma (OSCC) cells compared with human normal oral keratinocytes (HNOKs). • Overexpression of DAGLA in OSCCs controlled cell proliferation through cell-cycle progression. • The tumoral volume of the orlistat-treated group was clearly smaller than that of the control group in vivo. • DAGLA expression level might be a useful diagnostic indicator and a new therapeutic target for OSCCs. Diacylglycerol lipase alpha (DAGLA), which catalyzes the hydrolysis of diacylglycerol to 2-arachidonoylglycerol and free fatty acid, is required for axonal growth during the brain development and for retrograde synaptic signaling at mature synapses. So far, no information was found regarding the possible role of DAGLA in human tumorigenesis. Thus, the current study sought to clarify the contribution of DAGLA in oral squamous cell carcinomas (OSCCs) and assess the clinical possibilities for OSCC treatment. Using real-time quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry, we found a significant up-regulation of DAGLA in OSCCs compared with normal cells and tissues both at mRNA and protein expression levels. Knockdown models in OSCC-derived cell lines for DAGLA (siDAGLA) and treatment with a lipase inhibitor (orlistat) showed several depressed cellular functions, including cellular proliferation and migratory activities through cell-cycle arrest at G1 phase. Furthermore, we found that DAGLA-positive OSCC samples were correlated highly with the primary tumoral size. We concluded that DAGLA may be a key determinant in tumoral progression and might be a therapeutic target for OSCCs.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

TRIM24 promotes hepatocellular carcinoma progression via AMPK signaling

Highlights: • TRIM24 expression is up-regulated in HCC, increased along with the progression of HCC. • TRIM24 promotes cell proliferation and migration of HCC cell line. • TRIM24 promotes HCC progression through AMPK signaling. • TRIM24 negatively regulated AMPK expression in vitro and in vivo. • TRIM24 expression levels negatively correlated with AMPK in HCC clinical samples. Hepatocellular carcinoma (HCC) is one of the most common cancers diagnosed worldwide. However, the mechanism underlying HCC pathogenesis remains unknown. In the present study, TRIM24 was found increased in human HCC clinical samples and positively correlated with HCC tumor grade. Furthermore, TRIM24 knockdown inhibits proliferation and migration in a human HCC cell line in vitro while also inhibiting tumor growth in vivo. Mechanistically, TRIM24 appears to promote liver tumor development via AMPK signaling as AMPK knockdown alleviated the in vitro and in vivo effects of TRIM24 knockdown in a human HCC cell line. Taken together, these data enhance our understanding of HCC development in addition to highlighting TRIM24-regulated AMPK signaling as a potential therapeutic target for HCC treatment.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Knockdown of POLDIP2 suppresses tumor growth and invasion capacity and is linked to unfavorable transformation ability and metastatic feature in non-small cell lung cancer

Highlights: • Overexpression of POLDIP2 promoters transformation ability in H1299 cells. • Knockdown of POLDIP2 inhibits tumorigenicity and metastasis in vitro and in vivo. • Knockdown of POLDIP2 reduces cyclin D1, CDH2, Slug, and Twist expression. The main problem in the treatment of non-small cell lung cancer (NSCLC) is metastasis. Epithelial–mesenchymal transition (EMT) is known as the critical signaling in tumor progression, metastasis, and also the drug resistance. In this study, we reported a novel gene Polymerase delta-interacting protein 2 (POLDIP2) was downregulated in NSCLC tissues and first demonstrated that overexpression of POLDIP2 increased the anchorage-independent growth (AIG) and invasiveness of H1299 cells. In addition, we examined that knockdown of POLDIP2 in H1299 and A549 cells reduced tumorigenicity and metastatic capacity in vitro and also in vivo. Moreover, downregulation of the cell proliferation marker cyclin D1 and EMT markers CDH2, Slug, and Twist was showed in H1299 cells by POLDIP2 knockdown, suggesting that the inhibition of malignancy was affected by modulating key genes for tumor growth and invasiveness. Taken together, our study is the first study that demonstrated that POLDIP2 gene was function as an oncogene in NSCLC and implied the oncogenic ability might be through promoting cell proliferation or EMT.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Inhibition of miR-135b by SP-1 promotes hypoxia-induced vascular endothelial cell injury via HIF-1α

