Murine Cytomegalovirus Infection Induced miR-1929-3p Down-Regulation Promotes the Proliferation and Apoptosis of Vascular Smooth Muscle Cells in Mice by Targeting Endothelin A Receptor and Downstream NLRP3 Activation Pathway.

Our previous study demonstrated in vivo that mouse cytomegalovirus (MCMV) infection promoted vascular remodeling after downregulation of miR-1929-3p. This study aimed to investigate the role of miR-1929-3p/ETAR/NLRP3 pathway in mouse vascular smooth muscle cells (MOVAS) after MCMV infection. First, PCR was used to detect the success of the infection. Second, MOVAS were transfected with the miR-1929-3p mimic, inhibitor, and ETAR overexpressed adenovirus vector. Cell proliferation was detected using EdU, whereas apoptosis was detected using flow cytometry. The expression of miR-1929-3p and ETAR were detected using qRT-PCR. Western blot detected proteins of cell proliferation, apoptosis, and the NLRP3 inflammasome. Interleukin-1ß and interleukin-18 were determined using ELISA. The results revealed that after 48 h, MCMV infection promoted the proliferation of MOVAS when the MOI was 0.01. MCMV infection increased ETAR by downregulating miR-1929-3p. The miR-1929-3p mimic reversed the proliferation and apoptosis, whereas the miR-1929-3p inhibitor promoted this effect. ETAR overexpression further promoted MCMV infection by downregulating miR-1929-3p-mediated proliferation and apoptosis. MCMV infection mediates the downregulation of miR-1929-3p and the upregulation of ETAR, which activates NLRP3 inflammasome. In conclusion, MCMV infection promoted the proliferation of MOVAS, possibly by downregulating miR-1929-3p, promoting the upregulation of the target gene ETAR and activating NLRP3 inflammasome.

Low expression of miR-1929-3p mediates murine cytomegalovirus-induced fibrosis in cardiac fibroblasts via targeting endothelin a receptor/NLRP3 inflammasome pathway.

MicroRNAs are crucial in the development of myocardial remodeling in hypertension. Low miR-1929-3p expression induced by murine cytomegalovirus (MCMV) infection is closely related to hypertensive myocardial remodeling. This study investigated the molecular mechanism of miR-1929-3p-induced myocardial remodeling after MCMV infection. We modeled MCMV-infected mouse cardiac fibroblasts (MMCFs) as the primary cell model. First, MCMV infection reduced the expression of miR-1929-3p and increased the mRNA and protein expression of its target gene endothelin receptor type A (ETAR) in mouse cardiac fibroblasts (MCFs), which demonstrated an internal relationship with myocardial fibrosis (MF) based on high proliferation, phenotypic transformation (α-SMA), and collagen expression in MMCFs. The transfection of the miR-1929-3p mimic downregulated the high expression of ETAR and alleviated these adverse effects in MMCFs. Inversely, these effects were exacerbated by the miR-1929-3p inhibitor. Second, the transfection of endothelin receptor type A over-expressed adenovirus (adETAR) reversed these positive effects of the miR-1929-3p mimic on MF improvement. Third, the transfection of adETAR exhibited a strong inflammatory response in MMCFs with increased expression of NOD-like receptors pyrin domain containing 3 (NLRP3) and increased secretion of interleukin-18. However, we found that the ETAR antagonist BQ123 and the selected NLRP3 inflammasome inhibitor MCC950 effectively eliminated the inflammatory response induced by both MCMV infection and miR-1929-3p inhibitor. Moreover, the MCF supernatant was related to cardiomyocyte hypertrophy. Our findings suggest that MCMV infection promotes MF by inducing the downregulation of miR-1929-3p and the high expression of ETAR, which activates NLRP3 inflammasomes in MCFs.

The interplay of transcriptional coregulator NUPR1 with SREBP1 promotes hepatocellular carcinoma progression via upregulation of lipogenesis.

