siRNA Interference Research Articles

Abstract 4741: NUDT21 regulates PIGN through APA mechanism to affect the progression of MDS and AML

Abstract Introduction: PIGN gene is crucial in regulating mitotic integrity to maintain chromosomal stability and prevent MDS leukemic transformation and progression. Alternative Polyadenylation (APA) regulation and 3' RNA cleavage are 2 known vital processes in cellular DNA damage response to maintain genomic stability. NUDT21 gene encodes protein that is one subunit of the cleavage factor Im complex required for 3' RNA cleavage and APA processing. Although PIGN gene has six potential polyadenylation sites, the exact role of NUDT21 product on PIGN expression regulation has not been illustrated before. Methods: At first, we analyzed NUDT21 gene expression profile in AML, MDS, and normal control specimens and to plot the relationship between NUDT21 gene expression status and patient survival length from 5 datasets. We also used correlation regression analysis method to predict the gene expression correlations between NUDT21 gene and PIGN gene. Then, we used Caspas9 technology and vector overexpression technology to knockout or overexpress PIGN in HEK293 cells; RNA interference to knockdown the NUDT21 gene in HEK293, HL60, K562, THP-1,SKM-1 and MUTZ-1 cells. MTT and CCK8 methods were used to evaluate cell proliferation. Meanwhile, cell cycle, apoptosis, Reactive Oxygen Species (ROS) and GPI-anchored proteins (CD55, CD59, and CD235) expression profiles were evaluated by flowcytometry; Immunofluorescence experiment, RT-PCR were used to evaluate the impact of NUDT21 gene knockdown on PIGN expression. in vivo mice siRNA interference tests were conducted to verify the impact of NUDT21 knockdown on PIGN gene expression. RNA-Seq & Dapars analyses were conducted on mRNAs from 3 cell lines with various PIGN gene expression status to observe the effects of NUDT21 knockdown on PIGN 3'UTR processing. Results: NUDT21 expression increases in AML and high-risk MDS. NUDT21 expression level is negatively correlated with overall survival. NUDT21 knockdown decreases cell proliferation but increases cell differentiation via prolonging G1 phase, decreasing S phase, and increasing apoptosis. NUDT21 knockdown increases cell membrane GPI-anchoring proteins (CD55, CD59, and CD235) expressions; increases ROS and Caspas3 expressions but decreases CyclinD1 expression. NUDT21 knockout can increase PIGN expression, but PIGN expression status has no impact on NUDT21 expression. RT-PCR and WB results showed that NUDT21 knockdown can increase TP53 expression but decrease p21 expression. in vivo mice siRNA interference tests confirmed that NUDT21 knockdown increases PIGN gene expression. The RNA-Seq/Dapars analyses results showed that 3'UTR of PIGN transcript was significantly shortened and PIGN expression increased after NUDT21 knockdown. Conclusion: NUDT21 modulates PIGN gene expression through APA mechanism. PIGN polyadenylation may regulate cell proliferation status via modulating cell cycle and apoptosis. Citation Format: Jeffrey J. Pu, Fanggang Ren, Na Zhang, Shuo Li, Lan Zhang, Eric Miller, Guirong Wang, Hongwei Wang. NUDT21 regulates PIGN through APA mechanism to affect the progression of MDS and AML. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4741.

Asiatic acid improves insulin secretion of β cells in type 2 diabetes through TNF-α/Mfn2 pathway.

To investigate the effects and molecular mechanisms of asiatic acid on β-cell function in type 2 diabetes mellitus (T2DM). The T2DM model was established by high fat diet and streptozotocin injection in ICR mice, and the effects of asiatic acid on glucose regulation were investigated in model mice. The islets were isolated from palmitic acid-treated diabetic mice. ELISA was used to detect the glucose-stimulated insulin secretion, tumor necrosis factor (TNF)-α and interleukin (IL)-6. ATP assay was applied to measure ATP production, and Western blotting was used to detect protein expression of mature β cell marker urocortin (Ucn) 3 and mitofusin (Mfn) 2. The regulatory effects of asiatic acid on glucose-stimulated insulin secretion (GSIS) and Ucn3 expression were also investigated after siRNA interference with Mfn2 or treatment with TNF-α. Asiatic acid with the dose of 25 mg·kg－1·d－1 had the best glycemic control in T2DM mice and improved the homeostasis model assessment β index. Asiatic acid increased the expression of Mfn2 and Ucn3 protein and improved the GSIS function of diabetic β cells in vitro and in vivo (both P<0.05). Moreover, it improved the ATP production of islets of T2DM mice in vitro (P<0.05). Interfering Mfn2 with siRNA blocked the up-regulation of Ucn3 and GSIS induced by asiatic acid. Asiatic acid inhibited islet TNF-α content and increased Mfn2 and Ucn3 protein expression inhibited by TNF-α. Asiatic acid improves β cell insulin secretion function in T2DM mice by maintaining the β cell maturity, which may be related to the TNF-α/Mfn2 pathway.

