Constructing an artificial in vitro multi-enzyme cascade pathway to convert glycerol and CO2 into L-aspartic acid.

Developing utilization technologies for biomass resources, exploring their applications in the fields of energy and chemical engineering, holds significant importance for promoting sustainable development and constructing a green, low-carbon society. In this study, we designed a non-natural in vitro multi-enzyme system for converting glycerol and CO2 into L-aspartic acid (L-Asp). The coupled system utilized eight enzymes, including alditol oxidase (ALDO), catalase-peroxidase (CAT), lactaldehyde dehydrogenase (ALDH), glycerate 2-kinase (GK), phosphopyruvate hydratase (PPH), phosphoenolpyruvate carboxylase (PPC), L-aspartate dehydrogenase (ASPD), and polyphosphate kinase (PPK), to convert the raw materials into L-Asp in one-pot coupled with NADH and ATP regeneration. Under optimal reaction conditions, 18.6 mM of L-Asp could be produced within 2.0 h at a total enzyme addition of 4.85 mg/mL, demonstrating the high efficiency and productivity characteristics of the designed system. Our technological application provides new insights and methods for the development of biomass resource utilization technologies.

Cholesterol uptake in the intestine is regulated by the LASP1-AKT-NPC1L1 signaling pathway.

Cholesterol is essential for the stability and architecture of the plasma membrane and a precursor of bile acids and steroid hormones in mammals. Excess dietary cholesterol uptake leads to hypercholesterolemia and atherosclerosis and plays a role in cancer development. The role of actin-binding scaffolding protein LIM and SH3 protein 1 (LASP1) in cholesterol trafficking has not been investigated previously. Cholesterol levels, its uptake, and excretion were studied in mice deficient for low-density lipoprotein receptor and Lasp1 (Ldlr-/-Lasp1-/- mice) upon feeding a high-fat diet, and in LASP1-knockdown, differentiated human intestinal epithelial CaCo-2 cells. When compared with diet-fed Ldlr-/- control mice, Ldlr-/-Lasp1-/- mice displayed a reduction in serum cholesterol levels. Mechanistically, we identified a new role of LASP1 in controlling the translocation of the intestinal cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1) to the apical cell surface, which was limited in LASP1-knockdown human CaCo-2 enterocytes and in the intestine of Ldlr-/- Lasp1-/- compared with Ldlr-/- mice, linked to LASP1-pAKT signaling but not CDC42 activation. In line, a reduction in cholesterol reabsorption was noted in LASP1-knockdown CaCo-2 cells in vitro, and an enhanced cholesterol excretion via the feces was observed in Ldlr-/- Lasp1-/- mice. These data uncover a novel function of Lasp1 in cholesterol trafficking, promoting cholesterol reabsorption in the intestine. Targeting LASP1 locally could thus represent a novel targeting strategy to ameliorate hypercholesterolemia and associated diseases.NEW & NOTEWORTHY We here uncovered LASP1 as a novel regulator of the shuttling of the sterol transporter NPC1L1 to the cell surface in enterocytes to control cholesterol absorption. Accordingly, LASP1-deficient mice displayed lowered serum cholesterol levels under dietary cholesterol supplementation.

Inhibition of GLUD1 mediated by LASP1 and SYVN1 contributes to hepatitis B virus X protein-induced hepatocarcinogenesis.

Glutamate dehydrogenase 1 (GLUD1) is implicated in oncogenesis. However, little is known about the relationship between GLUD1 and hepatocellular carcinoma (HCC). In the present study, we demonstrated that the expression levels of GLUD1 significantly decreased in tumors, which was relevant to the poor prognosis of HCC. Functionally, GLUD1 silencing enhanced the growth and migration of HCC cells. Mechanistically, the upregulation of interleukin-32 through AKT activation contributes to GLUD1 silencing-facilitated hepatocarcinogenesis. The interaction between GLUD1 and AKT, as well as α-ketoglutarate regulated by GLUD1, can suppress AKT activation. In addition, LIM and SH3 protein 1 (LASP1) interacts with GLUD1 and induces GLUD1 degradation via the ubiquitin-proteasome pathway, which relies on the E3 ubiquitin ligase synoviolin (SYVN1), whose interaction with GLUD1 is enhanced by LASP1. In hepatitis B virus (HBV)-related HCC, the HBV X protein (HBX) can suppress GLUD1 with the participation of LASP1 and SYVN1. Collectively, our data suggest that GLUD1 silencing is significantly associated with HCC development, and LASP1 and SYVN1 mediate the inhibition of GLUD1 in HCC, especially in HBV-related tumors.

m6A-modified circ_0124554 promotes colorectal cancer progression and radioresistance through the miR-1184/LASP1 pathway.