Nuclear protein 1 (NUPR1) is a transcriptional coregulator that has been implicated in the development of various cancer types. In addition, de novo fatty acid synthesis plays a pivotal role in hepatocellular carcinoma (HCC) development. However, little is currently known on the role of NUPR1 in hepatocellular carcinoma. In this study, bioinformatics analysis was conducted to analyze the expression level, prognosis value and enriched pathways of NUPR1 in Liver Hepatocellular Carcinoma (LIHC). We found that NUPR1 was significantly upregulated in human hepatocellular carcinoma cells compared with normal hepatocytes from LIHC patients in TCGA cohorts and our patients. Kaplan-Meier analysis and COX proportional hazard progression model showed that high expression of NUPR1 was correlated with a poor prognosis of LIHC patients. CCK-8, EdU and colony formation assays were performed to explore the effect of NUPR1 on the proliferation of HCC cells, then wound healing and transwell migration assays were performed to evaluate the effects of NUPR1 on cell migration. Furthermore, subcutaneous xenograft models were established to study tumor growth. Results showed that NUPR1 overexpression correlated with a highly proliferative and aggressive phenotype. In addition, NUPR1 knockdown significantly inhibited hepatocellular carcinoma cell proliferation and migration in vitro and hindered tumorigenesis in vivo. Mechanistically, endogenous NUPR1 could interact with sterol regulatory element binding protein 1 (SREBP1) and upregulated lipogenic gene expression of fatty acid synthase (FASN), resulting in the accumulation of lipid content. Moreover, pharmacological or genetic blockade of the NUPR1-SREBP1/FASN pathway enhanced anticancer activity in vitro and in vivo. Overall, we identified a novel function of NUPR1 in regulating hepatocellular carcinoma progression via modulation of SREBP1-mediated de novo lipogenesis. Targeting NUPR1-SREBP1/FASN pathway may be a therapeutic alternative for hepatocellular carcinoma.

TXN inhibitor impedes radioresistance of colorectal cancer cells with decreased ALDH1L2 expression via TXN/NF-κB signaling pathway.

BACKGROUND: Colorectal cancer (CRC) is prevalent worldwide and is often challenged by treatment failure and recurrence due to resistance to radiotherapy. Here, we aimed to identify the elusive underlying molecular mechanisms of radioresistance in CRC. METHODS: Weighted gene co-expression network analysis was used to identify potential radiation-related genes. Colony formation and comet assays and multi-target single-hit survival and xenograft animal models were used to validate the results obtained from the bioinformatic analysis. Immunohistochemistry was performed to examine the clinical characteristics of ALDH1L2. Co-immunoprecipitation, immunofluorescence and flow cytometry were used to understand the molecular mechanisms underlying radioresistance. RESULTS: Bioinformatic analysis, in vitro, and in vivo experiments revealed that ALDH1L2 is a radiation-related gene, and a decrease in its expression induces radioresistance in CRC cells by inhibiting ROS-mediated apoptosis. Patients with low ALDH1L2 expression exhibit resistance to radiotherapy. Mechanistically, ALDH1L2 interacts with thioredoxin (TXN) and regulates the downstream NF-κB signaling pathway. PX-12, the TXN inhibitor, overcomes radioresistance due to decreased ALDH1L2. CONCLUSIONS: Our results provide valuable insights into the potential role of ALDH1L2 in CRC radiotherapy. We propose that the simultaneous application of TXN inhibitors and radiotherapy would significantly ameliorate the clinical outcomes of patients with CRC having low ALDH1L2.

[Corrigendum] MicroRNA­1929­3p participates in murine cytomegalovirus­induced hypertensive vascular remodeling through Ednra/NLRP3 inflammasome activation.