Early-life cadmium exposure elevates susceptibility to allergic asthma in ovalbumin-sensitized and challenged mice

Increasing evidence have demonstrated that early-life exposure to environmental toxicants elevates risk of allergic asthma. Cadmium (Cd) is widely present in the environment. The purposes of this study were to evaluate the impact of early-life Cd exposure on susceptibility to ovalbumin (OVA)-evoked allergic asthma. Newly weaned mice were subjected to a low concentration of CdCl2 (1 mg/L) by drinking water for 5 consecutive weeks. Penh value, an index of airway obstruction, was increased in OVA-stimulated and challenged pups. Abundant inflammatory cells were observed in the lung of OVA-exposed pups. Goblet cell hyperplasia and mucus secretion were shown in the airway of OVA-stimulated and challenged pups. Early-life Cd exposure exacerbated OVA-evoked airway hyperreactivity, Goblet cell hyperplasia and mucus secretion. The in vitro experiments showed that mucoprotein gene MUC5AC mRNA was upregulated in Cd-exposed bronchial epithelial cells. Mechanistically, endoplasmic reticulum (ER) stress-related molecules GRP78, p-eIF2α, CHOP, p-IRE1α and spliced XBP-1 (sXBP-1) were elevated in Cd-subjected bronchial epithelial cells. The blockade of ER stress, using chemical inhibitor 4-PBA or sXBP-1 siRNA interference, attenuated Cd-induced MUC5AC upregulation in bronchial epithelial cells. These results indicate that early-life Cd exposure aggravates OVA-induced allergic asthma partially through inducing ER stress in bronchial epithelial cells.

Semaphorin 4C regulates ovarian steroidogenesis through RHOA/ROCK1-mediated actin cytoskeleton rearrangement.

Semaphorins are a family of evolutionarily conserved morphogenetic molecules that were initially found to be associated with axonal guidance. Semaphorin 4C (Sema4C), a member of the fourth subfamily of semaphorins, has been demonstrated to play multifaceted and important roles in organ development, immune regulation, tumor growth, and metastasis. However, it is completely unknown whether Sema4C is involved in the regulation of ovarian function. We found that Sema4C was widely expressed in the stroma, follicles, and corpus luteum of mouse ovaries, and its expression was decreased at distinct foci in ovaries of mice of mid-to-advanced reproductive age. Inhibition of Sema4C by the ovarian intrabursal administration of recombinant adeno-associated virus-shRNA significantly reduced oestradiol, progesterone, and testosterone levels in vivo. Transcriptome sequencing analysis showed changes in pathways related to ovarian steroidogenesis and the actin cytoskeleton. Similarly, knockdown of Sema4C by siRNA interference in mouse primary ovarian granulosa cells or thecal interstitial cells significantly suppressed ovarian steroidogenesis and led to actin cytoskeleton disorganization. Importantly, the cytoskeleton-related pathway RHOA/ROCK1 was simultaneously inhibited after the downregulation of Sema4C. Furthermore, treatment with a ROCK1 agonist after siRNA interference stabilized the actin cytoskeleton and reversed the inhibitory effect on steroid hormones described above. In conclusion, Sema4C may play an important role in ovarian steroidogenesis through regulation of the actin cytoskeleton via the RHOA/ROCK1 signaling pathway. These findings shed new light on the identification of dominant factors involved in the endocrine physiology of female reproduction.

Excessive iodine induces thyroid follicular epithelial cells apoptosis by activating HIF-1α-mediated hypoxia pathway in Hashimoto thyroiditis

