Leucine-rich-alpha-2-glycoprotein 1 Expression Research Articles

Increased level of serum leucine-rich-alpha-2-glycoprotein 1 in patients with clear cell renal cell carcinoma

BackgroundCurrently, no useful serum markers exist for clear cell renal cell carcinoma (ccRCC), making early detection challenging as diagnosis relies solely on imaging tests. Radiation exposure is also a concern due to multiple required CT examinations during treatment. Renal cell carcinoma (RCC) histological types include ccRCC and non-clear cell RCC (non-ccRCC); however, treatment response to medications varies which necessitates accurate differentiation between the two. Therefore, we aimed to identify a novel serum marker of RCC. Increased LRG1 expression in the serum has been demonstrated in multiple cancer types. However, the expression of LRG1 expression in the serum and cancer tissues of patients with RCC has not been reported. Since ccRCC is a hypervascular tumor and LRG1 is capable of accelerating angiogenesis, we hypothesized that the LRG1 levels may be related to ccRCC. Therefore, we examined LRG1 expression in sera from patients with RCC.MethodsUsing an enzyme-linked immunosorbent assay, serum levels of leucine-rich-alpha-2-glycoprotein 1 (LRG1) were measured in 64 patients with ccRCC and 22 patients non-ccRCC who underwent radical or partial nephrectomy, as well as in 63 patients without cancer.ResultsMedian values of serum LRG1 and their inter-quartile ranges were 63.2 (42.8–94.2) µg/mL in ccRCC, 23.4 (17.7–29.6) µg/mL in non-ccRCC, and 36.0 (23.7–56.7) µg/mL in patients without cancer, respectively (ccRCC vs. non-ccRCC or patients without cancer: P < 0.001). C-reactive protein (CRP) levels (P = 0.002), anemia (P = 0.037), hypercalcemia (P = 0.023), and grade (P = 0.031) were independent predictors of serum LRG1 levels in ccRCC. To assess diagnostic performance, the area under the receiver operating characteristic curve of serum LRG1 was utilized to differentiate ccRCC from non-cancer and non-ccRCC, with values of 0.73 (95% CI, 0.64–0.82) and 0.91 (95% CI, 0.82–0.96), respectively.ConclusionsLRG1 served as a serum marker associated with inflammation, indicated by CRP, anemia, hypercalcemia, and malignant potential in ccRCC. Clinically, serum LRG1 levels may assist in differentiating ccRCC from non-ccRCC with excellent diagnostic accuracy.

LRG1 predicts the prognosis and is associated with immune infiltration in thyroid cancer: a bioinformatics study.

The diagnostic and prognostic value of the leucine-rich alpha-2-glycoprotein 1 (LRG1) gene in thyroid cancer remains unclear. Using the Cancer Genome Atlas (TCGA) database, we conducted a bioinformatics analysis to determine the role of LRG1 in thyroid cancer. Data from 512 patients with thyroid cancer and 59 normal individuals were collected from TCGA database. The Kruskal-Wallis test and logistic analysis were used to examine the relationship between LRG1 expression and clinicopathologic characteristics. Cox regression and Kaplan-Meier analysis were used to determine the predictive value of LRG1 on clinical outcomes. Single-sample gene set enrichment analysis (ssGSEA) was used to reveal associations between LRG1 expression and immune infiltration levels in thyroid cancer. LRG1 was highly expressed in thyroid cancer (P < 0.001) and could effectively distinguish tumor tissue (area under the curve = 0.875) from normal tissue. Moreover, LRG1 was significantly correlated with pathological N stage (odds ratio (OR) = 2.411 (1.659-3.505), P < 0.001). Kaplan-Meier survival analysis revealed that patients with high LRG1 expression had better overall survival (hazard ratio (HR) = 0.30, P = 0.038). Cox regression analysis indicated that pathological M stage was a risk factor for progression-free interval (HR = 5.964 (2.010-17.694), P < 0.001). Using ssGSEA, we found that LRG1 expression was positively correlated with the number of T helper 1 cells (R = 0.435, P < 0.001), dendritic cells (R = 0.442, P < 0.001), and macrophages (R = 0.459, P < 0.001). LRG1 may be an important biomarker for predicting the prognosis of thyroid cancer and represent a suitable target for immunotherapy associated with immune infiltration.