Highlights: • Expression of SP-1 and miR-135b in hypoxia-exposed HUVECs. • SP-1 loss affected cell proliferation and apoptosis in HUVECs exposed to hypoxia. • SP-1 loss attenuated hypoxia-induced levels of inflammatory factors and VEGF. • SP-1 loss increased miR-135b and reduced HIF-1α expression in HUVECs. • SP-1/miR-135b/HIF-1α was a novel axis in hypoxia-induced injury in HUVECs. Myocardial hypoxia-induced endothelial cell apoptosis contributes to cardiac dysfunction, such as myocardial infarction (MI), myocardial ischemia, and heart failure. Thus, it is important to investigate the molecular mechanisms of vascular endothelial cells (VECs) during exposure to hypoxia. SP-1 is an important regulator of cytokines associated with cell functions. We found that SP-1 expression increased in human umbilical vein endothelial cells (HUVECs) exposed to hypoxia by western blot. Then the SP-1 siRNA was transfected into HUVECs under hypoxic condition. MTT assay showed that hypoxia reduced the cell proliferation, but SP-1 siRNA reversed that. Transfection with si-SP-1 also reversed cell apoptosis and reactive oxygen species (ROS) production increased by hypoxia treatment. Moreover, inflammatory phenotype were increased in hypoxia induced HUVECs, including ICAM-1,VCAM-1 levels as well as TNFα, IL-6 and IL-1β secretion, and the si-SP-1 also reversed this effect of hypoxia. Additionally, si-SP-1 increased expression of miR-135b and reduced expression of hypoxia-inducible factor 1-α (HIF-1α), which is the target gene of miR-135b. To investigate the underlying mechanism of SP-1 on hypoxia induced HUVECs injury, the anti-miR-135b or HIF-1α agonist (CoCl{sub 2}) were used. Finally, the result indicated that both anti-miR-135b or CoCl{sub 2} treatment reversed the effects of SP-1 siRNA under hypoxia. In conclusion, the SP-1/miR-135b/HIF-1α axis may play a critical role in hypoxia-induced vascular endothelial injury. Our study thus provides novel insights into the role of this transcription factor and miRNAs in the pathogenesis of hypoxia-induced cardiac dysfunctions.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

High expression of enhancer RNA MARC1 or its activation by DHT is associated with the malignant behavior in bladder cancer

Highlights: • DHT treatment significantly elevated eMARC1 expression levels. • eMARC1 silencing impaired the androgenic effect of DHT in BCa cells. • DHT promoted the malignant behavior of BCa by activating eMARC1. Enhancer RNAs (eRNAs), a subclass of noncoding RNA from enhancers, have biological functions in gene expression. However, their potential role in bladder cancer (BCa) remains largely unknown. The present study investigated the functional role of androgen-associated androgen receptor (AR) mediated-eRNA MARC1 (eMARC1) in BCa progression. Cell proliferation, migration, and apoptosis of BCa cell lines (5637 and T24) with different eMARC1 expression levels or treated with 5α-dehydrotestosterone (DHT) were investigated. In the current study, we discovered that eMARC1 was highly expressed in BCa tissues and cell lines, and eMARC1 overexpression promoted the progression of BCa cells, while knockdown of eMARC1 suppressed tumorigenesis. DHT treatment significantly elevated eMARC1 expression levels, which also facilitated cell proliferation, motility, and inhibited cell apoptosis. We further found that eMARC1 silencing impaired the androgenic effect of DHT in BCa cells. These results suggested that eMARC1 exerted its effects on BCa cell progression, and DHT promoted bladder cancer progression by activating eMARC1.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

FPR1 gene silencing suppresses cardiomyocyte apoptosis and ventricular remodeling in rats with ischemia/reperfusion injury through the inhibition of MAPK signaling pathway