Following the publication of this article, the authors have realized that the images selected for the 'MCMV+miR­NC' and 'MCMV+miR­1929­3p' data panels (15 months), as featured in the lower of the two rows of data shown in Fig. 4A on p. 726, were derived from the same experimental group. The authors re­examined their original data, and discovered that an error had inadvertently been made on account of the similar file names of the two images. The corrected version of Fig. 4, featuring the correct data for the 'MCMV+miR­NC' experiment, is shown on the next page. The authors regret that this error was not picked up upon before the paper was sent to press, and thank the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish a corrigendum. Note that the error did not affect either the results or the conclusions reported in the study, and all the authors agree to this corrigendum. Furthermore, the authors regret any inconvenience caused to the readership. [the original article was published in International Journal of Molecular Medicine 47: 719-731, 2021; DOI: 10.3892/ijmm.2020.4829].

Integration of glucose and cardiolipin anabolism confers radiation resistance of HCC.

BACKGROUND AND AIMS: Poor response to ionizing radiation (IR) due to resistance remains a clinical challenge. Altered metabolism represents a defining characteristic of nearly all types of cancers. However, how radioresistance is linked to metabolic reprogramming remains elusive in hepatocellular carcinoma (HCC). APPROACH AND RESULTS: Baseline radiation responsiveness of different HCC cells were identified and cells with acquired radio-resistance were generated. By performing proteomics, metabolomics, metabolic flux, and other functional studies, we depicted a metabolic phenotype that mediates radiation resistance in HCC, whereby increased glucose flux leads to glucose addiction in radioresistant HCC cells and a corresponding increase in glycerophospholipids biosynthesis to enhance the levels of cardiolipin. Accumulation of cardiolipin dampens the effectiveness of IR by inhibiting cytochrome c release to initiate apoptosis. Mechanistically, mammalian target of rapamycin complex 1 (mTORC1) signaling-mediated translational control of hypoxia inducible factor-1α (HIF-1α) and sterol regulatory element-binding protein-1 (SREBP1) remodels such metabolic cascade. Targeting mTORC1 or glucose to cardiolipin synthesis, in combination with IR, strongly diminishes tumor burden. Finally, activation of glucose metabolism predicts poor response to radiotherapy in cancer patients. CONCLUSIONS: We demonstrate a link between radiation resistance and metabolic integration and suggest that metabolically dismantling the radioresistant features of tumors may provide potential combination approaches for radiotherapy in HCC.

Selenium supplementation improves nutrient intake and digestibility, and mitigates CH4 emissions from sheep grazed on the mixed pasture of alfalfa and tall fescue.

Low selenium (Se) in soil and forage can adversely affect on the quality of animal-derived foods, and hence on human health. Lambs grazed on mixed pastures of alfalfa (Medicago sativa) and tall fescue (Festuca arundinacea) were supplemented with five levels of Se [0, 3, 6, 9 and 12 µg/kg body weight (BW)]. The intake of dry matter (DM) and organic matter (OM) varied with the level of Se supplementation, with a peak at 6 µg Se per kg BW (p ≤ 0.05). Gross energy (GE) intake, digestive energy (DE) intake and metabolic energy (ME) intake were higher at 6 µg Se per kg BW than at other Se levels (p < 0.01); in addition, methane energy (CH4 -E) output was lower at 6 µg Se per kg BW. Supplementation with Se significantly increased nitrogen (N) intake, faecal N and urine N, for which the peak values were 20.2 g N/, 5.62 g N/day and 7.92 g N/day, respectively, at 6 µg Se per kg BW. Se intake, blood Se, faecal Se, urine Se and retained Se were negatively correlated with forage crude protein (CP) content (p < 0.001) but were positively correlated with the content of neutral detergent fibre (NDF) (p < 0.001) and acid detergent fibre (ADF) (p < 0.001). Thus, we recommend the addition of 6 µg Se per kg BW to sheep grazed on pastures in regions with low soil Se.

MicroRNA­1929­3p participates in murine cytomegalovirus­induced hypertensive vascular remodeling through Ednra/NLRP3 inflammasome activation.