Correlation between LRG1 and Adipokines in Psoriasis

Background and Objective. Patients with psoriasis may exhibit abnormal changes in serum adipokine levels, which are often related to disease severity of the disease. Leucine-rich alpha-2-glycoprotein 1 (LRG1) is an acute-phase inflammatory protein that may be linked to adipokines in psoriasis. In this study, we evaluated the differences in the expression of adipokines and LRG1 between patients with psoriasis and healthy individuals, analyzed the correlation between the expression of LRG1 and adipokines, and explored their relationship with psoriatic lesions. Methods. In this cross-sectional study, patients with psoriasis (n = 54) and healthy controls (n = 26) were enrolled, and their clinical characteristics were recorded. Fasting venous blood samples were collected from each participant. The serum concentrations of leptin, resistin, adiponectin, and LRG1 in each sample were measured using the enzyme-linked immunosorbent assay. Results. The study included 54 patients with psoriasis vulgaris and 26 healthy controls. The serum levels of LRG1, leptin, and resistin were significantly higher in patients with psoriasis than in healthy controls. Conversely, adiponectin levels were significantly lower in patients with psoriasis. The study showed that LRG1 expression was positively correlated with leptin and resistin expression but negatively correlated with adiponectin expression. Interestingly, only leptin, resistin, and LRG1 expression showed a linear correlation with the Psoriasis Area and Severity Index (PASI). When we categorized patients with psoriasis based on their LRG1 levels, we observed that the group with high LRG1 levels showed a higher PASI. Conclusions. We observed a significant correlation between LRG1 and adipokine expression in patients with psoriasis. In addition, the expression levels of LRG1, leptin, and resistin were observed to be correlated with the severity of psoriasis. We believe that the occurrence and development of psoriasis are collectively influenced by LRG1 and leptin/resistin expression.

Expression of LRG-1 in mice with hypertensive renal damage and its significance.

Long-term elevated blood pressure may lead to kidney damage, yet the pathogenesis of hypertensive kidney damage is still unclear. This study aims to explore the role and significance of leucine-rich alpha-2-glycoprotein-1 (LRG-1) in hypertensive renal damage through detecting the levels of LRG-1 in the serum and kidney of mice with hypertensive renal damage and its relationship with related indexes. C57BL/6 mice were used in this study and randomly divided into a control group, an angiotensin II (Ang II) group, and an Ang II+irbesartan group. The control group was gavaged with physiological saline. The Ang II group was pumped subcutaneously at a rate of 1.5 mg/(kg·d) for 28 days to establish the hypertensive renal damage model in mice, and then gavaged with equivalent physiological saline. The Ang II+irbesartan group used the same method to establish the hypertensive renal damage model, and then was gavaged with irbesartan. Immunohistochemistry and Western blotting were used to detect the expression of LRG-1 and fibrosis-related indicators (collagen I and fibronectin) in renal tissues. ELISA was used to evaluate the level of serum LRG-1 and inflammatory cytokines in mice. The urinary protein-creatinine ratio and renal function were determined, and correlation analysis was conducted. Compared with the control group, the levels of serum LRG-1, the expression of LRG-1 protein, collagen I, and fibronectin in kidney in the Ang II group were increased (all P<0.01). After treating with irbesartan, renal damage of hypertensive mice was alleviated, while the levels of LRG-1 in serum and kidney were decreased, and the expression of collagen I and fibronectin was down-regulated (all P<0.01). Correlation analysis showed that the level of serum LRG-1 was positively correlated with urinary protein-creatinine ratio, blood urea nitrogen, and blood creatinine level in hypertensive kidney damage mice. Serum level of LRG-1 was also positively correlated with serum inflammatory factors including TNF-α, IL-1β, and IL-6. Hypertensive renal damage mice display elevated expression of LRG-1 in serum and kidney, and irbesartan can reduce the expression of LRG-1 while alleviating renal damage. The level of serum LRG-1 is positively correlated with the degree of hypertensive renal damage, suggesting that it may participate in the occurrence and development of hypertensive renal damage.

