Insulin-like growth factor binding protein related protein 1 knockdown attenuates hepatic ?brosis via the regulation of MMPs/TIMPs in mice

Background: Previous research suggested that insulin-like growth factor binding protein related protein 1(IGFBPrP1), as a novel mediator, contributes to hepatic fibrogenesis. Matrix metalloproteinases(MMP) and tissue inhibitors of metalloproteinases(TIMP) play an essential role in hepatic fibrogenesis by regulating homeostasis and remodeling of the extracellular matrix(ECM). However, the interaction between IGFBPrP1 and MMP/TIMP is not clear. The present study was to knockdown IGFBPrP1 to investigate the correlation between IGFBPrP1 and MMP/TIMP in hepatic fibrosis. Methods: Hepatic fibrosis was induced by thioacetamide(TAA) in mice. Knockdown of IGFBPrP1 expression by ultrasound-targeted microbubble destruction-mediated CMB-shRNA-IGFBPrP1 delivery, or inhibition of the Hedgehog(Hh) pathway by cyclopamine treatment, was performed in TAA-induced liver fibrosis mice. Hepatic fibrosis was determined by hematoxylin and eosin and Sirius red staining. Hepatic expression of IGFBPrP1, α-smooth muscle actin( α-SMA), transforming growth factor β 1(TGF β1), collagen I, MMPs/TIMPs, Sonic Hedgehog(Shh), and glioblastoma family transcription factors(Gli1) were investigated by immunohistochemical staining and Western blotting analysis. Results: We found that hepatic expression of IGFBPrP1, TGF β1, α-SMA, and collagen I were increased longitudinally in mice with TAA-induced hepatic fibrosis, concomitant with MMP2/TIMP2 and MMP9/TIMP1 imbalance and Hh pathway activation. Knockdown of IGFBPrP1 expression, or inhibition of the Hh pathway, reduced the hepatic expression of IGFBPrP1, TGF β1, α-SMA, and collagen I and re-established MMP2/TIMP2 and MMP9/TIMP1 balance. Conclusions: Our findings suggest that IGFBPrP1 knockdown attenuates liver fibrosis by re-establishing MMP2/TIMP2 and MMP9/TIMP1 balance, concomitant with the inhibition of hepatic stellate cell activation, down-regulation of TGF β1 expression, and degradation of the ECM. Furthermore, the Hh pathway mediates IGFBPrP1 knockdown-induced attenuation of hepatic fibrosis through the regulation of MMPs/TIMPs balance.

Effects of transfected adenovirus-mediated transcription factor X-box binding protein 1 on hippocampal-derived neural stem cell proliferation and apoptosis under hypoxia

BACKGROUND： Neural stem cell （NSC） survival is closely associated with cell apoptosis in ischemic-hypoxic regions following transplantation. Numerous studies have revealed that X-box binding protein 1 （XBP1） is a transcription factor during endoplasmic reticulum unfolded protein response and is essential for cell survival, differentiation, and anti-apoptotic effects. OBJECTIVE： To determine the effects of the XBP1 gene on NSC proliferation and apoptosis under hypoxic conditions following XBP1 gene transfection into rat embryonic hippocampal NSCs using recombinant adenovirus vector. DESIGN, TIME AND SETTING： In vitro experiments were performed at the Laboratory of Cell Biology of Jilin University and Laboratory of Proteomics, Department of Neurology, Jilin University China from September 2008 to November 2009. MATERIALS： Recombinant adenovirus package XBP1 gene and Ad-XBPl-enhanced green fluorescent protein plasmid （Guangzhou Easywin BioMed Technology, China）, rabbit anti-XBP1 and its target gene estrogen receptor degradation-enhancing a-mannosidase-like protein （EDEM） glucose-regulated protein 78 （GRP78）, anti-apoptotic molecule Bcl-2 and proapoptotic molecule Bax polyclonal antibody （Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA）, and COCI2 （Sigma, St. Louis, MO, USA） were used in the present study. METHODS： Hippocampi from embryonic, Sprague Dawley rats on gestational day 16 were harvested for NSC isolation and cloning, followed by immunofluorescence for Nestin and sub-culturing. The recombinant adenovirus Ad-XBPl-enhanced green fluorescent protein plasmid was transfected into rat embryonic hippocampal NSCs, and then CoCl2 was applied to induce hypoxia. MAIN OUTCOME MEASURES： Cell quantification and 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide colorimetric assay were utilized to detect proliferation in XBPl-transfected NSCs for 7 consecutive days. Western blot assay was utilized to quantify XBP1 GRP78, EDEM, Bcl-2, and Bax expression. Flow cytometry was used to measure apoptosis. RESULTS： NSC proliferation was significantly enhanced following XBP1 gene transfection （P 〈 0.05）. Under hypoxic conditions, GRP78, EDEM, and Bcl-2 levels increased, but Bax levels decreased. In addition, NSC apoptosis decreased following transfection （P 〈 0.05）. CONCLUSION： The XBP1 gene was successfully transfected into rat embryonic hippocampal NSCs using a recombinant adenovirus vector. NSC proliferation following transfection, as well as anti-apoptotic effects under hypoxia, was significantly increased.

