SEARCH · Engineering Papers

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At least 433 records · Page 24

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DOE OSTI2022

Protocol for single-cell isolation and genome amplification of environmental microbial eukaryotes for genomic analysis

We describe environmental microbial eukaryotes (EMEs) sample collection, single-cell isolation, lysis, and genome amplification, followed by the rDNA amplification and OTU screening for recovery of high-quality species-specific genomes for de novo assembly. These protocols are part of our pipeline that also includes bioinformatic methods. The pipeline and its application on a wide range of phyla of different sample complexity are described in our complementary paper. In addition, this protocol describes optimized lysis, genome amplification, and OTU screening steps of the pipeline. For complete details on the use and execution of this protocol, please refer to Ciobanu et al. (2021).

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2013

Comparative proteome approach demonstrates that platelet-derived growth factor C and D efficiently induce proliferation while maintaining multipotency of hMSCs

This is the first study that comprehensively describes the effects of the platelet-derived growth factor (PDGF) isoforms C and D during in vitro expansion of human mesenchymal stem cells (hMSCs). Our results show that PDGFs can enhance proliferation of hMSCs without affecting their multipotency. It is of great value to culture and expand hMSCs in a safe and effective manner without losing their multipotency for manipulation and further development of cell-based therapies. Moreover, differential effects of PDGF isoforms have been observed on lineage-specific differentiation induced by BMP2 and Vitamin D3. Based on label-free LC-based quantitative proteomics approach we have furthermore identified specific pathways induced by PDGFs during the proliferation process, showing the importance of bioinformatics tools to study cell function. - Highlights: • PDGFs (C and D) significantly increased the number of multipotent undifferentiated hMSCs. • Enhanced proliferation did not impair the ability to undergo lineage-specific differentiation. • Proteomic analysis confirmed the overall signatures of the ‘intact’ cells.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

PTTG1, A novel androgen responsive gene is required for androgen-induced prostate cancer cell growth and invasion

Androgens (AR) play an important role in initiation and progression of prostate cancer. It has been shown that AR exert their effects mainly through the androgen-activated AR which binds to androgen response elements (AREs) in the regulatory regions of target genes to regulate the transcription of androgen-responsive genes, thus, identification of AR downstream target gene is critical to understand androgen function in prostate cancer. In this study, our results showed that androgen treatment of LNCaP cells induced PTTG1 expression, which was blocked by the androgen receptor antagonist, Casodex. Bioinformatics analysis and experiments using PTTG1 promoter deletion mutants showed that the PTTG1 promoter contains a putative androgen response element (ARE), which localizes in the −851 to −836 region of the promoter. Androgen activated androgen receptor (AR) binding to this ARE was confirmed by Chromatin immunoprecipitation (ChIP) assay. Furthermore, Knockdown of PTTG1 expression using short hairpin RNA significantly reduced androgen-induced LNCaP cell growth and invasion. In addition, we showed PTTG1 is highly expressed in metastasis prostate cancer tissue. These results suggest that PTTG1 is a novel downstream target gene of androgen receptor and take part in prostate cancer proliferation and metastasis. - Highlights: • Androgen treatment of LNCaP cells induced PTTG1 expression. • Knockdown of PTTG1 expression significantly reduced androgen-induced LNCaP cell growth and invasion. • PTTG1 is highly expressed in metastasis prostate cancer tissue. • PTTG1 is a novel downstream target gene of androgen receptor.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2016