Alterations of plasma exosomal proteins and motabolies are associated with the progression of castration-resistant prostate cancer

BackgroundCurrent diagnosis tools for prostate cancer (PCa) such as serum PSA detection and prostate biopsy cannot distinguish dormant tumors from invasive malignancies, either be used as prognosis marker for castration resistant prostate cancer (CRPC), the lethal stage of PCa patients. Exosomes have been widely investigated as promising biomarkers for various diseases. We aim to characterize the proteomic and metabolomic profile of exosomes and to evaluate their potential value for the diagnosis of PCa, especially CRPC. We also investigate the functions of some specific exosome biomarkers in the progression of CRPC.MethodsIntegrated proteomics and metabolomics analysis were performed for plasma-derived exosomes collected from tumor-free controls (TFC), PCa and CRPC patients. Expression of specific exosomal proteins were further validated by targeted 4D-parallel reaction monitoring (PRM) mass spectrometry among the three cohorts. Tissue distribution and functional role of exosomal protein LRG1 was studied in clinical PCa tissue samples and cell line models.ResultsThree potential exosomal protein markers were identified. The apolipoprotein E level in PCa samples was 1.7-fold higher than that in TFC (receiver operating characteristic value, 0.74). Similarly, the levels of exosome-derived leucine-rich alpha2-glycoprotein 1 (LRG1) and inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) in the CRPC group were 1.7 and 2.04 times, respectively, higher than those in the PCa group (ROC values, 0.84 and 0.85, respectively), indicating that LRG1 and ITIH3 could serve as predictive markers for CRPC. For metabolomic evaluation of exosomes, a series of differentially expressed metabolites were identified, and a combined metabolite panel showed ROC value of 0.94 for distinguishing PCa from TFC and 0.97 for distinguishing CRPC from PCa. Immunohistochemistry of tissue microarray showed that LRG1 protein was significantly upregulated in advanced prostate cancer and functional assay revealed that ectopic expression of LRG1 can significantly enhance the malignant phenotype of prostate cancer cells. More importantly, PCa cell derived LRG1-overexpressed exosomes remarkably promoted angiogenesis.ConclusionIntegration of proteomics and metabolomics data generated proteomic and metabolic signatures of plasma exosomes that may facilitate discrimination of CRPC from PCa and TFC patients, suggesting the potential of exosomal proteins and metabolites as CRPC markers. The study also confirmed the important role of exosomal protein LRG1 in PCa malignant progression.

PS-B01-5: TISSUE-SPECIFIC REGULATION OF LRG1 EXPRESSION IN MULTIPLE HYPERTENSION MODELS OF MOUSE

Object: Leucine-rich alpha-2-glycoprotein-1 (LRG1), which is firstly isolated from human plasma, has been reported to be related to several diseases as a biomarker or a pathogenic factor. Serum LRG1 is associated with the prognosis of diabetic nephropathy and cardiovascular diseases, and we previously demonstrated that LRG1 especially plays a pivotal role in the initial development of diabetic nephropathy. In addition, a previous study showed that increased plasma LRG1 levels in patients with type 2 diabetes mellitus was associated with increased systolic blood pressure (BP), arterial stiffness estimated by pulse wave velocity, and decreased endothelium-dependent vasodilation estimated by laser doppler flowmetry. However, the relationship between LRG1 and BP have remained substantially unknown. With respect to the function of LRG1, LRG1 is expressed in endothelial cells and promotes pathological angiogenesis by modulating TGF-beta signaling. LRG1 switches TGF-beta signaling from the Smad2/3 pathway to the Smad1/5/8 pathway. In addition, the activation of Smad2/3 signaling enhanced endothelial nitric oxide synthase (eNOS) production and promoted endothelium-dependent vasodilation. Suppressing eNOS activity reduces NO levels and leads to endothelial dysfunction and hypertension. Moreover, LRG1 is increased in response to inflammation and oxidative stress in endothelial cells. However, little is known about the relationship between LRG1 in endothelial cells and eNOS. Based on the previous findings, we hypothesized that LRG1 may inhibit eNOS production by potentially blocking the Smad2/3 pathway, which will promote to form a vicious cycle that progresses vascular endothelial damage and hypertension. As the first step to elucidate this hypothesis, we investigated LRG1 expression in various tissues including vascular endothelium in some hypertension models with endothelial dysfunction. Design and method: Male 10–12 weeks-aged C57/BL mice were divided into control and hypertension with endothelial dysfunction. BP was measured using a tail-cuff method during the experiment, and mice were sacrificed and their organs were collected at the end of the experiment. We examined the expression of LRG1, Smad2/3, Smad1/5/8 and NOS in the vascular endothelium, kidney, heart, adipose tissue by PCR, Western blot and immunohistochemical analyses. Results: We are currently conducting the experiments and will present our findings at this conference. Conclusion: We plan to present the results of tissue-specific regulation of LRG1 expression in some hypertension models of mice at this conference.

LRG1 in pancreatic cancer cells promotes inflammatory factor synthesis and the angiogenesis of HUVECs by activating VEGFR signaling.