Ischemia/reperfusion (I/R) injury, one of the leading health problems in the world, is defined as a cause of cardiomyocytes death. In the present study, we investigate the role of formyl peptide receptor 1 (FPR1) in cardiomyocyte apoptosis and ventricular remodeling of I/R injury rats and the underlying mechanism involving mitogen-activated protein kinase (MAPK) signaling pathway. The important differentially expressed genes (DEGs) in I/R injury were screened out and downstream pathways affected by DEGs were predicted. We grouped 90 rats into sham, I/R, NC siRNA, FRP1 siRNA, empty vector, and FRP1 vector groups and established a model of I/R injury in rats. CVF value, myocardial infarct areas and positive expression rate of FPR1 and MAPK were detected. Levels of FPR1 and MAPK pathway-related genes were determined by RT-qPCR and western blot analysis. MTT assay was performed to evaluate cell proliferation and flow cytometry to evaluate cell cycle progression and apoptosis. GSE19804 and GSE27262 were screened from Gene Expression Omnibus database. FPR1 was higher in patients with I/R injury and activate the MAPK signaling pathway. FRP1 gene silencing decreased CVF value, infarct area, apoptotic index, positive expression rates of FPR1 and MAPK, decreased FPR1, p38, ERK, JNK, MMP-2, TIMP-2, NF-kB, Bax, p-p38, p-ERK, and p-JNK levels, increased Bcl-2 level, promoted cell proliferation and cell cycle progression, and inhibited cell apoptosis rate. Overall, our study demonstrates that the silencing of FPR1 gene depresses inflammation, cardiomyocyte apoptosis and ventricular remodeling in rats with I/R injury through the suppressing the activation of the MAPK signaling pathway.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

AIM2 is a potential therapeutic target in human renal carcinoma and suppresses its invasion and metastasis via enhancing autophagy induction

Highlights: • Low AIM2 expression was correlated with RCC patient survival. • AIM2 suppressed the proliferation, migration and invasion of renal cancer cells. • AIM2 enhanced the expression of autophagy-related genes. • AIM2 exerts the anti-tumor effect via enhancing the induction of autophagy. Absent in melanoma 2 (AIM2) as a tumor suppressor is recently recognized to play an important role in many human solid tumors. However, the biological significance and function of AIM2 in renal cell carcinoma (RCC) remain unknown. In this study, we proposed to investigate the clinicopathological and prognostic significance of AIM2 in RCC patient specimens, to analyze the correlation between AIM2 expression and disease progression, and to further investigate the role and mechanism of AIM2 in malignant behaviors of renal carcinoma by cellular and animal models. The correlation analysis showed that low AIM2 expression was significantly correlated with lymph node metastasis, poor 5-year overall (P < 0.05) and disease-specific survival (P < 0.05) in RCC patients. Over-expression of AIM2 in 786-O or OSRC-2 cells suppressed the cell proliferation, migration and invasion as compared with control. The increased AIM2 expression enhanced the expression of autophagy-related genes (Bcl-2, Beclin-1, LC3-Ⅱ, and ATG-5) and increased the ratio of LC3-Ⅱ/LC3-Ⅰin renal carcinoma cells. Of note, the blockade of autophagy by 3-Methyladenine (3-MA) abrogated the inhibition of cell migration and invasion by over-expressing AIM2, indicated that AIM2 suppressed the malignant behaviors of renal carcinoma by enhancing the induction of autophagy. Furthermore, 3-MA abolished the prevention of tumor growth on nude mouse established by AIM2-OSRC-2 cells. Our results suggested that AIM2 might serve as a novel prognostic indicator as well as a potential therapeutic target for renal carcinoma treatment.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Fos-like antigen 2 (FOSL2) promotes metastasis in colon cancer