BACKGROUND: Circular RNAs (circRNAs) are believed to regulate the progression of various cancers including colorectal cancer (CRC). However, the role and mechanism of circ_0124554 in regulating the sensitivity of CRC to radiation remain unknown. METHODS: The RNA levels of circ_0124554, LIM and SH3 protein 1 (LASP1), and methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit (METTL3) were detected by quantitative real-time polymerase chain reaction. Protein expression was checked by western blot. Cell proliferation, apoptosis, migration, and invasion were investigated by 5-Ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry analysis, and transwell assay, respectively. The sensitivity of CRC cells to radiation was analyzed by cell colony formation assay. Xenograft mouse model assay was conducted to disclose the role of circ_0001023 in the sensitivity of tumors to radiation in vivo. The binding relationships among circ_0124554, miR-1184 and LASP1 were confirmed by a dual-luciferase reporter assay. m6A RNA immunoprecipitation assay was performed to identify the association of METTL3 with circ_0124554. RESULTS: Circ_0124554 expression was upregulated in CRC tissues and cells in comparison with normal colorectal tissues and cells. Circ_0124554 knockdown inhibited proliferation, migration and invasion and promoted apoptosis and radiosensitivity of CRC cells. Moreover, circ_0124554 depletion inhibited tumor formation and improved radiosensitivity in vivo. MiR-1184 was identified as a target miRNA of circ_0124554 and targeted LASP1. Additionally, LASP1 overexpression rescued circ_0124554 knockdown-mediated effects in CRC cells. METTL3 mediated m6A methylation of circ_0124554. Further, circ_0124554 overexpression attenuated METTL3 depletion-induced effects in CRC cells. CONCLUSION: m6A-modified circ_0124554 promoted CRC progression and radioresistance by inducing LASP1 expression through interaction with miR-1184.

Knockdown of long non-coding RNA LINC01123 plays a molecular sponge on miR-625-5p to inhibit the process of colorectal cancer cells via LASP1.

Colorectal cancer (CRC) at an advanced stage of cancer has a lower 5-year survival rate. Research on the molecular biological mechanisms of CRC is helpful for disease prevention and treatment. Long non-coding RNAs (lncRNAs) were shown to be suitable as therapeutic targets for CRC. Previously, our research team found that LINC01123 promoted proliferation and metastasis in CRC by regulating miR-625-5p and the LIM and SH3 protein 1 (LASP1). Therefore, this study speculated that the molecular sponge effect of LINC01123 on miR-625-5p affected the process of CRC via regulating LASP1. The LINC01123-silenced CRC cell models (using the LOVO and SW480 cells) and xenograft tumor models were established to verify the above conjecture. As a result, it was found that silencing LINC01123 inhibited viability, proliferation, metastasis, and invasion but promoted apoptosis in LOVO and SW480 cells. Additionally, the knockdown of LINC01123 inhibited the LASP1, N-cadherin, PCNA, and Bcl-2 protein levels and raised the E-cadherin, Bax, and Caspase-3 protein levels in vitro. Furthermore, it showed that LINC01123, as a molecular sponge, targeted the miR-625-5p/LASP1 axis. The results of the xenograft tumor assay further verified the above effects of LINCO1123-silenced on tumor growth in vivo. And the miR-625-5p mimics treatment promoted the aforementioned effects of silencing LINC01123 on CRC cells while overexpressing LASP1 has an antagonistic effect to silencing LINC01123. In conclusion, this study suggests that silencing LINC01123 inhibits the process of CRC via sponging to the miR-625-5p/LASP1 axis. This finding hopes to provide research fundamentals on the biological mechanism study of CRC.

Silenced LASP1 interacts with DNMT1 to promote TJP2 expression and attenuate articular cartilage injury in mice by suppressing TJP2 methylation.