MicroRNAs (miRNAs) play an important role in the development of vascular remodeling in essential hypertension (EH) by mediating the effects of human cytomegalovirus (HCMV) on the vascular system. Therefore, the aim of the present study was to investigate the effects of murine cytomegalovirus (MCMV) infection on blood pressure and vascular function in mice, in order to elucidate the role of miR­1929­3p in this process. For model development, 7­month­old C57BL/6J mice were infected with the Smith strain of MCMV, and MCMV DNA, IgG and IgM were detected. Subsequently, blood pressure was measured via the carotid artery, and the morphological changes of the aorta were evaluated by hematoxylin and eosin and Masson's trichrome staining. miR­1929­3p transfection was performed using an adeno­associated virus packaged vector and the changes in vascular structure were then observed. The levels of nitric oxide (NO) and endothelial NO synthase were also assessed with colorimetry. Vascular remodeling and expression of NLRP3 inflammasome pathway­related proteins were detected by immunohistochemistry and western blotting. Endothelin­1 (ET­1), interleukin (IL)­1ß and IL­18 were assayed by ELISA. The results revealed that MCMV infection increased the blood pressure, promoted vascular remodeling, caused endothelial cell injury, and downregulated miR­1929­3p. However, these effects were alleviated by miR­1929­3p overexpression, which downregulated endothelin A receptor (Ednra) and NLRP3 inflammasome, as well as endothelial injury­ and vascular remodeling­related genes. Taken together, the findings of the present study indicated that overexpression of miR­1929­3p may improve MCMV­induced vascular remodeling, possibly via the deactivation of the NLRP3 inflammasome by ET­1/Ednra.

Effects of Allium mongolicum Regel supplementation on the digestibility, methane production, and antioxidant capacity of Simmental calves in northwest China.

This study evaluated the effects of Allium mongolicum Regel (AM) supplementation on nitrogen (N) balance, ruminal fermentation, and antioxidant properties. Sixteen male calves were assigned randomly to four groups, and the four were added with 0 (CON), 200 mg/kg (body weight; BW) (Low AM; LA), 400 mg/kg (BW) (Middle AM; MA), or 800 mg/kg (BW) (High AM; HA) per day for each individual. AM was added on dry matter (DM) basis. The experiment lasted for 58 days. Supplementation of AM could significantly increase average daily gain, DM digestibility, acid detergent fiber digestibility, and retained N/Intake N. N digestibility and molar proportion of propionate in the MA and HA treatments were higher than that in the CON treatment (p < .05), respectively. AM supplementation significantly increased the molar concentration of total volatile fatty acid in the rumen fluid (p < .05). The ratio of acetate to propionate in the MA and HA groups was lower than that in the CON treatment (p < .05). Furthermore, AM supplementation significantly reduced methane (CH4 ) (p < .05) emissions. AM supplementation significantly increased the activities of superoxide dismutase. The MA group could significantly increase the activities of glutathione peroxidase and decrease the content of malondialdehyde. Our results indicated that AM supplementation could affect the nutrient digestibility, CH4 emission, and antioxidant capacity of Simmental calves.

miR-1929-3p Overexpression Alleviates Murine Cytomegalovirus-Induced Hypertensive Myocardial Remodeling by Suppressing Ednra/NLRP3 Inflammasome Activation.