Among different cancers, incidence and mortality of colorectal cancer (CRC) is one of the highest. KRAS mutation is one of the underlying features in the pathogenesis of CRC with CRC tumors harboring mutant KRAS exhibiting a more aggressive behavior compared to CRC tumors with wild type KRAS. We had earlier shown that the microRNA-143 (miR-143) replenishment not only chemosensitizers CRC cell line with mutant KRAS instead of wild-type KRAS gene, to paclitaxel-mediated cytotoxicity, but also inhibits cell migration and invasion ability. Hence, the study aimed to determine how miR-143 replenishment is inhibiting pre-metastatic behavior in CRC cells with mutant KRAS. Top ten mRNA targets of miR-143 as predicted by TargetScan were evaluated by qRT-PCR in LoVo cells which were performed mock transfection or miR-143 mimic transfection. Evaluation of the changes in cognate mRNA target(s) was done in 30 paired CRC tissue and tumor adjacent normal tissue specimens and in LoVo cells by western blot. Effect of the mRNA target on pro-metastatic behavior was assayed by gain- and loss-of-function studies using a combination of western blotting and in vitro cell proliferation and transwell migration/invasion assay in LoVo cells and in the normal colonic epithelium cell line FHC. In vivo effect of the cognate mRNA target on CRC metastasis was assayed by xenograft assay. Of the 10 predicted mRNA targets, FOSL2 (P < 0.05) and IGFBP5 (P > 0.05) was down regulated in LoVo cells transfected with the miR-143 mimic. FOSL2 mRNA levels were significantly downregulated in CRC tissue specimens compared with adjacent normal tissue (P < 0.05). Immunoblot analysis showed that FOSL2, but not IGFBP5, protein expression is down regulated in LoVo cells after the miR-143 mimic transfection. FOSL2 overexpression in the normal colonic epithelial cell line FHC or siRNA-mediated silencing in LoVo cells induced and repressed, respectively, pro-mesenchymal cell features. Whereas manipulation of FOSL2 expression did not have any effect on cell proliferation rates, silencing its expression inhibited cell migration and invasion ability in vitro. In addition, silencing of FOSL2 expression in the LoVo cells can significantly inhibited invasion of hepatic, while no effect was found for tumorigenic potential. Our results suggest that FOSL2 is a critical regulator of CRC metastasis and might be an important marker for prognostic in CRC patients.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Diacylglycerol kinase γ predicts prognosis and functions as a tumor suppressor by negatively regulating glucose transporter 1 in hepatocellular carcinoma

Highlights: • DGKγ expression was downregulated in hepatocellular carcinoma (HCC). • Lower DGKγ expression in HCC tumor tissues was an independent prognostic factor for poor post-surgical overall survival. • Cell proliferation and migration of HCC cells were suppressed by DGKγ ectopic expression. • DGKγ overexpression led to down regulation of GLUT1 protein level and AMPK activity. Diacylglycerol kinases (DGK) are a family of enzymes catalyzing the transformation of diacylglycerol into phosphatidic acid, which have been recognized as key regulators in cell signaling pathways. The role of DGKγ in human malignancies has seldom been studied. In this study, we investigated the role of DGKγ in hepatocellular carcinoma (HCC). We found that DGKγ was down-regulated in HCC tumor tissues and cell lines as compared to that in non-tumor tissues. The prognostic value of DGKγ expression was evaluated by Cox regression and Kaplan-Meier analyses. Lower DGKγ expression in tumor tissues was an independent prognostic factor for poor post-surgical overall survival. By using HDACs inhibitors treatment and ChIP-PCR, we discovered that histone H3 and H4 deacetylation mainly contributed to the downregulation of DGKγ expression. Functional studies revealed that ectopic expression of DGKγ inhibited cell proliferation and cell migration in HCC cells. Mechanism studies showed that DGKγ overexpression led to down regulation of GLUT1 protein level and AMPK activity, which result in glucose uptake suppression as well as lactate and ATP production declination. The decrease of GLUT1 level could be partially rescued by treatments with either DGK inhibitor and lysosome inhibitor, indicating DGKγ may down-regulate GLUT1 through its kinase activity and lysosome degradation process. Together, this study demonstrated that DGKγ plays a tumor suppressor role in HCC by negatively regulating GLUT1. DGKγ could be a novel prognostic indicator and therapeutic target for HCC.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Long noncoding RNA Nespas inhibits apoptosis of epileptiform hippocampal neurons by inhibiting the PI3K/Akt/mTOR pathway