This study aimed to investigate the roles of leucine-rich alpha-2-glycoprotein 1 (LRG1) in regulating angiogenesis during pancreatic cancer (PC) pathogenesis. LRG1 expression in tissues was detected by qRT-PCR and immunohistochemistry. LRG1 in BxPC-3 and Capan-2 cells was knocked down or overexpressed. Cell viability and the migration and invasion abilities of cells were analyzed using the Cell Counting Kit-8 (CCK-8) assay and Transwell system, respectively. Interleukin-1 beta (IL-1β), IL-18, and vascular endothelial growth factor A (VEGFA) contents in cell culture were measured by ELISA, and the angiogenesis of HUVECs was assessed by the in vitro tube formation assay. In vitro LRG1 expression in BxPC-3 and Capan-2 cells was determined using immunofluorescence. The results showed that LRG1 expression was significantly increased in pancreatic cancer tissues and cell lines. LRG1 knockdown inhibited the viability, migration, invasion, and IL-1β and IL-18 synthesis of BxPC-3 and Capan-2 cells. VEGFA synthesis in BxPC-3 and Capan-2 cells was also inhibited by LRG1 knockdown, which caused impaired tube formation of co-cultured HUVECs. LRG1 overexpression enhanced the viability, migration, and invasion of BxPC-3 and Capan-2 cells, also causing elevated tube formation of HUVECs and IL-1β and IL-18 synthesis in co-cultures of HUVECs and BxPC-3 or Capan-2 cells. Silencing of VEGF receptor (VEGFR) abrogated the enhanced tube formation and IL-1β and IL-18 synthesis in HUVECs co-cultured with BxPC-3 or Capan-2 cells overexpressing LRG1. In conclusion, LRG1, which is highly expressed in pancreatic cancer cells, promotes inflammatory factor synthesis and the angiogenesis of HUVECs though activating the VEGFR signaling pathway.

Colorectal cancer-associated fibroblasts promote metastasis by up-regulating LRG1 through stromal IL-6/STAT3 signaling

Cancer-associated fibroblasts (CAFs) have been shown to play a strong role in colorectal cancer metastasis, yet the underlying mechanism remains to be fully elucidated. Using CRC clinical samples together with ex vivo CAFs-CRC co-culture models, we found that CAFs induce expression of Leucine Rich Alpha-2-Glycoprotein 1(LRG1) in CRC, where it shows markedly higher expression in metastatic CRC tissues compared to primary tumors. We further show that CAFs-induced LRG1 promotes CRC migration and invasion that is concomitant with EMT (epithelial-mesenchymal transition) induction. In addition, this signaling axis has also been confirmed in the liver metastatic mouse model which displayed CAFs-induced LRG1 substantially accelerates metastasis. Mechanistically, we demonstrate that CAFs-secreted IL-6 (interleukin-6) is responsible for LRG1 up-regulation in CRC, which occurs through a direct transactivation by STAT3 following JAK2 activation. In clinical CRC tumor samples, LRG1 expression was positively correlated with CAFs-specific marker, α-SMA, and a higher LRG1 expression predicted poor clinical outcomes especially distant metastasis free survival, supporting the role of LRG1 in CRC progression. Collectively, this study provided a novel insight into CAFs-mediated metastasis in CRC and indicated that therapeutic targeting of CAFs-mediated IL-6-STAT3-LRG1 axis might be a potential strategy to mitigate metastasis in CRC.

LRG1 is an adipokine that mediates obesity-induced hepatosteatosis and insulin resistance.

Dysregulation in adipokine biosynthesis and function contributes to obesity-induced metabolic diseases. However, the identities and functions of many of the obesity-induced secretory molecules remain unknown. Here, we report the identification of leucine-rich alpha-2-glycoprotein 1 (LRG1) as an obesity-associated adipokine that exacerbates high fat diet-induced hepatosteatosis and insulin resistance. Serum levels of LRG1 were markedly elevated in obese humans and mice compared with their respective controls. LRG1 deficiency in mice greatly alleviated diet-induced hepatosteatosis, obesity, and insulin resistance. Mechanistically, LRG1 bound with high selectivity to the liver and promoted hepatosteatosis by increasing de novo lipogenesis and suppressing fatty acid β-oxidation. LRG1 also inhibited hepatic insulin signaling by downregulating insulin receptor substrates 1 and 2. Our study identified LRG1 as a key molecule that mediates the crosstalk between adipocytes and hepatocytes in diet-induced hepatosteatosis and insulin resistance. Suppressing LRG1 expression and function may be a promising strategy for the treatment of obesity-related metabolic diseases.