To investigate the regulatory mechanisms and effects of LIM and SH3 protein 1 (LASP1) on osteoarthritis (OA). IL-1ß was used to induce OA in cell models. Viability and apoptosis of chondrocytes were assessed. The expressions of tumor necrsis factor-α (TNF-α) and IL-6 were measured by ELISA kit, and Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were performed to test the expression of related proteins. The STRING database was used to predict the relationship between LASP1 and DNA methyltransferase 1 (DNMT1). The tight junction protein 2 (TJP2) and Gene Expression Omnibus data were analyzed for differential OA genes. Methylation-specific PCR detected methylation of the TJP2 promoter region, and chromatin immunoprecipitation detected the enrichment of DNMT1 in the TJP2 promoter region. Safranin O-Fast Green staining and hematoxylin and eosin staining were used to determine the OARSI score and evaluate the pathological conditions of the joint tissues. LASP1 was highly expressed in IL-1ß-induced cell models. Silencing of LASP1 promoted chondrocyte proliferation and expression of Collagen II and Aggrecan and inhibited chondrocyte apoptosis, inflammatory factors, and matrix metalloprotein expression. TJP2 is weakly expressed in OA models, and LASP1 promotes methylation of the TJP2 promoter region by interacting with DNMT1. Silencing of LASP1 attenuated IL-1ß-induced chondrocyte degeneration by promoting TJP2 expression. Similarly, silencing LASP1 promotes TJP2 expression to alleviate articular cartilage injury in mice with OA. Silencing of LASP1 inhibited the methylation of the TJP2 promoter region by interacting with DNMT1, thereby alleviating articular cartilage damage in OA mice.

Circular RNA circSLC7A11 contributes to progression and stemness of laryngeal squamous cell carcinoma via sponging miR-877-5p from LASP1.

Background: Laryngeal squamous cell carcinoma (LSCC) belongs to tumors of head and neck. Circular RNA circSLC7A11 functions as oncogenes in various tumors. However, the role of circSLC7A11 in LSCC remains largely unknown. Here, we aimed to clarify the circSLC7A11 function in LSCC. Methods: Relevance between circSLC7A11 expressions and LSCC clinicopathological was checked using chi-square. Relevance between circSLC7A11 expressions and LSCC patients' survival time was validated using Kaplan-Meier analysis. CircSLC7A11 expressions in LSCC tissues and cells were determined using quantitative real-time PCR. CircSLC7A11 functions in LSCC were examined by Cell Counting Kit-8, EdU analysis, Western blot, flow cytometry, sphere formation assay, and Transwell analysis. Meanwhile, circSLC7A11 mechanism in LSCC was determined using dual-luciferase reporter analysis, RNA pull-down, RNA Immunoprecipitation. Results: CircSLC7A11 was highly expressed in LSCC, and high circSLC7A11 expressions were interrelated to the TNM stage. Also, LSCC patients with high circSLC7A11 owned shorter overall survival. Functional studies revealed that circSLC7A11 knockdown reduced LSCC cell proliferation, migration, invasion, stemness characteristics, and enhanced cell apoptosis. Mechanistic study data corroborated that circSLC7A11 targeted miR-877-5p, miR-877-5p targeted LASP1. LASP1 was negatively interrelated to miR-877-5p and was positively interrelated to circSLC7A11 in LSCC tissues. Also, circSLC7A11 knockdown reduced the LASP1 levels, and miR-877-5p inhibitor co-transfection reversed this reduction. Rescue assays further demonstrated that circSLC7A11 accelerated LSCC through miR-877-5p/LASP1. Conclusion: CircSLC7A11 exerted oncogenic functions in LSCC by miR-877-5p/LASP1, hinting that circSLC7A11 was a novel biomarker for LSCC.

LASP1, CERS6, and Actin Form a Ternary Complex That Promotes Cancer Cell Migration.

CERS6 is associated with metastasis and poor prognosis in non-small cell lung cancer (NSCLC) patients through d18:1/C16:0 ceramide (C16 ceramide)-mediated cell migration, though the detailed mechanism has not been elucidated. In the present study, examinations including co-immunoprecipitation, liquid chromatography, and tandem mass spectrometry analysis were performed to identify a novel binding partner of CERS6. Among the examined candidates, LASP1 was a top-ranked binding partner, with the LIM domain possibly required for direct interaction. In accord with those findings, CERS6 and LASP1 were found to co-localize on lamellipodia in several lung cancer cell lines. Furthermore, silencing of CERS6 and/or LASP1 significantly suppressed cell migration and lamellipodia formation, whereas ectopic addition of C16 ceramide partially rescued those phenotypes. Both LASP1 and CERS6 showed co-immunoprecipitation with actin, with those interactions markedly reduced when the LASP1-CERS6 complex was abolished. Based on these findings, it is proposed that LASP1-CERS6 interaction promotes cancer cell migration.