BackgroundHashimoto thyroiditis (HT) is considered the most common autoimmune thyroid disease. A growing body of evidence suggests that HT incidence correlates with excessive iodine intake. We should probe the effects of excessive iodine intake in HT development and its possible mechanism.Methods and resultsThe study recruited 20 patients: 10 with HT and 10 with nodular goiter. We detected the expression of an apoptosis-related protein caspase-3 by immunohistochemistry. In vitro study, we explored the proliferation and apoptosis status in thyroid follicular cells (TFCs) stimulated with different iodine concentrations by MTT and flow cytometry. Then we performed RNA sequence analysis of Nthy-ori3-1 cells treated for 48 h with KI to probe the underlying mechanism. Finally, we used RT-PCR and siRNA interference to verify the results. We identified apoptosis in thyroid tissue obtained from HT patients coincides with the increase of caspase-3 levels. In vitro study, iodine suppressed proliferation of TFCs and promoted TFCs apoptosis in a dose-dependent manner with regulating caspase-3 activation. HIF-1α-NDRG1 mediated hypoxia pathway activation promoted the transmission of essential apoptosis signals in TFCs.ConclusionOur study confirmed that excessive iodine adsorption activates the HIF-1α-mediated hypoxia pathway to promote apoptosis of TFCs, which may be an important risk factor contributing to HT development.

RNF6 activates TGF-β1/c-Myb pathway to promote EMT in esophageal squamous cell carcinoma.

This study aimed to investigate RING-Finger Protein 6 (RNF6) expression in esophageal squamous cell carcinoma (ESCC) cells and whether it affects cell proliferation, invasion, and migration by regulating the TGF-β1/c-Myb pathway. TCGA database was used to analyze RNF6 expression in normal tissues and esophageal cancer tissues. Kaplan-Meier method was used to examine the correlation between RNF6 expression and patient prognosis. SiRNA interference vector and RNF6 overexpression plasmid were constructed, and RNF6 was transfected into Eca-109 and KYSE-150 esophageal cancer cell line. In vitro scratch assay and Transwell assay were conducted to investigate the effects of RNF6 on the migration and invasion of Eca-109 and KYSE-150 cells. RT-PCR detected the expression of Snail, E-cadherin, and N-cadherin, and TUNEL detected the apoptosis of cells. RNF6 up-regulation promoted the progression of esophageal cancer and predicted poor prognosis. RNF6 also enhanced the migration and invasion of ESCC cells in vitro. RNF6 silencing inhibited the migration and invasion of ESCC cells. TGF-β inhibitors reversed the oncogenic effects of RNF6. RNF6 regulated the migration and invasion of ESCC cells by activating the TGF-β pathway. RNF6/TGF-β1 promoted esophageal cancer progression through c-Myb. RNF6 promotes the proliferation, invasion, and migration of ESCC cells possibly by activating the TGF-β1/c-Myb pathway and affects the progression of ESCC.

Shikonin and cisplatin synergistically overcome cisplatin resistance of ovarian cancer by inducing ferroptosis via upregulation of HMOX1 to promote Fe2+ accumulation

BackgroundCisplatin-based chemotherapy often results in ovarian cancer (OC) chemical resistance and treatment failure. The combination of natural compounds with platinum-based agents is a new strategy for overcoming cisplatin resistance. At present, the synergistic effects and mechanism of combination of shikonin and cisplatin to overcome cisplatin resistance in OC are still unknown. PurposeThis study was to evaluate the synergistic effects of shikonin and cisplatin on cisplatin-resistant OC cells and to assess the underlying molecular basis for these effects. MethodsCell counting kit-8 assay, colony-formation assay, proteomic analysis, reactive oxygen species (ROS) detection, lipid peroxidation (LPO) detection, Fe2+ detection, western blot, and quantitative real-time reverse transcription PCR (qRT-PCR) were performed to evaluate the effects of shikonin and cisplatin on cisplatin-resistant OC cells. Underlying mechanisms of action were investigated in vitro using small molecule inhibitors and siRNA. In vivo, the effect of shikonin and cisplatin combination on tumor growth in BALB/c nude mice was evaluated, with tumor immunohistochemical (IHC) staining performed to detect ferroptosis-related proteins. ResultsIn vitro, shikonin and cisplatin were shown to synergistically reduce the viability of cisplatin-resistant OC cells. Proteomic results demonstrated that the combination of the two drugs induced a ferroptotic process, as evidenced by increased levels of ROS, LPO, and Fe2+, with downregulation of glutathione peroxidase 4 (GPX4). Heme oxygenase 1 (HMOX1) inhibition and siRNA interference attenuated the combined effect of the two drugs on cell viability. Accumulation of Fe2+ was attenuated by siRNA interference of HMOX1. In vivo, combination treatment significantly inhibited the growth of subcutaneous tumors in BALB/c nude mice and increased the expression of ferroptosis-related proteins in tumor tissue. ConclusionWe report for the first time that the co-treatment of shikonin and cisplatin overcomes cisplatin resistance in OC through ferroptosis. Mechanistic analysis reveals the co-treatment induces ferroptosis through upregulation of HMOX1 that promotes Fe2+ accumulation.