Shikonin regulates C-MYC and GLUT1 expression through the MST1-YAP1-TEAD1 axis

The general mechanism underlying the tumor suppressor activity of the Hippo signaling pathway remains unclear. In this study, we explore the molecular mechanisms connecting the Hippo signaling pathway with glucose metabolism. We have found that two key regulators of glycolysis, C-MYC and GLUT1, are targets of the Hippo signaling pathway in human leukemia cells. Our results revealed that activation of MST1 by the natural compound shikonin inhibited the expression of GLUT1 and C-MYC. Furthermore, RNAi experiments confirmed the regulation of GLUT1 and C-MYC expression via the MST1-YAP1-TEAD1 axis. Surprisingly, YAP1 was found to positively regulate C-MYC mRNA levels in complex with TEAD1, while it negatively regulates C-MYC levels in cooperation with MST1. Hence, YAP1 serves as a rheostat for C-MYC, which is regulated by MST1. In addition, depletion of MST1 stimulates lactate production, whereas the specific depletion of TEAD1 has an opposite effect. The inhibition of lactate production and cellular proliferation induced by shikonin also depends on the Hippo pathway activity. Finally, a bioinformatic analysis revealed conserved TEAD-binding motifs in the C-MYC and GLUT1 promoters providing another molecular data supporting our observations. In summary, regulation of glucose metabolism could serve as a new tumor suppressor mechanism orchestrated by the Hippo signaling pathway. - Highlights: • Shikonin inhibits C-MYC and GLUT1 expression in MST1 and YAP1 dependent manner. • YAP1-TEAD1 interaction activates C-MYC and GLUT1 expression. • MST1 in cooperation with YAP1 inhibits C-MYC and GLUT1 expression. • MST1-YAP1-TEAD1 axis regulates lactate production by leukemic cells. • MST1 and YAP1 proteins block proliferation of leukemic cells.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

MicroRNA-140-5p attenuated oxidative stress in Cisplatin induced acute kidney injury by activating Nrf2/ARE pathway through a Keap1-independent mechanism

Oxidative stress was predominantly involved in the pathogenesis of acute kidney injury (AKI). Recent studies had reported the protective role of specific microRNAs (miRNAs) against oxidative stress. Hence, we investigated the levels of miR140-5p and its functional role in the pathogenesis of Cisplatin induced AKI. A mice Cisplatin induced-AKI model was established. We found that miR-140-5p expression was markedly increased in mice kidney. Bioinformatics analysis revealed nuclear factor erythroid 2-related factor (Nrf2) was a potential target of miR-140-5p, We demonstrated that miR-140-5p did not affect Kelch-like ECH-associated protein 1 (Keap1) level but directly targeted the 3′-UTR of Nrf2 mRNA and played a positive role in the regulation of Nrf2 expression which was confirmed by luciferase activity assay and western blot. What was more, consistent with miR140-5p expression, the mRNA and protein levels of Nrf2, as well as antioxidant response element (ARE)-driven genes Heme Oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase l (NQO1) were significantly increased in mice kidney tissues. In vitro study, Enforced expression of miR-140-5p in HK2 cells significantly attenuated oxidative stress by decreasing ROS level and increasing the expression of manganese superoxide dismutase (MnSOD). Simultaneously, miR-140-5p decreased lactate dehydrogenase (LDH) leakage and improved cell vitality in HK2 cells under Cisplatin-induced oxidative stress. However, HK2 cells transfected with a siRNA targeting Nrf2 abrogated the protective effects of miR-140-5p against oxidative stress. These results indicated that miR-140-5p might exert its anti-oxidative stress function via targeting Nrf2. Our findings showed the novel transcriptional role of miR140-5p in the expression of Nrf2 and miR-140-5p protected against Cisplatin induced oxidative stress by activating Nrf2-dependent antioxidant pathway, providing a potentially therapeutic target in acute kidney injury. - Highlights: • miR-140-5p expression was up-regulated in Cisplatin induced AKI. • miR-140-5p directly targeted the 3′-UTR of Nrf2 mRNA. • miR-140-5p activated the Nrf2/ARE signaling pathway and served as an early protective response against oxidative stress.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

MiR-3202 protects smokers from chronic obstructive pulmonary disease through inhibiting FAIM2: An in vivo and in vitro study