LRG1 promotes epithelial-mesenchymal transition of retinal pigment epithelium cells by activating NOX4.

To investigate the effect of leucine-rich-alpha-2-glycoprotein 1 (LRG1) on epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE) cells, and to explore the role of NADPH oxidase 4 (NOX4). RPE cells (ARPE-19 cell line) were treated with transforming growth factor-β1 (TGF-β1) to induce EMT. Changes of the mRNA and protein expression levels of LRG1 were tested in the TGF-β1 treated cells. The recombinant human LRG1 protein (rLRG1) and siRNA of LRG1 were used to establish accumulation of exogenous LRG1 model and the down-regulation of LRG1 model in ARPE-19 cells respectively, and to detect EMT-related markers including fibronectin, α-smooth muscle actin (α-SMA) and zonula occludens-1 (ZO-1). The mRNA and protein expression level of NOX4 were measured according to the above treatments. VAS2870 was used as a NOX4 inhibitor in rLRG1-treated cells. EMT-related markers were detected to verify the effect of NOX4 in the process of EMT. TGF-β1 promoted the expression of LRG1 at both the mRNA and protein levels during the process of EMT which showed the up-regulation of fibronectin and α-SMA, as well as the down-regulation of ZO-1. Furthermore, the rLRG1 promoted EMT of ARPE-19 cells, which manifested high levels of fibronectin and α-SMA and low level of ZO-1, whereas knockdown of LRG1 prevented EMT by decreasing the expressions of fibronectin and α-SMA and increasing the expression of ZO-1 in ARPE-19 cells. Besides, the rLRG1 activated and LRG1 siRNA suppressed NOX4 expression. EMT was inhibited when VAS2870 was used in the rLRG1-treated cells. These results for the first time demonstrate that LRG1 promotes EMT of RPE cells by activating NOX4, which may provide a novel direction to explore the mechanisms of subretinal fibrosis.

LRG1 promotes hypoxia-induced cardiomyocyte apoptosis and autophagy by regulating hypoxia-inducible factor-1α

ABSTRACT Cardiomyocyte apoptosis and autophagy play important roles in acute myocardial infarction (AMI), but the effect of leucine-rich alpha-2-glycoprotein 1 (LRG1) on the apoptosis and autophagy of H9c2 has not yet been reported. It was found through differential gene analysis and LASSO analysis that LRG1 was the key gene in AMI. In this study, western blot was applied to detect the protein expression of Bax, Bcl2, LC3, p62, LRG1 and hypoxia-inducible factor-1α (HIF-1α); CCK-8 assay was employed to detect cell viability; Annexin V-FITC/PI staining was adopted to evaluate apoptosis, and immunofluorescence assay was applied to detect autophagy. Under hypoxia conditions in H9c2 cells, LRG1 protein levels were increased, the cell activity was decreased, and apoptosis and autophagy were promoted; the downregulated LRG1 significantly enhanced cell viability but inhibited apoptosis and autophagy. When knocking down HIF-1α in the overexpressed LRG1 cells, the effects of LRG1 were reversed under hypoxia condition. In conclusion, LRG1/HIF-1α promoted H9c2 cell apoptosis and autophagy in hypoxia, potentially providing new ideas for the determination and treatment of AMI. Abbreviation: LRG1: Leucine-rich alpha-2-glycoprotein 1; LRR: leucine-rich repeat; HIF-1α: Hypoxia-inducible factor-1α; AMI: acute myocardial infarction

Intracellular leucine-rich alpha-2-glycoprotein-1 competes with Apaf-1 for binding cytochrome c in protecting MCF-7 breast cancer cells from apoptosis.