Identification of EMP1 as a critical gene for cisplatin resistance in ovarian cancer by using integrated bioinformatics analysis.

Cisplatin resistance is among the main reasons for the poor prognosis of ovarian cancer (OC) patients. Until now, effective biomarkers for predicting cisplatin resistance in OC and specific drugs for reversing this resistance are lacking. This study identified the critical gene associated with cisplatin resistance in OC and provided a potential target for overcoming this resistance. Differentially expressed genes between cisplatin-resistant and -sensitive OCs were identified by screening public datasets. Survival analysis was conducted to screen prognosis-related DEGs. CIBERSORT, ESTIMATE, and immune checkpoint genes were used to assess the association between EMP1 expression and tumor microenvironment features. CTRP and GDSC databases were employed to analyze the correlation between EMP1 expression and cisplatin resistance. Furthermore, immunohistochemistry, qPCR, Western blotting, siRNA interference, and the CCK8 assay were performed to verify the role of EMP1 in cisplatin resistance in vitro. Finally, xenograft mouse models were generated to further confirm the role of EMP1 in cisplatin resistance in vivo. EMP1 was identified as a critical gene associated with cisplatin resistance in OC. According to bioinformatics analyses, increased EMP1 expression was linked to higher stromal/ESTIMATE scores as well as greater ICG expression levels. The in vitro experiments showed that EMP1 was highly expressed in cisplatin-resistant OC tissues and cells, and silencing this EMP1 expression enhanced OC cell sensitivity to cisplatin. Finally, in vivo experiments confirmed that EMP1 promotes tumor growth and cisplatin resistance. EMP1 can act as a predictive biomarker for cisplatin resistance in OC and as a potential therapeutic target.

IL-10 enhances cell-to-cell communication in chondrocytes via STAT3 signaling pathway

Gap junction intercellular communication (GJIC) allows the transfer of material, message and energy between cells, which influences cell behaviors including cell proliferation, migration, differentiation and apoptosis and determines cell fate. Interleukin-10 (IL-10), a versatile cytokine, attracts more and more attention in the cartilage pathology such as osteoarthritis (OA) due to its potential in anti-inflammation and wound repair. However, whether IL-10 can mediate GJIC in chondrocytes remains elusive. In the current study, we aimed to explore the role of IL-10 on GJIC and its underlying mechanism. We found that IL-10 can promote GJIC in living chondrocytes. IL-10-enhanced GJIC in chondrocytes was dependent on the up-regulation of connexin 43 (Cx43). Knockdown experiment based on siRNA interference then confirmed that IL-10-enhanced GJIC required participation of IL-10 receptor 1 (IL-10R1). IL-10 activated signal transducer and activator of transcription 3 (STAT3) signaling and promoted the nuclear accumulation of p-STAT3 through IL-10 receptor 1. Inhibitor experiment further confirmed the importance of STAT3 signaling in IL-10-mediated GJIC. Taking together, our results provided a thorough process of IL-10-modulated cell-to-cell communication in chondrocytes and established a bridge between inflammatory factor, IL-10, and GJIC, which can increase our understanding about the physiology and pathology of cartilage.

Cbx2 is a functional target of the let-7 family in the gonad of Japanese flounder (Paralichthys olivaceus).

As a key member of the miRNA family, the role and target gene of the let-7 family in the gonad of Japanese flounder (Paralichthys olivaceus) is unclear. Chromobox homolog 2 (CBX2) is one of the core components of the polycomb group complex (PcG) and significantly influences gonadal development. The deletion of CBX2 can lead to sex reversal in mammals. Therefore, exploring the relationship between the let-7 family and cbx2 is crucial to clarify the role played by the let-7 family in the gonad of Japanese flounder. We predicted and verified the target interaction between the let-7 family and cbx2. The results showed that cbx2 was a direct target of let-7d, let-7e, let-7g, let-7j, and let-7b. Among them, let-7d, let-7e, let-7g, and let-7j exhibited an extremely significant targeting relationship with cbx2 (p<0.001). Taking let-7g as an example, we further investigated the regulatory role between let-7g and cbx2 in the gonad by miRNA overexpression and inhibition experiments in primary testis cells. The results revealed that let-7g could negatively regulate cbx2 at the level of primary testis cells. And the expression of sf1 (steroidogenic factor 1) was also significantly decreased after the interference of cbx2 siRNA. This suggests that the let-7 family may be involved in the Japanese flounder gonadal development via targeting cbx2.

Reduced FANCE Confers Genomic Instability and Malignant Behavior by Regulating Cell Cycle Progression in Endometrial Cancer.