Highlights: • Smoking decreased level of miR-3202 both in COPD patients and normal donors. • The miR-3202 influenced the release of inflammatory factors through targeting FAIM2 in T lymphocytes. • Abnormal expression of miR-3202 in T lymphocytes damaged human bronchial epithelial cells. Previous study found the variable miR-3202 as a potential biomarker in smoker with or without chronic obstructive pulmonary disease (COPD). This study aims to identify the molecular involvement of miR-3202 in the pathophysiology of COPD. Level of miR-3202 in blood sample of non-smoker non-COPD(C), smoker without COPD(S), smoker with stable COPD(S-COPD) and smoker with acute exacerbation COPD(AE-COPD) was observed by quantitative real-time PCR. By bioinformatics prediction, Fas apoptotic inhibitory molecule 2 (FAIM2) was identified as a potential target of miR-3202. In vitro, human bronchial epithelial (HBE) cells and cigarette smoke extract (CSE) stimulated T lymphocytes were co-cultured. Cell proliferation and apoptosis of HBE cells were determinated. In vivo, rats were exposed in cigarette smoke for 30 days and expression of miR-3202 and FAIM2 in bronchia were detected. Results showed that The miR-3202 was down-regulated in S, S-COPD and AE-COPD group when compared with C group. Decreased level of miR-3202 was also observed in CSE treated T lymphocyte. Additionally, CSE stimulation increased INF-γ and TNF-α levels and FAIM2 expression whereas inhibited Fas and FasL expressions in T lymphocytes. However, these effects were significantly suppressed by miR-3202 overexpression and enhanced by miR-3202 inhibitor. Likely to exogenous miR-3202, FAIM2 knockdown significantly inhibited HBE cells apoptosis, as well as inhibited INF-γ and TNF-α levels. In COPD rats model, miR-3202 was reduced while FAIM2 was up-regulated accordingly. Here, results suggest that high level miR-3202 in T lymphocytes may protect epithelial cells through targeting FAIM2. MiR-3202 might be used as a notable biomarker of COPD.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Long non-coding RNA UCA1 upregulation promotes the migration of hypoxia-resistant gastric cancer cells through the miR-7-5p/EGFR axis

Highlights: • Two novel hypoxia-resistant gastric cancer (HRGC) cell lines were established. • UCA1 was upregulated in HRGC cells, which promoted their migration. • UCA1 directly interacted with miR-7-5p. • UCA1 suppressed the degradation of EGFR mRNA by sponging miR-7-5p. A variety of solid tumors are surrounded by a hypoxic microenvironment, which is known to be associated with high metastatic capability and resistance to various clinical therapies, contributing to a poor survival rate for cancer patients. Although the majority of previous studies on tumor-associated hypoxia have focused on acute hypoxia, chronic hypoxia more closely mimics the actual hypoxic microenvironment of a tumor. In this study, two novel hypoxia-resistant gastric cancer (HRGC) cell lines which could grow normally in 2% oxygen were established. The long non-coding RNA UCA1 was upregulated in HRGC cells, which promoted their migration. Bioinformatics analysis and a luciferase reporter assay showed that miR-7-5p could bind to specific sites of UCA1 to regulate the target EGFR through competitive endogenous RNA function. UCA1 directly interacted with miR-7-5p and decreased the binding of miR-7-5p to the EGFR 3′-untranslated region, which suppressed the degradation of EGFR mRNA by miR-7-5p. Therefore, long-term hypoxia induced UCA1 to promote cell migration by enhancing the expression of EGFR. This study thus reveals a new mechanism by which a hypoxic microenvironment promotes tumor metastasis, and highlights UCA1 as a potential biomarker for predicting the metastasis of gastric cancer to guide clinical treatment.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

microRNA-212-induced protection of the heart against myocardial infarction occurs via the interplay between AQP9 and PI3K/Akt signaling pathway

Myocardial infarction (MI) is defined as the irreversible death of heart muscle that occurs secondary to prolonged lack of oxygen supply, which has resulted in millions of death worldwide. This study was conducted with aims of investigating how microRNA-212 (miR-212) and the inhibition of aquaporin-9 (AQP9) through the activation of the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway plays a role in the prevention of MI. The relationship between miR-212 and AQP9 was determined with the use of bioinformatics combined with the dual luciferase reporter gene assay. Next, the MI model was established and the cardiomyocytes were transfected with different mimic, inhibitor and siRNAs to investigate the specific activity of miR-212, AQP9 and the PI3K/Akt signaling pathway in MI. The expression of miR-212, AQP9, PI3K, Akt, VEGF, Bax, and B cell lymphoma 2 (Bcl-2), along with cell apoptosis were determined using reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blot analysis and flow cytometry. Based on the results, AQP9 was verified as the direct target gene of miR-212. MI led to a decrease in miR-212 expression and an increase in AQP9 expression. It was also found that miR-212 activated the PI3K/Akt signaling pathway in MI through the inhibition of AQP9 expression. The overexpression of miR-212 or silencing AQP9 decreased cardiomyocytes apoptosis. These findings indicated that the overexpression of miR-212 inhibited AQP9 by activating the PI3K/Akt signaling pathway, thus decreasing cardiomyocytes apoptosis, promoting vascular regeneration and alleviating ventricular remodeling in rats with MI.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Hepatitis B virus upregulates host microRNAs that target apoptosis-regulatory genes in an in vitro cell model