L-asparaginase activity and anti-L-asparaginase antibody as biomarkers in estimating PEG-asp-related anaphylaxis risk in childhood acute lymphoblastic leukemia patients

Background: L-Asparaginase (L-asp), the unconjugated form of polyethylene glycol-conjugated L-asparaginase (PEG-asp), regulates T cell stimulation, antibody production, and lysosomal protease activity to mediate PEG-asp-related anaphylaxis. This study aimed to investigate the relation of L-asp activity and anti-L-asp antibody with anaphylaxis risk and non-anaphylaxis adverse reaction risk in childhood acute lymphoblastic leukemia (ALL) patients who underwent PEG-asp contained therapy. Methods: In total, 170 childhood ALL patients underwent PEG-asp-contained treatment and their L-asp activity and anti-L-asp antibody were detected on the 7th day after treatment initiation. Results: There were 27 (15.9%) patients who had PEG-asp-related adverse reaction: 17 (10.0%) patients experienced PEG-asp-related anaphylaxis and 14 (8.2%) patients experienced PEG- asp-related non-anaphylaxis adverse reaction. Moreover, L-asp activity was negatively related to anti-L-asp antibody in childhood ALL patients (P<0.001). Elevated L-asp activity was associated with the absence of PEG-asp-related anaphylaxis (P<0.001), PEG-asp-related non-anaphylaxis adverse reaction (P=0.004), and PEG-asp-related adverse reaction (P<0.001). However, the anti- L-asp antibody displayed opposite trend similar to L-asp activity. Receiver operating characteristic (ROC) curve analyses exhibited L-asp activity and anti-L-asp antibody exhibited superior predictive values in estimating PEG-asp-related anaphylaxis risk with area under curve (AUC) of 0.955 and 0.905, respectively compared to PEG-asp-related non-anaphylaxis adverse reaction risk with AUC of 0.730 and 0.675, respectively. Besides, patients with de novo disease, higher risk stratification, and allergic history showed trends linked with PEG-asp-related anaphylaxis risk. Conclusion: The monitoring of L-asp activity and anti-L-asp antibody maybe useful for early estimation and prevention of PEG-asp-related anaphylaxis in childhood ALL management (AU)

L-asparaginase activity and anti-L-asparaginase antibody as biomarkers in estimating PEG-asp-related anaphylaxis risk in childhood acute lymphoblastic leukemia patients.

BACKGROUND: L-Asparaginase (L-asp), the unconjugated form of polyethylene glycol-conjugated L-asparaginase (PEG-asp), regulates T cell stimulation, antibody production, and lysosomal protease activity to mediate PEG-asp-related anaphylaxis. This study aimed to investigate the relation of L-asp activity and anti-L-asp antibody with anaphylaxis risk and non-anaphylaxis adverse reaction risk in childhood acute lymphoblastic leukemia (ALL) patients who underwent PEG-asp contained therapy. METHODS: In total, 170 childhood ALL patients underwent PEG-asp-contained treatment and their L-asp activity and anti-L-asp antibody were detected on the 7th day after treatment initiation. RESULTS: There were 27 (15.9%) patients who had PEG-asp-related adverse reaction: 17 (10.0%) patients experienced PEG-asp-related anaphylaxis and 14 (8.2%) patients experienced PEG- asp-related non-anaphylaxis adverse reaction. Moreover, L-asp activity was negatively related to anti-L-asp antibody in childhood ALL patients (P<0.001). Elevated L-asp activity was associated with the absence of PEG-asp-related anaphylaxis (P<0.001), PEG-asp-related non-anaphylaxis adverse reaction (P=0.004), and PEG-asp-related adverse reaction (P<0.001). However, the anti- L-asp antibody displayed opposite trend similar to L-asp activity. Receiver operating characteristic (ROC) curve analyses exhibited L-asp activity and anti-L-asp antibody exhibited superior predictive values in estimating PEG-asp-related anaphylaxis risk with area under curve (AUC) of 0.955 and 0.905, respectively compared to PEG-asp-related non-anaphylaxis adverse reaction risk with AUC of 0.730 and 0.675, respectively. Besides, patients with de novo disease, higher risk stratification, and allergic history showed trends linked with PEG-asp-related anaphylaxis risk. CONCLUSION: The monitoring of L-asp activity and anti-L-asp antibody maybe useful for early estimation and prevention of PEG-asp-related anaphylaxis in childhood ALL management.