Leucine-rich alpha-2-glycoprotein-1 (LRG1) has been shown to compete with apoptosis activating factor-1 (Apaf-1) for binding cytochrome c (Cyt c) and could play a role in inhibition of apoptosis. Employing MCF-7 breast cancer cells, we report that intracellular LRG1 does protect against apoptosis. Thus, cells transfected with the lrg1 gene and expressing higher levels of LRG1 were more resistant to hydrogen peroxide-induced apoptosis than parental cells, while cells in which LRG mRNA was knocked down by short hairpin (sh) RNA-induced degradation were more sensitive. The amount of Cyt c co-immunoprecipitated with Apaf-1 from the cytosol of apoptotic cells was inversely related to the level of LRG1 expression. In lrg1-transfected cells partially-glycosylated LRG1 was found in the cytosol and there was an increase in cytosolic Cyt c in live lrg1-transfected cells relative to parental cells. However, apoptosis was not spontaneously induced because Cyt c was bound to LRG1 and not to Apaf-1. Cyt c was the only detectable protein co-immunoprecipitated with LRG1. Following hydrogen peroxide treatment degradation of LRG1 allowed for induction of apoptosis. We propose that intracellular LRG1 raises the threshold of cytoplasmic Cyt c required to induce apoptosis and, thus, prevents onset of the intrinsic pathway in cells where Cyt c release from mitochondria does not result from committed apoptotic signaling. This mechanism of survival afforded by LRG1 is likely to be distinct from its extracellular survival function that has been reported by several research groups.

Prognostic Value of LRG1 in Breast Cancer: A Retrospective Study

Background: High expression of leucine-rich alpha-2-glycoprotein 1 (LRG1) is closely related to angiogenesis, which may play an important role in promoting invasion and metastasis. However, the current literature has yet to clarify the clinical significance of LRG1 in breast cancer. Objectives: The purpose of this work was to validate the correlation between LRG1 expression and prognosis in early breast cancer. Methods: We utilized an LRG1 detection agent in 330 cases of early breast cancer. The correlation of LRG1 expression with clinicopathological features, patient recurrence, and survival was investigated. Results: Compared with adjacent tissue samples, an elevated expression of LRG1 was observed in breast cancer samples. Moreover, LRG1 expression is associated with the number of lymphatic metastases and TNM pathological stage (p = 0.000, p = 0.000, respectively). For disease-free survival (DFS), the Kaplan-Meier curve indicated a poorer prognosis for the group with high LRG1 levels compared with the low LRG1 group (p = 0.000). A similar result was found for overall survival (OS; p = 0.000). The multivariate Cox regression indicated that LRG1 was still associated with DFS (HR 2.090, 95% CI 1.205–3.625, p = 0.009) and OS (HR 2.112, 95% CI 1.167–3.822, p = 0.013). The histological grade, TNM pathological stage, and molecular subtype were identified as independent risk factors affecting OS. Conclusion: In the malignant progression of breast cancer, high LRG1 levels are associated with lymphatic metastasis, histological grade, poor DFS, and poor OS. This study validates the use of LRG1 as a potential prognosis biomarker for early breast cancer.

MicroRNA-497 elevation or LRG1 knockdown promotes osteoblast proliferation and collagen synthesis in osteoporosis via TGF-β1/Smads signalling pathway.

MicroRNAs (miRNAs) have been corroborated to engage in the process of cellular activities in osteoporosis. However, few researches have been conducted to expose the integrated role of miR‐497, leucine‐rich alpha‐2‐glycoprotein‐1 (LRG1) and transforming growth factor beta 1 (TGF‐β1)/Smads signalling pathway in osteoporosis. Thereafter, the study is set out to delve into miR‐497/LRG1/TGF‐β1/Smads signalling pathway axis in osteoporosis. Osteoporosis bone tissues and normal bone tissues were collected. Rat osteoporosis models were constructed via ovariectomy. Model rats were injected with restored miR‐497 or depleted LRG1 to explore their roles in osteoporosis. Rat osteoblasts were extracted from osteoporosis rats and transfected with restored miR‐497 or depleted LRG1 for further verification. MiR‐497 and LRG1 expression in femoral head tissues and osteoblasts of osteoporosis rats were detected. TGF‐β1/Smads signalling pathway‐related factors were detected. MiR‐497 was poorly expressed while LRG1 was highly expressed and TGF‐β1/Smads signalling pathway activation was inhibited in osteoporosis. MiR‐497 up‐regulation or LRG1 down‐regulation activated TGF‐β1/Smads signalling pathway, promoted collagen type 1 synthesis and suppressed oxidative stress in femoral head tissues in osteoporosis. MiR‐497 restoration or LRG1 knockdown activated TGF‐β1/Smads signalling pathway, promoted viability and suppressed apoptosis of osteoblasts in osteoporosis. Our study suggests that miR‐497 up‐regulation or LRG1 down‐regulation promotes osteoblast viability and collagen synthesis via activating TGF‐β1/Smads signalling pathway, which may provide a novel reference for osteoporosis treatment.