Highlights: • Hepatitis B virus changes the expression of host microRNAs in HepG2 cells. • MiR-192 and miR-194 regulate apoptosis. • MiR-192 and miR-194 target the important apoptosis-regulators BIM and cFLIP. • Hepatitis B virus upregulates microRNAs that affect apoptosis in vitro. Chronic hepatitis B (CHB) infection increases the risk of developing severe liver disease including cirrhosis and hepatocellular carcinoma (HCC). As microRNAs may modulate host – virus interactions, we here investigated if hepatitis B virus (HBV) infection modulate microRNA expression using an in vitro HepG2 cell model system with inducible HBV replication. We found that HBV replication was associated with upregulation of miR-192-5p, miR-194-5p and miR-215-5p, of which miR-192-5p and miR-215-5p have identical seed sequences. Bioinformatics analyses revealed a significant enrichment of potential target genes involved in apoptosis signaling of all three microRNAs. In line with this, transfection with a mimic of miR-192-5p suppressed the protein level of pro-apoptotic BIM and reduced endoplasmic reticulum (ER) stress-induced apoptosis in HepG2 cells. In contrast, transfection with a mimic of miR-194-5p downregulated the anti-apoptotic proteins SODD and cFLIP, and sensitized HepG2 cells to both ER stress- and cytokine-induced apoptosis. In conclusion, our study suggests that HBV upregulates the expression of miR-192-5p and miR-194-5p in the host cell. These microRNAs target important apoptosis-regulatory proteins, and may thus contribute to the development of HBV-related liver disease.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

MiR-940 promotes the proliferation and migration of gastric cancer cells through up-regulation of programmed death ligand-1 expression

Although anti-programmed death ligand-1 (PD-L1) therapy has shown light in treatment of gastric cancer, only a limited number of patients respond to the treatment. In addition to its immunosuppressive effect, PD-L1 is involved in other functions of tumor cells. Previously study showed that PD-L1 promoted EMT in lung cancer cells. However, the other effect and role of PD-L1 in gastric cancer remains unclear. In the present study, we first demonstrated that PD-L1 promoted the proliferation and migration in gastric cancer cell lines. We found that another STAT family member, STAT5a, is involved in regulating the expression of PD-L1 in gastric cancer. Additionally, Cbl-b interacted and ubiquitated STAT5a, down-regulated the expression of STAT5a and PD-L1. Moreover, bioinformatics predictions and experimental data showed that Cbl-b is a target gene of the microRNA miR-940. We further found that miR-940 promoted the proliferation and migration of gastric cancer in vivo and in vitro. Taken together, our findings suggest that miR-940/Cbl-b/STAT5a axis regulated the expression of PD-L1, which promotes the proliferation and migration of gastric cancer cells.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

HPRT promotes proliferation and metastasis in head and neck squamous cell carcinoma through direct interaction with STAT3

Highlights: • The higher expression of HPRT in HNSCC tissue was related to poor prognosis of patients. • Overexpression of HPRT increased the gene expression of epithelial mesenchymal transition markers. • HPRT directly interacted with STAT3. • Knocking down HPRT significantly decreased tumour growth and enhanced the anticancer effect against HNSCC xenografts. Increasing effort has been put into finding novel molecular pathways to improve the efficiency of EGFR inhibitors against head and neck squamous cell cancer (HNSCC). In this study, we performed data mining and bioinformatically analysed RNA-Seq data downloaded from TCGA and confirmed that higher expression of HPRT in HNSCC tissue was related to poor prognosis of patients. Then, we conducted in vitro and in vivo loss- and gain-of-function experiments to demonstrate the role of HPRT in HNSCC cell lines. Overexpression of HPRT increased the gene expression of epithelial mesenchymal transition markers via direct interaction with STAT3. Knocking down HPRT significantly decreased tumour growth and enhanced the anticancer effect of EGFR inhibitors against HNSCC xenografts. In conclusion, HPRT is a binding partner of STAT3 that promotes EMT and proliferation. Our findings support HPRT as a promising prognostic indicator and potential therapeutic target for HNSCC.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Endothelium-specific endothelin-1 expression promotes pro-inflammatory macrophage activation by regulating miR-33/NR4A axis