MicroRNAs (miRNAs) play crucial roles in the development of essential hypertension (EH). Previously, we found that the expression of miR-1929-3p was decreased in C57BL/6 mice with hypertension induced by murine cytomegalovirus (MCMV). In this study, we explored the role of miR-1929-3p in hypertension myocardial remodeling in MCMV-infected mice. First, we measured MCMV DNA and host IgG and IgM after infection and determined the expression of miR-1929-3p and its target gene endothelin A receptor (Ednra) mRNA in the myocardium of mice. Then, we performed invasive blood pressure (BP) monitoring. Heart-to-body weight ratio (HW/BW%), along with mRNA levels of B-type natriuretic peptide (BNP) and beta myosin heavy chain (ß-MHC), revealed myocardial remodeling. Hematoxylin/eosin and Masson's trichrome staining indicated morphological changes in the myocardium. Cardiac function was assessed via echocardiography. Moreover, MCMV-infected mice were injected with recombinant adeno-associated virus- (rAAV-) miR-1929-3p overexpression vector. Immunohistochemistry and western blotting showed the expression of Ednra and the activation of NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome. And enzyme-linked immunosorbent assay (ELISA) revealed the concentrations of endothelin-1 (ET-1), interleukin-1ß (IL-1ß), and interleukin-18 (IL-18). In this study, we found that decreased expression of miR-1929-3p in MCMV-infected mice induced high BP and further development of myocardial remodeling cardiac function injury through increased expression of Ednra. Strikingly, overexpression of miR-1929-3p ameliorated these pathological changes of the heart. The positive effect was shown to be associated with inhibition of NLRP3 inflammasome activation and decreased expression of key proinflammatory cytokine IL-1ß. Collectively, these results indicate that miR-1929-3p overexpression may effectively alleviate EH myocardial remodeling by suppressing Ednra/NLRP3 inflammasome activation in MCMV-infected mice.

Endoplasmic reticulum stress induces apoptosis of arginine vasopressin neurons in central diabetes insipidus via PI3K/Akt pathway.

AIMS: Central diabetes insipidus (CDI), a typical complication caused by pituitary stalk injury, often occurs after surgery, trauma, or tumor compression around hypothalamic structures such as the pituitary stalk and optic chiasma. CDI is linked to decreased arginine vasopressin (AVP) neurons in the hypothalamic supraoptic nucleus and paraventricular nucleus, along with a deficit in circulating AVP and oxytocin. However, little has been elucidated about the changes in AVP neurons in CDI. Hence, our study was designed to understand the role of several pathophysiologic changes such as endoplasmic reticulum (ER) stress and apoptosis of AVP neurons in CDI. METHODS: In a novel pituitary stalk electric lesion (PEL) model to mimic CDI, immunofluorescence and immunoblotting were used to understand the underlying regulatory mechanisms. RESULTS: We reported that in CDI condition, generated by PEL, ER stress induced apoptosis of AVP neurons via activation of the PI3K/Akt and ERK pathways. Furthermore, application of N-acetylcysteine protected hypothalamic AVP neurons from ER stress-induced apoptosis through blocking the PI3K/Akt and ERK pathways. CONCLUSION: Our findings showed that AVP neurons underwent apoptosis induced by ER stress, and ER stress might play a vital role in CDI condition through the PI3K/Akt and ERK pathways.

Transcriptional profiling of the developing rat brain reveals that the most dramatic regional differentiation in gene expression occurs postpartum.

Neural development involves the expression of ensembles of regulatory genes that control the coordinate and region-specific expression of a host of other genes, resulting in the unique structure, connectivity, and function of each brain region. Although the role of some specific genes in neural development has been studied in detail, we have no global view of the orchestration of spatial and temporal aspects of gene expression across multiple regions of the developing brain. To this end, we used transcriptional profiling to examine expression levels of 9955 genes in the hypothalamus, hippocampus, and frontal cortex across seven stages of postnatal development and up to four stages of prenatal development in individual male rats (six per group). The results reveal dramatic changes across development in >97% of the neurally expressed genes. They also uncover a surprising degree of regional differentiation occurring after birth and through the first 2 weeks of life. Cluster analysis identifies 20 clusters of transcripts enriched in genes related to particular functions, such as DNA metabolism, nuclear function, synaptic vesicle transport, myelination, and neuropeptide hormone activity. Thus, groups of genes with related functions change in the brain at specific times, possibly marking critical periods for each function. These findings can broadly serve as a backdrop for studying the role of individual genes in neural development. They also underscore the importance of early postnatal life in the rat, which corresponds to late gestation in the human, as a critical late phase of neural organization and differentiation, even in subcortical regions.