Introduction: Fanconi anemia complementation group E (FANCE) is a subunit of fanconi anemia (FA) pathway and plays a key role in repairing DNA interstrand cross-links (ICLs) damage. We investigate detailed functions and mechanisms of FANCE in endometrial cancer (EC). Methods: FANCE protein and RNA expression in EC and non-cancerous tissues were detected by Western blotting (WB), immunohistochemistry (IHC), and real-time polymerase chain reaction (RT-PCR) assays. Using lentiviral transfection and siRNA interference techniques, we constructed overexpressing FANCE (OE-FANCE) and FANCE-knockdown (FANCE-KD) EC cells. We then investigated DNA damage repair capacity of FANCE in EC cells including comet assay and γH2AX immunofluorescence assay. In vitro assays including CCK8, EDU and colony formation for chemoresistance and proliferation, transwell assay for metastasis were performed. Flow cytometer assay, cell cycle synchronization for cell cycle progression and EC cells RNA sequencing were determined. Finally, in vivo mouse models were used to detect tumor growth. Results: We found FANCE RNA and protein expression was significantly decreased in endometrioid adenocarcinoma (EAC) compared with normal and atypical hyperplasia endometrium. FANCE promoted the repair of ICL damage and double-strand break (DSB) in OE-FANCE EC cells. Furthermore, FANCE increased drug resistance in OE-FANCE EC cells by upregulating FA pathway and homologous recombination (HR) associated proteins. FANCE inhibited cell proliferation and metastasis through G2/M cell cycle arrest in vitro and vivo. FANCE participated in regulating several pathways. Conclusion: The study demonstrates the reduction of FANCE expression leads to genomic instability, thereby promoting the development of EC by regulating cell cycle.

TFEB coordinates autophagy and pyroptosis as hepatotoxicity responses to ZnO nanoparticles

Zinc oxide nanoparticles (ZnO NPs) have drawn serious concerns about their biotoxicity due to their extensive applications in biological medicine, clinical therapeutic, daily chemical production, food and agricultural additives. In our present study, we clarified hepatotoxic mechanism of ZnO NPs through investigating the crosstalk between autophagy and pyroptosis, a remaining enigma in hepatocyte stimulated by ZnO NPs. Based on the effects of autophagy intervention by Rapamycin (Rap) and 3-Methyladenine (3-MA), and the observation of pyroptosis morphology and related indexes, the autophagy and pyroptosis simultaneously initiated by ZnO NPs were interrelated and the autophagy characterized by autophagosome production and increased expression of autophagy proteins was identified as a protective response of ZnO NPs against pyroptosis. According to the analysis of protein expression and fluorescence localization, the NLRP3 inflammasome assemble and the classical Caspase-1/GSDMD-dependent pyroptosis induced by ZnO NPs was modulated by autophagy. In this process, the adjustment of TFEB expression and nuclear translocation by gene knockout and gene overexpression, further altered the tendency of ZnO NPs-induced pyroptosis via the regulation of autophagy and lysosomal biogenesis. The knockout of TFEB gene exacerbated the pyroptosis via autophagy elimination and lysosome inhibition. While the alleviation of NLRP3 generation and pyroptosis activation was observed after treatment of TFEB gene overexpression. Additionally, the siRNA interference confirmed that TRAF-6 was involved in the TFEB-mediated global regulation of autophagy-lysosome-pyroptosis in response to ZnO NPs. Accordingly, pyroptosis induced by ZnO NPs in hepatocyte could be significantly avoided by TFEB-regulated autophagy and lysosome, further providing new insights for the risk assessment and therapeutic strategy.

Circadian clock genes Bmal1 and Period may regulate nocturnal spawning by controlling sex hormone secretion in razor clam Sinonovacula constricta