The Clinical Prognostic Value of LRG1 in Esophageal Squamous Cell Carcinoma.

Leucine-rich-alpha-2-glycoprotein1 (LRG1) is a new oncogene-related gene, which has been proven important for the development and poor prognosis of human cancers. However, whether it participates in esophageal squamous cell carcinoma (ESCC) progression remains unclear. To investigate the expression level and functional influence of LRG1 in ESCC. The expression of LRG1 was evaluated on the mRNA and protein level in ESCC patients. Then, correlation of LRG1 expression with clinicpathological variables was analyzed in ESCC. Besides, to clarify the biological function of LRG1, Eca109 and KYSE150 cells were transfected with LRG1 shRNA, the cell viability, clonal efficiency, apoptosis and invasion assays in vitro were performed. LRG1 was significantly over-expressed in ESCC and related to deeper invasion depth (T stage) and distal metastasis (M stage). Kaplan-Meier analysis indicated that LRG1 up-regulation in ESCC was closely correlated to worse clinical survival (overall survival and progression-free survival), all P<0.001. LRG1 was confirmed to be an independent poor premonitory indicator for clinical outcomes in ESCC through the univariate and multivariate analyses. Down-regulation of LRG1 in ESCC cells markedly suppressed cell proliferation and invasion, stimulated apoptosis (all p <0.01). LRG1 might play a significant role in the progression of ESCC, and could be served as a promising prognostic prediction for ESCC patients.

Mechanical strain promotes skin fibrosis through LRG-1 induction mediated by ELK1 and ERK signalling

Biomechanical force and pathological angiogenesis are dominant features in fibro-proliferative disorders. Understanding the role and regulation of the mechanical microenvironment in which pathological angiogenesis occurs is an important challenge when investigating numerous angiogenesis-related diseases. In skin fibrosis, dermal fibroblasts and vascular endothelial cells are integral to hypertrophic scar formation. However, few studies have been conducted to closely investigate their relationship. Here we show, that leucine-rich-alpha-2-glycoprotein 1 (LRG-1) a regulator of pathological angiogenesis, links biomechanical force to angiogenesis in skin fibrosis. We discover that LRG-1 is overexpressed in hypertrophic scar tissues, and that depletion of Lrg-1 in mouse skin causes mild neovascularization and skin fibrosis formation in a hypertrophic scarring model. Inhibition of FAK or ERK attenuates LRG-1 expression through the ELK1 transcription factor, which binds to the LRG-1 promoter region after transcription initiation by mechanical force. Using LRG-1 to uncouple mechanical force from angiogenesis may prove clinically successful in treating fibro-proliferative disorders.

1388-P: Umbilical Cord Blood-Derived Exosomal LRG1 and ECM1 Induce Abnormal Placental Angiogenesis in Women with Gestational Diabetes Mellitus

Background: Gestational diabetes mellitus (GDM) is characterized by a high risk of fetal macrosomia and placenta hypervascularization. There is a need to investigate the underlying mechanism to explain these associate complications. Exosomes had been known participating in the regulation of various angiogenic processes. However, the effects of exosomes from cases of GDM on placental vascular network formation remain unclear. Methods and Results: Capillary branching per placental villi and protein level of CD31 were identified to be increased in GDM. Exosomes derived from umbilical cord blood (UE) enhanced endothelial cell vascularization significantly, especially in GDM pregnancies. The proteome of 10 normal exosomes and 10 GDM exosomes showed different profiles and uncovered two up-regulated proteins in GDM cases, namely leucine-rich alpha-2-glycoprotein-1 (LRG1) and extracellular matrix protein 1 (ECM1). Genetically engineered exosomes that overexpressed LRG1 or ECM1 resulted in hyper vascularization, including proliferation and migration. Conclusions: Our results demonstrate for the first time the expression of LRG1 and ECM1 in normal and GDM UE and their role in regulating pathological placental angiogenesis. These findings support the potential role of LRG1 and ECM1 in monitoring placental pathologies in this increasingly prevalent condition and novel insights into the causes. Disclosure J. Yao: None. T. Duan: None. K. Wang: None. Funding National Natural Science Foundation of China; National Key Research and Development Program of China

LRG‑1 enhances the migration of thyroid carcinoma cells through promotion of the epithelial‑mesenchymal transition by activating MAPK/p38 signaling.