Exosomal circCOL1A2 from cancer cells accelerates colorectal cancer progression via regulating miR-665/LASP1 signal axis.

Circular RNAs (circRNAs) have been demonstrated to exert pivotal functions in cancer progression but are poorly understood in colorectal cancer (CRC). This work intends to investigate the effect and mechanism of a novel cirRNA (circCOL1A2) in CRC. Exosomes were identified via transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to analyze the levels of genes and proteins. Proliferation, migration, and invasion were detected via cell counting kit-8 (CCK8), 5-Ethynyl-2'-deoxyuridine (EDU), and transwell experiments. RNA pull-down, luciferase reporter, and RNA immunoprecipitation (RIP) assays were performed to assess the binding between genes. Animal studies were carried out to evaluate the function of circCOL1A2 in vivo. We found that circCOL1A2 was highly expressed in CRC cells. And circCOL1A2 was packaged from cancerous cells into exosomes. The proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) properties were inhibited after the reduction of exosomal circCOL1A2. Mechanism studies proved the binding of miR-665 with circCOL1A2 or LASP1 Rescue experiments validated the reverse effects of miR-665 knockdown on circCOL1A2 silencing and LASP1 overexpression on miR-665. Animal studies further confirmed the oncogenic function of exosomal circCOL1A2 in CRC tumorigenesis. In conclusion, exosomal circCOL1A2 sponges miR-665 to enhance LASP1 expression and modulated CRC phenotypes. Thus, circCOL1A2 might be a valuable therapeutic target for CRC, offering novel insight into CRC treatment.

METTL14 promotes fibroblast-like synoviocytes activation via the LASP1/SRC/AKT axis in rheumatoid arthritis.

The objective of this study is to explore the specific roles of a crucial N6-methyladenosine (m6A) methyltransferase, methyltransferase-like 14 (METTL14), in fibroblast-like synoviocytes (FLSs) activation of rheumatoid arthritis (RA). RA rat model was induced by administering intraperitoneally collagen antibody alcohol. Primary fibroblast-like synoviocytes (FLSs) were isolated from joint synovium tissues in rats. shRNA transfection tools were used to downregulate METTL14 expression in vivo and vitro. The injury of joint synovium was shown by hematoxylin and eosin (HE) staining. The cell apoptosis of FLSs was determined by flow cytometry. The levels of IL-6, IL-18, and C-X-C motif chemokine ligand (CXCL)10 in serum and culture supernatants were measured by ELISA kits. The expressions of LIM and SH3 domain protein 1 (LASP1), p-SRC/SRC, and p-AKT/AKT in FLSs and joint synovium tissues were determined by Western blots. The expression of METTL14 was greatly induced in the synovium tissues of RA rats compared with normal control rats. Compared with sh-NC-treated FLSs, METTL14 knockdown significantly increased cell apoptosis, inhibited cell migration and invasion, and suppressed the production of IL-6, IL-18, and CXCL10 induced by TNF-α. METTL14 silencing suppresses the expression of LASP1 and the activation of Src/AKT axis induced by TNF-α in FLSs. METTL14 improves the mRNA stability of LASP1 through m6A modification. In contrast, these were reversed by LASP1 overexpression. Moreover, METTL14 silencing clearly alleviates FLSs activation and inflammation in a RA rat model. These results suggested that METTL14 promotes FLSs activation and related inflammatory response via the LASP1/SRC/AKT signaling pathway and identified METTL14 as a potential target for treating RA.

Identification of miR-1-3p, miR-143-3p and miR-145-5p association with bone metastasis of Gleason 3+4 prostate cancer and involvement of LASP1 regulation.