The circadian clock is an endogenous regulation mechanism that coordinates biological processes with daily changes, which are regulated by circadian clock genes. Bmal1 and Period are key circadian clock genes and their roles in reproductive development have been widely studied. The spawning time of Sinonovacula constricta is limited to the night even under external artificial stimulation, and it might be regulated by the internal circadian clock. In this study, the heart rate of S. constricta was higher between 20:00-04:00 at night and lower between 12:00-16:00 during the day, and the sex hormone contents were the highest at 00:00 and the lowest at 18:00 (P &amp;lt; 0.01). Therefore, these obvious changes in the circadian rhythm indicate that S. constricta is a nocturnal animal. The open reading frame (ORF) of Bmal1 comprises 1944 bp encoding 647 aa, while the ORF of Period comprises 3111 bp encoding 1036 aa. Bmal1 and Period were both expressed in four tissues, but they had opposite rhythmic expression patterns. Bmal1 expression was higher at 00:00-06:00 and lower at 12:00-18:00, and Period expression was opposite, thereby suggesting that Bmal1 and Period are involved in positive and negative pathways regulated by the circadian clock, respectively. Strong protein fluorescence signals of Bmal1 and Period proteins were observed in mature oocytes, spermatids, hepatocytes, and epithelial cells of siphons. After siRNA interference, the expression of both Bmal1 and Period significantly decreased (P &amp;lt; 0.01), and the sex hormone contents decreased significantly from 3 to 7 days in the siRNA treatment groups (P &amp;lt; 0.01). Therefore Bmal1 and Period may regulate nocturnal spawning by controlling sex hormone secretion. These findings provide a theoretical basis for understanding the molecular mechanism related to spawning, and may facilitate the artificial propagation of mollusks.

Roles of MEF2A and HOXA5 in the transcriptional regulation of the bovine FoxO1 gene

The Forkhead box factor 1 (FoxO1) gene plays a vital role in the growth and development of skeletal muscle. In the present study, expression analysis of the bovine FoxO1 gene exhibited the highest expression in longissimus dorsi muscle followed by its expression in adipose tissue. Moreover, high mRNA expression of FoxO1 gene was found in differentiated bovine myoblasts and adipocytes at day 6 of induced differentiation (p < 0.05). The regulatory pattern of the bovine FoxO1 gene was investigated through screening and dual-luciferase activity of the 1.7 kb 5′UTR (untranslated region) within pGL3-basic vector and a core promoter region was explored at (-285/-27) upstream of the transcription start site. The transcription factors (TFs) MEF2A and HOXA5 within the core promoter region (-285/-27) were found as the regulatory cis-acting element. The siRNA interference of the TFs, chromatin immunoprecipitation (ChIP) assay, and site-directed mutation validated that MEF2A and HOXA5 binding occurs in the region -285/-27 bp and performs an essential role in the transcriptional regulation of bovine FoxO1 gene. These findings explored the regulatory network mechanism of the FoxO1 gene in skeletal muscle development and adipogenesis for the bovine breed improvement program.

Phenolipid JE improves metabolic profile and inhibits gluconeogenesis via modulating AKT-mediated insulin signaling in STZ-induced diabetic mice

Phenolipids are characteristic phytochemicals of Syzygium genus. However, the antidiabetic potential and underlying molecular mechanism of these components are not fully elucidated. Herein, we studied the anti-diabetic effects of jambone E (JE), a phenolipid from S. cumini, with in vitro and in vivo models. Data from current study showed that JE enhanced glucose consumption and uptake, promoted glycogen synthesis, and suppressed gluconeogenesis in insulin resistant (IR)-HepG2 cells and primary mouse hepatocytes. JE also attenuated streptozotocin-induced hyperglycemia and hyperlipidemia in type 1 diabetic (T1D) mice. Eleven metabolites (e.g. trimethylamine n-oxide, 4-pyridoxic acid, phosphatidylinositol 39:4, phenaceturic acid, and hippuric acid) were identified as potential serum biomarkers for JE’s antidiabetic effects by an untargeted metabolomics approach. The further molecular mechanistic study revealed that JE up-regulated phosphorylation levels of protein kinase B (AKT), glycogen synthase kinase 3 beta, and forkhead box O1 (FoxO1), promoted nuclear exclusion of FoxO1 whilst decreased gene expression levels of peroxisome proliferator-activated receptor gamma coactivator-1 alpha, phosphoenolpyruvate carboxykinase and glucose 6-phosphatase in IR-HepG2 cells and T1D mice. Our data suggested that JE might be a potent activator for AKT-mediated insulin signaling pathway, which was confirmed by the usage of AKT inhibitor and AKT-target siRNA interference, as well as the cellular thermal shift assay. Findings from the current study shed light on the anti-diabetic effects of phenolipids in the Syzygium species, which supports the use of medicinal plants in the Syzygium genus for potential pharmaceutical applications.