Highlights: • Nespas is downregulated and inhibits hippocampal neuron apoptosis in epileptic mouse models. • Nespas binds with miR-615–3p in epileptiform hippocampal neurons. • MiR-615–3p targets Psmd11 in epileptiform hippocampal neurons. • Nespas inhibits hippocampal neuron apoptosis in epilepsy by upregulating Psmd11 and inhibiting the PI3K/Akt/mTOR pathway. Epilepsy is one of the most common neurological diseases with spontaneous recurrent seizures. Long noncoding RNAs (lncRNAs) are crucial modulators in numerous diseases, including epilepsy. However, the functional role and potential mechanism of lncRNA Nespas in epilepsy remain unknown. Our study clarified that Nespas was underexpressed in epileptiform hippocampal tissues and neurons. Furthermore, Nespas promoted hippocampal neuron viability and proliferation, and inhibited hippocampal neuron apoptosis. Mechanistically, Nespas interacted with microRNA 615–3p (miR-615–3p) in epileptiform hippocampal neurons. 26S proteasome non-ATPase regulatory subunit 11 (Psmd11) was a downstream target of miR-615–3p, and Nespas elevated Psmd11 expression via competitively binding to miR-615–3p in epileptiform hippocampal neurons. In addition, rescue assays suggested that Nespas promoted hippocampal neuron viability and proliferation, and suppressed hippocampal neuron apoptosis by upregulation of Psmd11. Furthermore, Nespas suppressed the PI3K/Akt/mTOR pathway via upregulating Psmd11 in epileptiform hippocampal neurons. This report explored the function and regulatory mechanism of Nespas in epileptiform hippocampal neurons for the first time. Our findings revealed that Nespas suppressed the apoptosis of epileptiform hippocampal neurons by inhibiting the PI3K/Akt/mTOR pathway via upregulation of Psmd11 at a miR-615–3p dependent way, indicating that Nespas may offer a new direction for the treatment of epilepsy.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Ubiquitin-specific protease 14 promotes radio-resistance and suppresses autophagy in oral squamous cell carcinoma

Highlights: • USP14 promotes radioresistance of OSCC cells. • USP14 elevates radioresistance of OSCC cells by suppressing apoptosis. • USP14 modulates radioresistance in OSCC by regulating autophagy. Oral squamous cell carcinoma (OSCC) is a common malignant tumor in the world. Radiotherapy is one of the standard therapies for patients with OSCC, but its clinical efficiency is limited due to radioresistance. In this study, we identified a mechanism of such resistance regulated by Ubiquitin-specific protease 14 (USP14). USP14 expression was significantly increased in clinical OSCC tissue samples and cell lines, and OSCC patients with high USP14 expression predicted poor overall survival rate. Additionally, a negative correlation between USP14 and LC3B was observed in patients with OSCC. We then found that irradiation (IR)-reduced cell survival of OSCC cells lines was further decreased when USP14 was knocked down. However, USP14 over-expression significantly promoted the cell viability of OSCC cells after IR treatment. Colony formation analysis confirmed thatafter IR treatment,USP14 knockdown markedly decreased the proliferation of OSCC cells, but over-expressing USP14 significantly up-regulated the proliferative activity of OSCC cells. Furthermore, DNA damage caused by IR was enhanced by USP14 knockdown, while been suppressed in OSCC cells with USP14 over-expression. Additionally, IR-inducedapoptosis was further promoted by USP14 knockdown in OSCC cells, which was, however, significantly abolished by USP14 over-expression.Moreover, our in vivo studies showed that IR-reduced tumor growth and tumor weight were further enhanced by USP14 knockdown in OSCC tumor-bearing nude mice. Finally, we found that USP14 knockdown could promote IR-induced autophagy by increasing LC3BII and γH2AX expression levels in IR-treated OSCC cells. However, this event was markedly abolished by ATG5 knockdown, subsequently restoring the cell proliferation in IR-incubated OSCC cells.Finally, we found that USP14-mediated apoptosis was autophagy-dependent in IR-treated OSCC cells. Taken together, these findings suggested that suppressing USP14 could alleviateradioresistancein OSCC both in vitro and in vivo by inducing apoptosis and autophagy, and thus could be served as a promising therapeutic strategy for OSCC treatment.

60 APPLIED LIFE SCIENCES↗