Xin-Hua Xiao

Identification and characterization of novel spliced variants of PRMT2 in breast carcinoma

Protein N‐arginine methyltransferases (PRMTs) participate in a number of cellular processes, including cell growth, nuclear/cytoplasmic protein shuttling, differentiation, RNA splicing and post‐transcriptional regulation. PRMT2 (also known as HRMT1L1) is clearly involved in lung function, the inflammatory response, apoptosis promotion, Wnt signaling and leptin signaling regulation through different mechanisms. In this study, we report the molecular and cell biological characterization of three novel PRMT2 splice variants isolated from breast cancer cells and referred to as PRMT2α, PRMT2β and PRMT2γ. Compared with the wild‐type PRMT2, these variants lack different motifs and therefore generate distinct C‐terminal domains. Confocal microscopy scanning revealed a distinct intracellular localization of PRMT2 variants, suggesting that the alternatively spliced C‐terminus of PRMT2 can directly influence its subcellular localization. Our findings reveal that these variants are capable of binding to estrogen receptor alpha (ERα) both in vitro and in vivo, and the N‐terminal regions of these variants contribute to ERα–PRMT2 interactions. Furthermore, these variants were proved to be able to enhance ERα‐mediated transactivation activity. Luciferase reporter assays showed that PRMT2s could modulate promoter activities of the ERα‐targeted genes of Snail and E‐cadherin. In addition, PRMT2 silencing could enhance 17β‐estradiol‐induced proliferation by regulating E2F1 expression and E2F1‐responsive genes in ERα‐positive breast cancer cells. Real‐time PCR and immunohistochemistry showed that overall PRMT2 expression was upregulated in breast cancer tissues and significantly associated with ERα positivity status both in breast cancer cell lines and breast cancer tissues. We speculate that PRMT2 and its splice variants may directly modulate ERα signaling and play a role in the progression of breast cancer.

Adiponectin protects endothelial cells from the damages induced by the intermittent high level of glucose

Globular adiponectin (gAd) has anti-atherogenic effects on the vascular wall. Intermittent hyperglycemia induces endothelial cells (ECs) injury but the physiological factors that may protect against ECs damage are largely unknown. In the present study, we investigated the effect of gAd on ECs dysfunction induced by intermittent high glucose. The gAd significantly attenuated intermittent high glucose-induced apoptosis and oxidative stress in human umbilical vein endothelial cells. This was achieved by decreasing caspase-3 and 3-nitrotyrosine protein expression, increasing nitric oxide (NO) secretion and phosphorylation of Akt, AMPK, and endothelial nitric oxide synthase protein expression. Pretreatment with a phosphatidylinositol 3’ kinase (PI3K) inhibitor, LY294002, partly reversed adiponectin’s anti-apoptotic effect. Taken together, our results indicate that gAd acts as a critical physiological factor which protects against fluctuating high glucose-induced endothelial damage. It may act via attenuating apoptosis and increasing synthesis of NO through both the PI3K/AKT and AMPK signaling pathway to reduce oxidative stress and cell apoptosis.

Identification and expression analysis of a novel transcript of the human PRMT2 gene resulted from alternative polyadenylation in breast cancer

The arginine N-methyltransferase 2 protein (PRMT2, also known as HRMT1L1) is thought to act as a coactivator of ERα. The present results show the occurrence of a novel transcript by alternative polyadenylation in the human PRMT2 gene. We demonstrated that the newly identified intron-retaining PRMT2L2 transcript is functionally intact, efficiently translated into protein in vivo. PRMT2 and PRMT2L2 mRNA expression profiles overlap with the distribution of ERα, with the strongest abundance in estrogen target tissues. Transient co-transfection assays demonstrated that PRMT2L2 enhance ERα-mediated transactivation activity of ERE-Luc in a ligand-dependent manner. Confocal microscopy scanning revealed a distinct intra-cellular localization of their fusion proteins, suggesting that the C-terminal region absent in PRMT2L2 is critical for the localization. Statistical analysis further showed that both PRMT2 and PRMT2L2 mRNAexpressions were up-regulated in breast cancer tissues, and significantly associated with ERα positivity status. Thus, post-transcriptional processing mechanism as alternative polyadenylation and splicing may play a crucial role in regulating human PRMT2 gene expression.

Zinc alpha2 glycoprotein alleviates palmitic acid-induced intracellular lipid accumulation in hepatocytes.