The hallmark of atherogenesis is characterized as endothelial dysfunction and subsequent macrophage activation. Although our previous study has demonstrated that endothelin-1 (ET-1) plays an important role in atherogenesis, the underlying mechanism remains deeply investigation. Enhanced atherosclerotic plaques were observed in endothelium-specific ET-1 overexpression ApoE{sup −/-} mice (eET-1/ApoE{sup −/-}) concomitant with increased secretion of pro-inflammatory adhesion molecules and cytokines. The conditional media used for culturing human umbilical vein endothelial cells (HUVECs) with AdET-1 infection and subjected to OX-LDL stimulation, was collected and utilized for bone marrow-derived macrophages (BMDMs) culturing. RT-PCR analysis showed increased genes expression related to classical M1 macrophages but decreased alternative activated M2 macrophages genes expression in macrophage culturing with the conditional media. Furthermore, consistent regulations of macrophage polarization were observed using isolated exosomes from the conditional media. More importantly, we noticed that miR-33 was enriched in the exosomes derived by HUVECs with AdET-1 infection, while bioinformatics analysis further indicated that miR-33 directly targeted NR4A and miR-33/NR4A axis was required for the effect of endothelial-specific ET-1 overexpression on pro-inflammatory macrophage activation. By contrast, such effects could be reversed by ET-1 knockdown. Taken together, our study indicated that the exosomes derived by HUVECs with AdET-1 infection can transfer miR-33 to macrophages and subsequently promote pro-inflammatory macrophage activation by directly targeting to NR4A. These evidences clearly revealed that miR-33/NR4A axis was the important mechanism underlying the effect of ET-1 on macrophage activation and indicated that ET-1 may act as a promising target for atherosclerosis management.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

BAP1 antagonizes WWP1-mediated transcription factor KLF5 ubiquitination and inhibits autophagy to promote melanoma progression

Accumulating evidence revealed the abnormal expression of KLF5 in human cancers while its role in melanoma remains uncharacterized. This study aimed to explore the role of KLF5 in the proliferation and metastasis of melanoma. Bioinformatics analysis was performed to detect WWP1, BAP1 and KLF5 expression in melanoma, followed by expression determination on clinical tissues from melanoma patients and cancer cells. The cancer cells were infected with lentivirus expressing KLF5 or BAP1 while PI3K, AKT and mTOR expression was detected and autophagy was observed. Treated cells were injected to mice when tumor growth was measured and autophagy-related protein was detected. Plasmids expressing WWP1 and Ub-K48 were co-transfected into treated melanoma cells while immunoprecipitation assay was performed to determine the interaction among KLF5, WWP1, and BAP1. WWP1 was poorly expressed in melanoma cells and tissues whereas KLF5 was highly expressed and was positively correlated to poor prognosis. KLF5 promoted melanoma cell malignant phenotypes as well as inhibited autophagy. Interestingly, KLF5 contributed to activation of PI3K-AKT-mTOR signaling pathway, thereby inhibiting autophagy in melanoma cells. WWP1 mediated K48-linked ubiquitination of KLF5 to promote its degradation, and BAP1 antagonized this modification and stabilized KLF5 protein expression. Besides, BAP1 promoted KLF5-mediated growth of melanoma in vivo. Taken altogether, BAP1 antagonized WWP1-mediated ubiquitination of KLF5 to inhibit autophagy and promote melanoma development.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Protective effect of SIRT6 on cholesterol crystal-induced endothelial dysfunction via regulating ACE2 expression