Leucine-rich-alpha-2-glycoprotein 1 (LRG-1) has been reported to be associated with multiple malignancies. However, its participation in thyroid carcinoma progression remains unclear. In the present study, the biological function and underlying molecular mechanisms of LRG-1 in thyroid carcinoma were investigated. It was found that LRG-1 was overexpressed in thyroid carcinoma tissues, and high LRG-1 expression predicted poor patient survival and late tumor stage. As shown in the mouse xenograft study, knockdown of LRG-1 significantly attenuated thyroid cancer growth in vivo. Based on wound healing, Transwell, proliferation and apoptosis assays, it was found that the knockdown of LRG-1, using shLRG-1, inhibited cell migration and invasion, but did not affect proliferation and apoptosis in thyroid cancer cells. Furthermore, LRG-1 also induced epithelial-mesenchymal transition (EMT) in thyroid carcinoma cells. Western blot analysis revealed that this tumor-promoting bioactivity of LRG-1 was attributed to its selective activation of MAPK/p38 signaling. All of these findings indicate that LRG-1 plays a deleterious role in the progression of thyroid carcinoma. LRG-1 may serve as a promising biomarker for predicting prognosis in thyroid carcinoma patients, and LRG-1-based therapy may be developed into a novel strategy for the treatment of thyroid carcinoma.

LRG-1 promotes pancreatic cancer growth and metastasis via modulation of the EGFR/p38 signaling

BackgroundThe abnormal expression of leucine-rich-alpha-2-glycoprotein 1 (LRG-1) is reported to be associated with multiple malignancies, but its role in the progression of pancreatic ductal adenocarcinoma (PDAC) remains to be determined.MethodsThe expression of LRG-1 was assessed in PDAC tissues by RT-PCR, Western blot and immunohistochemistry. LRG-1-silenced or overexpressed cell lines were constructed using shRNA or LRG-1-overexpressing plasmids. EdU incorporation assay, Transwell invasion and wound-healing assays were performed to evaluate the proliferation, invasion and migration of PDAC cells. In addition, protein expression of the mitogen-activated protein kinase (MAPK) pathway was detected using Western blot. Finally, Co-immunoprecipitation assay was conducted in search of the potential interaction between LRG-1 and epidermal growth factor receptor (EGFR).ResultsThe expression of LRG-1 in PDAC tissue was significantly higher than that in adjacent normal tissue, and high LRG-1 expression predicted poor survival and a late tumor stage. In addition, LRG-1 markedly promoted the viability, proliferation, migration and invasion of PDAC cells in vitro and facilitated tumor growth in vivo. More importantly, we revealed that these bioactivities of LRG-1 might result from its selective interaction with EGFR, which might further activate the p38/MAPK signaling pathways.ConclusionLRG-1 may prove to be a promising biomarker for predicting prognosis of PDAC patients. Inhibition of LRG-1 or its downstream pathway could be a potential therapeutic target for the treatment of PDAC.

Leucine-Rich Alpha-2-Glycoprotein1 Gene Interferes with Regulation of Apoptosis in Leukemia KASUMI-1 Cells.

BackgroundLeukemia cells have strong proliferation and anti-apoptosis capabilities. The purpose of this study was to investigate the effect of silencing the leucine-rich alpha-2-glycoprotein1 (LRG1) gene, which was found to regulate tumor proliferation and apoptosis in acute myeloid leukemia (AML) cell lines.Material/MethodsPlasmid interference technique was used to silence the LRG1 gene in the KASUMI-1 cell line. The cell counting kit-8 (CCK-8) assay was used to test the effect of transduction on cell viability. Cell cycle and apoptosis were detected by flow cytometry. Western blot and quantitative real-time polymerase chain reaction (RT-qPCR) were applied to detect the expression levels of proteins and mRNA, respectively.ResultsKASUMI-1 cells with the CD34+CD38− phenotype were sorted by flow cytometry. After transfection of the siLRG1 plasmid, the level of LRG1 expression was downregulated and cell viability was reduced. Silencing of LRG1 gene blocked KASUMI-1 cells in G0/G1 phase and promoted apoptosis. Further experiments found that LRG1 gene silencing significantly downregulated cell cycle-associated proteins and anti-apoptotic proteins, while upregulating pro-apoptotic proteins. Downregulation of LRG1 gene expression also inhibits signal transduction of the JAK-STAT pathway.ConclusionsLRG1 gene silencing regulates the expression of cyclin and apoptosis-related proteins to reduce cell viability and promote apoptosis, probably through inhibition of the JAK-STAT pathway.