Zinc alpha2 glycoprotein (ZAG) plays an important role in stimulating fat mobilization and lipolysis in adipose tissue, but its role in hepatic lipid metabolism remains unclear. Palmitic acid (PA) was used to stimulate HepG2 cells with ZAG overexpression or ZAG knock down (shRNA). Overexpression of ZAG significantly inhibited lipogenesis, promoted lipolysis and fatty acid β-oxidation, and attenuated PA-induced intracellular fat accumulation. Moreover, ZAG overexpression dramatically stimulated adiponectin expression in HepG2 cells. In contrast, knockdown of ZAG notably inhibited fatty acid β-oxidation, increased lipogenesis and lipid accumulation. Collectively, these data suggest that ZAG has the potential to alleviate hepatosteatosis, making it a promising therapeutic target for fatty liver.

MicroRNA-32 promotes calcification in vascular smooth muscle cells: Implications as a novel marker for coronary artery calcification

Cardiovascular calcification is one of the most severe outcomes associated with cardiovascular disease and often results in significant morbidity and mortality. Previous reports indicated that epigenomic regulation of microRNAs (miRNAs) might play important roles in vascular smooth muscle cell (VSMC) calcification. Here, we identified potential key miRNAs involved in vascular calcification in vivo and investigated the role of miR-32-5p (miR-32). According to microarray analysis, we observed increased expression of miR-125b, miR-30a, and miR-32 and decreased expression of miR-29a, miR-210, and miR-320 during the progression of vascularcalcification. Additionally, gain- and loss-of-function studies of miR-32 confirmed promotion of VSMC calcification in mice through the enhanced expression of bonemorphogenetic protein-2, runt-related transcription factor-2(RUNX2), osteopontin, and the bone-specific phosphoprotein matrix GLA protein in vitro. Moreover, miR-32 modulated vascularcalcification progression by activating phosphoinositide 3-kinase (PI3K)signaling and increasing RUNX2 expression and phosphorylation by targeting the 3′-untranslated region of phosphatase and tensin homolog Mrna (PTEN) in mouse VSMCs. Furthermore, we detected higher miR-32 levels in plasmafrom patients with coronary artery disease with coronary artery calcification (CAC) as compared with levels observed in non-CAC patients (P = 0.016), further confirming miR-32 as a critical modulator and potential diagnostic marker for CAC.

Wnt/β-catenin signaling pathway and lipolysis enzymes participate in methylprednisolone induced fat differential distribution between subcutaneous and visceral adipose tissue.

Glucocorticoids (GCs) are well known to induce fat distribution, which is consistent with the central adiposity phenotype seen in Cushings syndrome. GCs have been proposed to be both adipogenic and lipolytic in action within adipose tissues. Different adipogenic and lipolytic effects between subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) are likely to play a role in GCs induced fat differential distribution. Wnt/β-catenin signaling pathway is one of the most important regulators in adipogenesis. Adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) are the major lipases contributing to lipolysis. In the present study, we measured fat depot masses and the expression of Wnt/β-catenin signaling pathway and lipolytic enzymes of female Sprague-Dawley rats treated with or without methylprednisolone. We assessed the roles of Wnt/β-catenin signaling pathway and lipolytic enzymes in fat differential distribution between SAT and VAT. Our data suggested that methylprednisolone could inhibit Wnt/β-catenin signaling pathway in SAT and VAT, increase the expression of ATGL and HSL in SAT, and decrease the expression of ATGL and HSL in VAT. The differential expression of lipolysis enzymes induced by methylprednisolone between SAT and VAT might play a crucial role in fat distribution. Those findings would offer novel insights into the mechanisms of GCs induced fat distribution.

GABARAPL1 acts as a potential marker and promotes tumor proliferation and metastasis in triple negative breast cancer

GABAA-receptor-associated protein like-1 (GABARAPL1) is involved in a variety of cancers. The purpose of this study was to investigate the expression, prognostic roles and functions of GABARAPL1 in triple negative breast cancer (TNBC). Quantitative real-time PCR (qRT-PCR) showed that GABARAPL1 was up regulated in both TNBC cell lines and clinical TNBC tissues. High GABARAPL1 expression level was associated with shorter overall survival (OS) and disease free survival (DFS). Furthermore, inhibition of GABARAPL1 suppressed cell proliferation, tumorigenesis, invasion and metastasis, and induced cell apoptosis. We found that metadherin (MTDH) was a downstream target of GABARAPL1. Inhibition of GABARAPL1 suppressed the mRNA and protein expression of MTDH, and overexpression of MTDH could reverse the effects of GABARAPL1 inhibition, which meant GABARAPL1 performed its function partly through MTDH. Our findings demonstrate that GABARAPL1 acts as a tumor promoter in TNBC partly through MTDH. Targeting at GABARAPL1 could be a potential therapeutic strategy for TNBC.