Highlights: • ACE2 was involved in CC-induced endothelial dysfunction. • SIRT1 and SIRT6 contributed to regulating the protein expression of ACE2 in CC-stimulated endothelial cells. • SIRT6 regulated ACE2 with intranuclear p-ATF2, but not affecting p-ATF2 expression in the cytoplasm. Sirtuins are a family of highly conserved nicotinamide adenine dinucleotide (NAD{sup +})-dependent enzymes. Among the sirtuins, SIRT1 and SIRT6 participate in the regulation of endothelial functions and play significant roles in the physiological and pathological processes of cardiovascular diseases (CVD). Recently, our study found that minute cholesterol crystals (CC) can be endocytosed by endothelial cells and further impair endothelial functions. Since previous studies have reported that angiotensin-converting enzyme (ACE2) involves Angiotensin (Ang) II-induced inflammation in endothelial cells, this study was designed to investigate the role of SIRT1 and SIRT6 in CC-induced variation of ACE2 expression and the related mechanism between SIRT6 and ACE2. We found that ACE2 is involved in CC-induced endothelial dysfunction, which inhibits decreases in nitric oxide (NO) level and endothelial nitric oxide synthase (eNOS) activity and increases in inflammatory factors and adhesion molecules. Besides, SIRT1 and SIRT6 regulated the protein expression of ACE2 in CC-stimulated human umbilical vein endothelial cells (HUVECs). Moreover, bioinformatics analysis from the Enrichr database indicated that activating transcription factor 2 (ATF2), is highly correlated with genes that significantly upregulated after infection with the SIRT6 adenovirus vector. In CC-induced HUVECs, ACE2 expression was up-regulated in cells transfected with ATF2 siRNA. However, further mechanism studies revealed that overexpression of SIRT6 decreases the accumulation of p-ATF2 in the nucleus, but did not affect p-ATF2 expression in the cytoplasm. Taken together, these data indicated that SIRT6 regulates ACE2 might via inhibiting the accumulation of nucleus p-ATF2 in CC-induced endothelial dysfunction.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

LncRNA DLEU2 promotes cervical cancer cell proliferation by regulating cell cycle and NOTCH pathway

Highlights: • LncRNA DLEU2 is upregulated and related to tumor topography in cervical cancer. • LncRNA DLEU2 promotes cervical cancer cell proliferation. • LncRNA DLEU2 promotes the progression of the cell cycle. • LncRNA DLEU2 inhibits the activity of the Notch signaling pathway. Long noncoding RNAs (lncRNAs) are known to play a crucial role in the onset and progression of cervical cancer (CC). Here, the results of RNA microarray and RNA-sequencing dataset analysis showed that lncRNA DLEU2 was significantly upregulated in CC tissues. Clinicopathologic analysis indicated that lncRNA DLEU2 was closely related to tumor topography. Functional experiments and bioinformatics analysis revealed that lncRNA DLEU2 promoted CC cell proliferation and accelerated the cell cycle. Mechanistically, lncRNA DLEU2 promoted the progression of the cell cycle and inhibited the activity of the Notch signaling pathway by inhibiting p53 expression. Additionally, lncRNA DLEU2 probably interacted with ZFP36 Ring Finger Protein (ZFP36) to inhibit the expression of p53. In conclusion, this study revealed the function of lncRNA DLEU2 in CC tumorigenesis, suggesting new therapeutic targets in CC.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

sMicroRNA-28-5p acts as a metastasis suppressor in gastric cancer by targeting Nrf2

The transcription factor nuclear factor (erythroid-2)-related factor 2 (Nrf2) can principally serve a mode of protection for both the normal cells and cancer cells from cellular stress, and elevates cancer cell survival. microRNA-28 (miR-28) has been involved in the regulation of Nrf2 expression in breast epithelial cells. However, no comprehensive analysis has been conducted regarding the function of miR-28-5p regulating Nrf2 in gastric cancer (GC). In this study, we aimed to evaluate their interaction and biological roles in the migration and invasion of GC cells. The expression of Nrf2 in the cancer tissues harvested from 42 patients with GC was examined by an array of molecular techniques comprising of Immunohistochemical staining, RT-qPCR and Western blot analysis. Kaplan-Meier method was adopted for analysis of the correlation of Nrf2 with the prognosis of GC patients. Interaction between miR-28-5p and Nrf2 was determined using the bioinformatics analysis and dual luciferase reporter gene assay. Gain- and loss-of-function studies of miR-28-5p and Nrf2 were conducted to elucidate their effects on GC cell migration, invasion and metastasis, as well as expression pattern of several epithelial-mesenchymal transition (EMT)-related proteins. Results indicated that the expression pattern of Nrf2 was significantly upregulated in GC tissues and indicative of poor prognosis of GC patients. miR-28-5p was verified to target Nrf2 and downregulate its expression. GC cells with overexpression of miR-28-5p or Nrf2 knockdown exhibited a marked reduction in the migrated and invasive abilities, along with the N-cadherin expression yet an increase of E-cadherin expression. Furthermore, miR-28-5p exerted an inhibitory function on the metastatic and tumorigenicity of GC cells. In conclusion, miR-28-5p is a comprehensive tumor suppressor that inhibits GC cell migration and invasion through repressing the Nrf2 expression. Therefore, miR-28-5p may serve as a potential biomarker for the prognosis of GC and a novel therapeutic target in advanced GC.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