Gleason Score (GS) 3 + 4 prostate cancer (PCa) is heterogeneous in clinical course and molecular features. Risk stratification of indolent and aggressive PCa with GS 3 + 4 is critical, especially those with bone metastasis (BM) potential. Microarray-based microRNA(miRNA) profiling with eight PCa cases with or without BM was used to screen the candidate miRNAs associated with BM. Transwell and MTS assays were used to characterize the function of miRNAs and target gene LASP1. RT-qPCR and immunohistochemistry assays were utilized to illustrate the clinical significance of miRNAs and target gene in a cohort of 309 Chinese PCa cases. In the current study, we identified that miR-1-3p, miR-143-3p and miR-145-5p are associated with BM of GS 3 + 4 PCa. Through functional experiments, we show that miR-1-3p/143-3p/145-5p promotes proliferation and migration of PCa in vitro. LASP1 was predicted as the common target of these three miRNAs which was further confirmed by a luciferase assay. Overexpression of LASP1 was correlated with higher GS, higher pathological stage, and the presence of metastasis by immunohistochemistry. siRNA knockdown of LASP1 significantly suppressed proliferation and migration, whereas overexpression of LASP1 promoted it. Bioinformatics analysis revealed the involvement of Wnt signaling pathway in LASP1 mediated function. LASP1 may activate Wnt signaling by interacting with ß-catenin. In all, we suggest that miR-1-3p/143-3p/145-5p are associated with BM of Gleason 3 + 4 PCa. LASP1 is the common target of these miRNAs and may active Wnt signaling by interacting with ß-catenin.

Effects and Mechanism of LASP1 on Proliferation, Migration, and Apoptosis of Human Colorectal Cancer LOVO Cells / 肿瘤防治研究

Objective To explore the effects and mechanism of LASP1 gene expression on the proliferation, migration, and invasion of human colorectal cancer (LOVO) cells. Methods LASP1 overexpression plasmids and LASP1 interference plasmids were constructed and transfected to LOVO cells. qRT-PCR was used to detect LASP1 mRNA expression and validate the transfection. MTT method and Tunel staining were used to detect cell proliferation and apoptosis, respectively, and scratch test and Transwell test were employed to determine the migration and invasion abilities of cells. Western blot was applied to analyze the expression of LASP1, p-FAK/FAK, and p-AKT/AKT protein in cells. Results The plasmids were successfully transfected. LASP1 overexpression increased the proliferation, migration, and invasion of LOVO cells, decreased the apoptosis, and increased LASP1, p-FAK/FAK, p-AKT/AKT protein expression (P < 0.01). LASP1 knockdown reduced the proliferation, migration, and invasion of LOVO cells, increased the apoptosis, and decreased LASP1, p-FAK/FAK, and p-AKT/AKT protein expression (P < 0.01). Conclusion LASP1 positively regulates the FAK/AKT signaling pathway to promote the proliferation, migration, and invasion of LOVO cells.

Environmental enrichment ameliorates anxiety and depression-like behaviors in paternally stressed offspring mice through upregulation of hippocampal LASP1 / 西安交通大学学报(医学版)

【Objective】 To explore the role of environmental enrichment (EE) in paternal stress-induced anxiety and depression-like behaviors in offspring and its potential mechanisms. 【Methods】 Male C57BL/6 mice (F0) were treated with unpredictable chronic mild stress (UCMS) and subsequently mated with normal females to obtain F1 offspring mice. The standard environment (SE) and enriched environment (EE) were administered to F1-UCMS offspring mice during their early life (3-5 weeks of age). Anxiety-like behaviors were detected by open field test (OFT) and elevated plus-maze test (EPM); depression-like behaviors were detected via forced swimming test (FST) and sucrose preference test (SPT) at the age of 8 weeks. The expressions of LIM and SH3 domain protein 1 (LASP1) in the hippocampus of adult F1 offspring mice were detected by Real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting. 【Results】 Compared to F1 offspring of normal paternal (F1-Nor), F1 offspring mice of the stressed paternal (F1-UCMS) showed significantly anxiety-like behavior with reduced percentage of time spent in the central region of OFT and in the open arm of EPM (P<0.05); mice from the F1-UCMS group showed a significantly increased percentage of immobility in FST and a reduced percentage of sugar consumption in SPT (P<0.01), which demonstrated significant depression-like behaviors. Compared to the SE group, mice in the EE group had an increased percentage of time spent in the central region of the OFT [males: (7.44±0.75)% vs. (14.93±1.74)%, P<0.01; females: (8.89±1.06)% vs. (15.10±1.82)%, P<0.05] and an increased percentage of time in the open arm of EPM [males: (8.09±1.05)% vs. (14.15±1.88)%, P<0.05; females: (9.13±1.14)% vs. (14.04±1.37)%, P<0.05]. This indicated that EE ameliorated anxiety-like behavior in F1-UCMS mice with paternal stress. Compared to the SE group, mice in the EE group had an decreased percentage of immobility in FST [males: (58.63±4.51)% vs. (42.15±3.81)%, P<0.05; females: (57.96±4.19)% vs. (43.25±4.22)%, P<0.05] and an increased percentage of sugar consumption in SPT [males: (50.38±3.47)% vs. (70.39±3.12)%, P<0.01; females: (52.42±2.84)% vs. (69.99±3.55)%, P<0.01]. This indicated that EE ameliorated depression-like behavior in F1-UCMS mice with paternal stress. Hippocampal LASP1 expression was reduced in SE group compared to F1-Nor group (males: P<0.01; females: P<0.05), while LASP1 was increased in EE group compared to SE group (P<0.05) detected by RT-qPCR and Western blotting. 【Conclusion】 EE ameliorates paternal stress-induced anxiety and depression-like behaviors in F1-UCMS mice, and the mechanism may be associated with increased hippocampal LASP1 expression in F1-UCMS mice.