Zinc alpha2 glycoprotein protects against obesity-induced hepatic steatosis

Background/AimNonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, impaired insulin sensitivity, and chronic low-grade inflammation. Our previous studies indicated that zinc alpha2 glycoprotein (ZAG) alleviates palmitate (PA)-induced intracellular lipid accumulation in hepatocytes. This study is to further characterize the roles of ZAG on the development of hepatic steatosis, insulin resistance (IR), and inflammation.MethodsZAG protein levels in the livers of NAFLD patients, high-fat diet (HFD)-induced or genetically (ob/ob) induced obese mice, and in PA-treated hepatocytes were determined by western blotting. C57BL/6J mice injected with an adenovirus expressing ZAG were fed HFD for indicated time to induce hepatic steatosis, IR, and inflammation, and then biomedical, histological, and metabolic analyses were conducted to identify pathologic alterations in these mice. The molecular mechanisms underlying ZAG-regulated hepatic steatosis were further explored and verified in mice and hepatocytes.ResultsZAG expression was decreased in NAFLD patient liver biopsy samples, obese mice livers, and PA-treated hepatocytes. Simultaneously, ZAG overexpression alleviated intracellular lipid accumulation via upregulating adiponectin and lipolytic genes (FXR, PPARα, etc.) while downregulating lipogenic genes (SREBP-1c, LXR, etc.) in obese mice as well as in cultured hepatocytes. ZAG improved insulin sensitivity and glucose tolerance via activation of IRS/AKT signaling. Moreover, ZAG significantly inhibited NF-ĸB/JNK signaling and thus resulting in suppression of obesity-associated inflammatory response in hepatocytes.ConclusionsOur results revealed that ZAG could protect against NAFLD by ameliorating hepatic steatosis, IR, and inflammation.

Decreased miR-17-92 cluster expression level in serum and granulocytes preceding onset of antithyroid drug-induced agranulocytosis

PurposeWe aimed to determine changes in miR-17-92 cluster expression in serum and granulocytes from patients with antithyroid drug (ATD)-induced agranulocytosis.MethodsIn this study, real-time polymerase chain reaction (PCR) was used to detect serum miR-17-92 expression levels in 20 ATD-induced agranulocytosis and 16 control patients. Importantly, dynamic changes in neutrophil counts from granulocytopenia to agranulocytosis were observed in 6 of the 20 patients. miR-17-92 expression levels in granulocytes of those six patients under the granulocytopenia condition were measured and compared with corresponding granulocyte samples after recovery. Additionally, the expression levels of these miRNAs in patients with type I or type II bone marrow characteristics were analyzed, and the correlation between miR-17-92 and serum free thyroxine level was analyzed.ResultsWe found that levels of miR-17-92 expression decreased in both serum and pre-agranulocytosis granulocytes from patients with ATD-induced agranulocytosis compared with those in serum and granulocytes from both recovered patients and control patients. However, no difference among patients with either type of bone marrow characteristics was observed, and no correlation between serum miR-17-92 and free thyroxine levels was found.ConclusionIn ATD-induced agranulocytosis, expression of the miR-17-92 cluster is reduced in both serum and granulocytes, though this alteration does not correlate with bone marrow characteristics or thyroid function.

Identification of a novel mutation of NR0B1 in a patient with X-linked adrenal hypoplasia and symptomatic treatment.

Abstract Background: X-linked congenital adrenal hypoplasia (X-linked AHC) is characterized by acute onset of primary adrenal insufficiency in infancy or early childhood and hypogonadotropic hypogonadism (HH) at puberty. Mutations in NR0B1, the gene located on Xp21.3 and encoding an orphan nuclear receptor named DAX1, are responsible for this disease. Methods: The entire coding region of the NR0B1 gene of a 14-year-old X-linked AHC proband as well as his family members was sequenced. Clinical and endocrine evaluations with symptomatic treatment results were recorded. Results: DNA sequencing revealed a missense mutation (c.383-384 insA) in exon 1, which resulted in a novel frameshift mutation, thereby resulting in a truncated protein (p.Leu129 Pro fs*137). The therapeutic trail with an observation period of 20 weeks showed an effective improvement in symptoms of hypogonadism with human chorionic gonadotropin (HCG) administration, including a rapid improvement of serum testosterone level, descending of testicles as well as enlargement of testicles and growth of penis. Conclusions: Our study identified a novel frameshift mutation of the NR0B1 gene in a proband with X-linked AHC/HH and further expanded the number of NR0B1 mutations reported in the literature. Moreover, the symptomatic treatment observation provided referential evidence in the treatment of X-linked AHC associated hypogonadism and bilateral inguinal cryptorchidism.