PD-L1 induces macrophage polarization toward the M2 phenotype via Erk/Akt/mTOR

Highlights: • PD-L1 treatment upregulates CD206 but not CD86 expression. • PD-L1-induced DEGs are related to the immune response, mitochondrial function, and metabolism. • Nivolumab, LY294002, U0126, and rapamycin inhibit the PD-L1-stimulated CD206 expression. • PD-L1 promotes M2 polarization via Erk/Akt/mTOR. PD-L1 (programmed death-ligand 1) is the ligand of PD-1 (programmed cell death protein 1) and regulates inhibitory immune responses. It is well known that PD-L1 suppresses T cell function via binding to PD-1. However, little is known about the role of the PD-1/PD-L1 axis in macrophage polarization. According to previous studies, the function of the PD-1/PD-L1 axis in macrophage polarization is controversial, and the underlying mechanism has not been fully elucidated. Thus, we treated THP-1-derived macrophages with human PD-L1 Fc to determine the role of the PD-1/PD-L1 axis in macrophage polarization. To further explore the mechanism, we performed RNA sequencing and used specific inhibitors to identify the implicated signalling pathways. In this study, we found that PD-L1 induces the upregulation of CD206 expression, which is inhibited by nivolumab, LY294002, U0126, and rapamycin. Evaluation of differentially expressed genes (DEGs) and bioinformatics analysis indicated that PD-L1 also induces the upregulation of the expression of genes that maintain mitochondrial function and mediate metabolic switching. In addition, we did not detect PD-L1-induced CD86 alterations, indicating that PD-L1 treatment has no significant influence on M1 polarization. Taken together, these results suggest that PD-L1 binds to PD-1 and promotes M2 polarization accompanied by mitochondrial function enhancement and metabolic reprogramming via Erk/Akt/mTOR. This study elucidates the role of PD-L1 in macrophage polarization and verifies the underlying mechanisms for the first time. Considering that aberrantly upregulated PD-L1 expression contributes to a wide variety of diseases, targeting PD-L1-mediated macrophage polarization is a prospective therapeutic strategy for both neoplastic and nonneoplastic diseases.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

NPRL2 reduces the niraparib sensitivity of castration-resistant prostate cancer via interacting with UBE2M and enhancing neddylation

In this study, we explored the regulatory effects of nitrogen permease regulator 2-like (NPRL2) on niraparib sensitivity, a PARP inhibitor (PARPi) in castrate-resistant prostate cancer (CRPC). Data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) program were retrospectively examined. Gene-set enrichment analysis (GSEA) was conducted between high and low NRPL2 expression prostate adenocarcinoma (PRAD) cases in TCGA. CCK-8 assay, Western blot analysis of apoptotic proteins, and flow cytometric analysis of apoptosis were applied to test niraparib sensitivity. Immunofluorescent (IF) staining and co-immunoprecipitation (co-IP) were conducted to explore the proteins interacting with NPRL2. Results showed that the upregulation of a canonical protein-coding transcript of NPRL2 (ENST00000232501.7) is associated with an unfavorable prognosis. Bioinformatic analysis predicts a physical interaction between NPRL2 and UBE2M, which is validated by a following Co-IP assay. This interaction increases NPRL2 stability by reducing polyubiquitination and proteasomal degradation. Depletion of NPRL2 or UBE2M significantly increases the niraparib sensitivity of CRPC cells and enhances niraparib-induced tumor growth inhibition in vivo. NPRL2 cooperatively enhances UBE2M-mediated neddylation and facilitates the degradation of multiple substrates of Cullin-RING E3 ubiquitin ligases (CRLs). In conclusion, this study identified a novel NPRL2-UBE2M complex in modulating neddylation and niraparib sensitivity of CRPC cells. Therefore, targeting NPRL2 might be considered as an adjuvant strategy for PARPi therapy.

60 APPLIED LIFE SCIENCES↗