NUDT21 Modulates Pign through APA Mechanism to Affect the Progression of MDS and AML

Introduction: Previous studies indicated that PIGN gene is crucial in regulating mitotic integrity to maintain chromosomal stability and prevent MDS leukemic transformation and progression. Alternative Polyadenylation (APA) regulation and 3' RNA cleavage are 2 known vital processes in cellular DNA damage response to maintain genomic stability. NUDT21 gene encodes protein that is one subunit of the cleavage factor Im complex required for 3' RNA cleavage and APA processing. Although PIGN gene has six potential polyadenylation sites, the exact role of NUDT21 product on PIGN expression regulation has not been illustrated before. In this study, we assessed NUDT21 product regulation of PIGN expression via APA in MDS and AML patient samples, cell lines, and in mouse. Methods: At first, we used bioinformatics methods to analyze NUDT21 gene expression profile in AML, MDS, and normal control specimens and to plot the relationship between NUDT21 gene expression status and patient survival length from 5 datasets. We also used correlation regression analysis method to predict the gene expression correlations between NUDT21 gene and PIGN gene. Then, we used Caspas9 technology and vector overexpression technology to knockout or overexpress PIGN in HEK293 cells; RNA interference to knockdown the NUDT21 gene in HEK293, HL60, K562, THP-1,SKM-1 and MUTZ-1 cells. MTT and CCK8 methods were used to evaluate cell proliferation. Meanwhile, cell cycle, apoptosis, Reactive Oxygen Species (ROS) and GPI-anchored proteins (CD55, CD59, and CD235) expression profiles were evaluated by flowcytometry; Immunofluorescence experiment, RT-PCR were used to evaluate the impact of NUDT21 gene knockdown on PIGN expression. in vivo mice siRNA interference tests were conducted to verify the impact of NUDT21 knockdown on PIGN gene expression. RNA-Seq & Dapars analyses were conducted on mRNAs from THP-1,SKM-1 and MUTZ-1 cell lines with various PIGN gene expression status to observe the effects of NUDT21 knockdown on PIGN 3'UTR processing. Results:NUDT21 expression increases in mononuclear cells from AML and high-risk MDS. NUDT21 expression level is negatively correlated with the overall survival. NUDT21 knockdown decreases cell proliferation but increases cell differentiation via prolonging G1 phase, decreasing S phase, and increasing apoptosis. NUDT21 knockdown increases cell membrane GPI-anchoring proteins (CD55, CD59, and CD235) expressions; increases ROS and Caspas3 expressions but decreases CyclinD1 expression. NUDT21 knockout can increase the PIGN expression, but PIGN expression status has no impact on NUDT21 expression. RT-PCR and WB results showed that NUDT21 knockdown can increase TP53 expression but decrease p21 expression. in vivo mice siRNA interference tests confirmed that NUDT21 knockdown increases PIGN gene expression (Figure). The RNA-Seq/Dapars analyses results showed that 3'UTR of PIGN transcript was significantly shortened and PIGN expression increased after NUDT21 knockdown. Conclusion:NUDT21 modulates PIGN gene expression through APA mechanism. PIGN polyadenylation modulation may regulate cell cycle and cell proliferation through NUDT21-PIGN-P21- Cyclin D1 pathway and may regulate cell apoptosis through NUDT21-PIGN-TP53-ROS-caspas3 pathway. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Adaptation of African swine fever virus to porcine kidney cells stably expressing CD163 and Siglec1.

African swine fever virus (ASFV) is a complex large DNA enveloped virus that causes African swine fever (ASF) with a fatality rate of up to 100%, seriously threatening the global swine industry. Due to the strict cell tropism of ASFV, there is no effective in vitro cell line, which hinders its prevention and control. Herein, we analyzed genome-wide transcriptional profiles of ASFV-susceptible porcine alveolar macrophages (PAMs) and non-susceptible cell lines PK15 and 3D4-21, an found that PAM surface pattern recognition receptors (PRRs) were significantly higher and common differential genes were significantly enriched in phagocytosis compared with that observed in PK15 and 3D4-21 cell lines. Therefore, endocytosis functions of host cell surface PRRs may play key roles in ASFV infection in vitro. ASFV was found to be infective to PK15 and 3D4-21 cell lines overexpressing CD163 and Siglec1, and to the PK15S1-CD163 cell line stably expressing CD163 and Siglec1. However, the PK15 and 3D4-21 cell lines overexpressing CD163 or Siglec1 alone were not infectious. Simultaneous interference of CD163 and Siglec1 in PAMs with small interfering RNA (siRNA) significantly reduced the infectivity of ASFV. However, siRNA interference of CD163 and Siglec1 respectively did not affect ASFV infectivity. ASFV significantly inhibited IFN expression levels in PAMs and PK15S1-CD163 cells, but had no effect on PK15 and 3D4-21 cell lines. These results indicate that CD163 and Siglec1 are key receptors for ASFV-infected host cells, and both play a synergistic role in the process of ASFV infection. ASFV inhibits IFN expression in susceptible cells, thereby downregulating the host immune response and evading the immune mechanism. The discovery of the ASFV receptor provides novel ideas to study ASFV and host cell interactions, pathogenic mechanisms, development of receptor blockers, vaccine design, and disease resistance breeding.