Hepatitis B virus X protein increases LASP1 SUMOylation to stabilize HER2 and facilitate hepatocarcinogenesis.

The hepatitis B virus (HBV) X protein (HBX), a viral macromolecule, plays a vital role in the development of HBV-related hepatocellular carcinoma (HCC). Increased expression of HER2 is linked to HBV infection, and HBX is responsible for HER2 upregulation in HCC. Nevertheless, the underlying molecular mechanisms are not yet fully understood. In the study, we discovered that HBX promoted HER2 expression to facilitate the sensitization of the insulin signaling pathway and enhance the growth and migration of HCC cells. Mechanistically, the viral protein enhanced the stability of HER2 by preventing its ubiquitination-mediated proteasomal degradation through LASP1, which could bind to HER2. Furthermore, increased SUMOylation of LASP1 contributed to the upregulation of HER2 and the interaction of LASP1 with HER2. In addition, RANBP2 and RANGAP1 were found to interact with LASP1 and promote SUMOylation of LASP1 to upregulate HER2 expression in HBX-associated hepatoma cells. In summary, our work provides a novel insight into hepatocarcinogenesis mediated by HBX and estimates the detailed mechanisms related to the increase in HER2 regulated by the viral protein, which might help provide a theoretical basis for identifying novel targets for HBV-positive HCC treatment.

LASP1 in Cellular Signaling and Gene Expression: More than Just a Cytoskeletal Regulator.

LIM and SH3 protein 1 was originally identified as a structural cytoskeletal protein with scaffolding function. However, recent data suggest additional roles in cell signaling and gene expression, especially in tumor cells. These novel functions are primarily regulated by the site-specific phosphorylation of LASP1. This review will focus on specific phosphorylation-dependent interaction between LASP1 and cellular proteins that orchestrate primary tumor progression and metastasis. More specifically, we will describe the role of LASP1 in chemokine receptor, and PI3K/AKT signaling. We outline the nuclear role for LASP1 in terms of epigenetics and transcriptional regulation and modulation of oncogenic mRNA translation. Finally, newly identified roles for the cytoskeletal function of LASP1 next to its known canonical F-actin binding properties are included.

Network pharmacology and experimental analysis to reveal the mechanism of Dan-Shen-Yin against endothelial to mesenchymal transition in atherosclerosis.

Atherosclerosis is a chronic inflammatory disease characterized by the formation of plaque and endothelial dysfunction. Under pro-inflammatory conditions, endothelial cells adopt a mesenchymal phenotype by a process called endothelial-to-mesenchymal transition (EndMT) which plays an important role in the pathogenesis of atherosclerosis. Dan-Shen-Yin (DSY) is a well-known traditional Chinese medicine used in the treatment of cardiovascular disease. However, the molecular mechanism whereby DSY mitigates atherosclerosis remains unknown. Therefore, we employed a network pharmacology-based strategy in this study to determine the therapeutic targets of DSY, and in vitro experiments to understand the molecular pharmacology mechanism. The targets of the active ingredients of DSY related to EndMT and atherosclerosis were obtained and used to construct a protein-protein interaction (PPI) network followed by network topology and functional enrichment analysis. Network pharmacology analysis revealed that the PI3K/AKT pathway was the principal signaling pathway of DSY against EndMT in atherosclerosis. Molecular docking simulations indicated strong binding capabilities of DSY's bioactive ingredients toward PI3K/AKT pathway molecules. Experimentally, DSY could efficiently modify expression of signature EndMT genes and decrease expression of PI3K/AKT pathway signals including integrin αV, integrin ß1, PI3K, and AKT1 in TGF-ß2-treated HUVECs. LASP1, which is upstream of the PI3K/AKT pathway, had strong binding affinity to the majority of DSY's bioactive ingredients, was induced by EndMT-promoting stimuli involving IL-1ß, TGF-ß2, and hypoxia, and was downregulated by DSY. Knock-down of LASP1 attenuated the expression of integrin αV, integrin ß1, PI3K, AKT1 and EndMT-related genes induced by TGF-ß2, and minimized the effect of DSY. Thus, our study showed that DSY potentially exerted anti-EndMT activity through the LASP1/PI3K/AKT pathway, providing a possible new therapeutic intervention for atherosclerosis.

CERS6-AS1 Facilitates Oncogenesis and Restrains Ferroptosis in Papillary Thyroid Carcinoma by Serving as a ceRNA through miR-497-5p/LASP1 Axis.

OBJECTIVE: The purpose of this study was to explore the function and mechanism of LncRNA CERS6-AS1 on papillary thyroid cancer. MATERIALS: CERS6-AS1, miR-497-5p, and LASP1 expression in papillary thyroid cancer tissues and cells were detected by RT-PCR. The relationship between CERS6-AS1 expression and clinical characteristics was analyzed, and overall survival was evaluated via Kaplan-Meier analysis. Cell activity was tested by cell counting kit-8, cell reactive oxygen species was detected by DCFH-DA method, and cell iron ion was detected by iron analysis kit. The relationship among CERS6-AS1, miR-497-5p, and LASP1 was confirmed by luciferase reporter gene detection, RNA pull-down detection, and RIP detection. The expression of related proteins was assessed by western blot or immunohistochemistry. RESULTS: High level of CERS6-AS1 and LASP1 was detected in papillary thyroid cancer tissues and cells and predicted poor prognosis. In contrast, miR-497-5p was decreased in papillary thyroid cancer tissues and cells, which was positively correlated with prognosis. Silencing CERS6-AS1 suppressed cell viability and increased ferroptosis in papillary thyroid cancer. LASP1 was modulated by CERS6-AS1 through sponging miR-497-5p. Up-regulation of LASP1 or silencing miR-497-5p could weaken the effect of CERS6-AS1 on papillary thyroid cancer cells. Silencing CERS6-AS1 restrained the growth of xenografted tumors. CONCLUSION: Our findings demonstrated that down-regulation of CERS6-AS1 reduced cell viability and amplified cell ferroptosis by modulating the miR-497-5p/LASP1 axis in papillary thyroid cancer.

Downregulation of long noncoding RNA breast cancer anti-estrogen resistance 4 inhibits cell proliferation, invasion, and migration in esophageal squamous cell carcinoma by regulating the microRNA-181c-5p/LIM and SH3 protein 1 axis.

Recently, abnormal expression of long non-coding RNAs (lncRNAs) has been observed in esophageal squamous cell carcinoma (ESCC). In various human cancers, breast cancer anti­estrogen resistance 4 (BCAR4) was reported to be highly expressed, while the biological roles of BCAR4 in ESCC remain unclear. In ESCC cells and tissues, BCAR4 and microRNA -181c-5p (miR-181c-5p) expression, and phosphorylated signal transducer and activator of transcription (p-STAT3) and COX2 expression were evaluated by real-time reverse transcription PCR (qRT-PCR) and Western blot analysis. Cell function was evaluated by colony formation, CCK-8 assay, transwell and flow cytometer assays. Interactions between BCAR4 and miR-181c-5p, as well as miR-181c-5p and LIM and SH3 protein 1 (LASP1) were evaluated by RIP and luciferase reporter assay. ESCC cell malignancy with inhibition of BCAR4 was confirmed by a tumor xenograft model in vivo. In both ESCC tissues and cell lines, BCAR4 was upregulated. Downregulation of BCAR4 effectively induced cell apoptosis and inhibited invasion and migration in vitro, and reduced tumorigenesis in nude mice. BCAR4 was a sponge of miR-181c-5p to upregulate LASP1. Moreover, knockdown of BCAR4 and overexpression of miR-181c-5p inhibited the activation of the STAT3/COX2 signaling, which was reversed by overexpression of LASP1. In conclusion, BCAR4 promotes ESCC tumorigenesis by targeting the miR-181c-5p/LASP1 axis, which may act as a treatment and diagnosis biomarker for ESCC.