Swinhoeic acid from Potentilla fragarioides ameliorates high glucose-induced oxidative stress and accumulation of ECM in mesangial cells via Keap1-dependent activation of Nrf2

ABSTRACT Objectives Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus. Oxidative stress resulting from high glucose promotes accumulation of ECM and development of DN. Activation of Nrf2 could attenuate oxidative stress and following accumulation of ECM. To find novel therapy for DN, we explored the effects of swinhoeic acid from Potentilla fragarioides on mesangial cells under high glucose and underlying mechanisms. Methods CCK-8 and BrdU incorporation assays for survival of mesangial cells gave the concentration of swinhoeic acid in following investigations. ROS, MDA, SOD and CAT were determined. And ECM proteins and their upstream regulators TGF-β1 and CTGF were detected using ELISA assays. Activation of Nrf2 was explored by immunofluorescence staining together with luciferase reporter assay. To demonstrate the role of Nrf2 activation, siRNA interference was performed. And co-immunoprecipitation assay was used to elucidate swinhoeic acid affects the interaction between Keap1 and Nrf2. Results Swinhoeic acid at 10 and 20 μM attenuated oxidative stress and accumulation of ECM in mesangial cells under high glucose. Itactivated Nrf2 in a Keap1-dependent manner, which was involved in its effects. Conclusion Swinhoeic acid ameliorates oxidative stress and accumulation of ECM resulting from high glucose in mesangial cells via activating Nrf2 in Keap1-dependent manner.

Expression of TCF3 in Burkitt's Lymphoma and Its Proliferative Effect and Prognostic Significance

To observe the mutation and expression of TCF3 gene in Burkitt's lymphoma (BL), and explore its effect on the proliferation of BL cells and clinical efficacy and prognosis. The mutation and expression of TCF3 in tumor tissues from BL patients were observed by the second-generation sequencing and real-time quantitative PCR. The proliferation and apoptosis of lymphoma cells after TCF3 knocked down were observed by siRNA interference technique and CCK-8 method. Survival analysis was used to observe the relationship between TCF3 mutation and the treatment efficacy and prognosis of BL patients. There were high frequency mutation rate (mutation rate was 23.7%) and high expression of TCF3 in BL patients. After TCF3 knocked down, cell proliferation was inhibited and apoptosis was promoted. In TCF3-siRNA group and control group, the cell proliferation rate at 48 h was (50.2±5.9)% and (96.6±11.4)%, and apoptosis rate was 30.1% and 1.5%, respectively, which showed significantly different between the two groups (P<0.001, P=0.005). The complete remission rate of patients with TCF3 mutation was low. The complete remission rate of mutant group and wild-type group was 44.4% and 82.8%, respectively (P=0.023). The 2-year progression-free survival rate and overall survival rate of the patients with TCF3 mutation was 55.6% and 61.0%, respectively, which was lower than 83.2% and 85.2% of the patients without mutation, but the differences were not statistically significant. There are mutation and abnormal expression of TCF3 in patients with BL. Patients with TCF3 mutations have low remission rate and poor prognosis.

Extracellular vesicles of Clonorchis sinensis promote IL-6 and TNF-α secretion via the Toll-like receptor 9-mediated ERK pathway in biliary epithelial cells

Clonorchis sinensis is closely associated with cholangitis, cholecystitis, biliary fibrosis and cholangiocarcinoma. The present study elucidated the role of extracellular vesicles of C. sinensis (CsEVs) in activating Toll-like receptor 9 (TLR9) and regulating inflammatory responses. The results showed that TLR9 expression was increased in the livers of C. sinensis-infected mice. CsEVs were cup-shaped or saucer-shaped and 80–120 nm in diameter. CsEVs activated TLR9 and promoted IL-6 and TNF-α expression in mouse biliary epithelial cells (BECs), and TLR9 siRNA interference reduced the secretion of the two cytokines. CsEV stimulation promoted the phosphorylation of ERK, p38, AKT, and p65, and TLR9 siRNA interference regulated the phosphorylated ERK, AKT and p65 levels. The ERK inhibitor decreased the CsEVs-induced IL-6 and TNF-α secretion. The present study elucidated for the first time that CsEVs induced IL-6 and TNF-α production in BECs via the TLR9-mediated ERK pathway.