Interaction between insulin-like growth factor binding protein-related protein 1 and transforming growth factor beta 1 in primary hepatic stellate cells

BACKGROUND: We previously showed that insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) is a novel mediator in liver fibrosis. Transforming growth factor beta 1 (TGF beta 1) is known as the strongest effector of liver fibrosis. Therefore, we aimed to investigate the detailed interaction between IGFBPrP1 and TGF beta 1 in primary hepatic stellate cells (HSCs). METHODS: We overexpressed TGF beta 1 or IGFBPrP1 and inhibited TGF beta 1 expression in primary HSCs for 6, 12, 24, 48, 72, and 96 hours to investigate their interaction and observe the accompanying expressions of a-smooth muscle actin (alpha-SMA), collagen I, fibronectin, and phosphorylated-mothers against decapentaplegic homolog 2/3 (p-Smad2/3). RESULTS: We found that the adenovirus vector encoding the TGF beta 1 gene (AdTGF beta 1) induced IGFBPrP1 expression while that of alpha-SMA, collagen I, fibronectin, and TGF beta 1 increased gradually. Concomitantly, AdIGFBPrP1 upregulated TGF beta 1, alpha-SMA, collagen I, fibronectin, and p-Smad2/3 in a time-dependent manner while IGFBPrP1 expression was decreased at 96 hours. Inhibition of TGF beta 1 expression reduced the IGFBPrP1-stimulated expression of alpha-SMA, collagen I, fibronectin, and p-Smad2/3. CONCLUSIONS: These findings for the first time suggest the existence of a possible mutually regulation between IGFBPrP1 and TGF beta 1, which likely accelerates liver fibrosis progression. Furthermore, IGFBPrP1 likely participates in liver fibrosis in a TGF beta 1-depedent manner, and may act as an upstream regulatory factor of TGF beta 1 in the Smad pathway.

Y-box binding protein 1 augments sorafenib resistance via the PI3K/Akt signaling pathway in hepatocellular carcinoma

BACKGROUND Sorafenib is the first-line treatment for patients with advanced hepatocellular carcinoma(HCC).Y-box binding protein 1(YB-1)is closely correlated with tumors and drug resistance.However,the relationship between YB-1 and sorafenib resistance and the underlying mechanism in HCC remain unknown.AIM To explore the role and related mechanisms of YB-1 in mediating sorafenib resistance in HCC.METHODS The protein expression levels of YB-1 were assessed in human HCC tissues and adjacent nontumor tissues.Next,we constructed YB-1 overexpression and knockdown hepatocarcinoma cell lines with lentiviruses and stimulated these cell lines with different concentrations of sorafenib.Then,we detected the proliferation and apoptosis in these cells by terminal deoxynucleotidyl transferase dUTP nick end labeling,flow cytometry and Western blotting assays.We also constructed a xenograft tumor model to explore the effect of YB-1 on the efficacy of sorafenib in vivo.Moreover,we studied and verified the specific molecular mechanism of YB-1 mediating sorafenib resistance in hepatoma cells by digital gene expression sequencing(DGE-seq).RESULTS YB-1 protein levels were found to be higher in HCC tissues than in corresponding nontumor tissues.YB-1 suppressed the effect of sorafenib on cell proliferation and apoptosis.Consistently,the efficacy of sorafenib in vivo was enhanced after YB-1 was knocked down.Furthermore,KEGG pathway enrichment analysis of DGEseq demonstrated that the phosphoinositide-3-kinase(PI3K)/protein kinase B(Akt)signaling pathway was essential for the sorafenib resistance induced by YB-1.Subsequently,YB-1 interacted with two key proteins of the PI3K/Akt signaling pathway(Akt1 and PIK3R1)as shown by searching the BioGRID and HitPredict websites.Finally,YB-1 suppressed the inactivation of the PI3K/Akt signaling pathway induced by sorafenib,and the blockade of the PI3K/Akt signaling pathway by LY294002 mitigated YB-1-induced sorafenib resistance.CONCLUSION Overall,we concluded that YB-1 augments sorafenib resistance through the PI3K/Akt signaling pathway in HCC and suggest that YB-1 is a key drug resistance-related gene,which is of great significance for the application of sorafenib in advanced-stage HCC.

Golgi localization and dynamics of hyaluronan binding protein 1 (HABP1/p32/C1QBP) during the cell cycle

Hyaluronan binding protein 1 (HABP1) is a negatively charged multifunctional mammalian protein with a unique structural fold. Despite the fact that HABP1 possesses mitochondrial localization signal, it has also been localized to other cellular compartments. Using indirect immunofluorescence, we examined the sub-cellular localization of HABP1 and its dynamics during mitosis. We wanted to determine whether it distributes in any distinctive manner after mitotic nuclear envelope disassembly or is dispersed randomly throughout the cell. Our results reveal the golgi localization of HABP1 and demonstrate its complete dispersion throughout the cell during mitosis. This distinctive distribution pattern of HABP1 during mitosis resembles its ligand hyaluronan, suggesting that in concert with each other the two molecules play critical roles in this dynamic process.

Special AT-rich sequence-binding protein 1 promotes cell growth and metastasis in colorectal cancer

AIM: To evaluate the expression of special AT-rich sequence-binding protein 1 (SATB1 ) gene in colorectal cancer and its role in colorectal cancer cell proliferation and invasion.METHODS: Immunohistochemistry was used to detect the protein expression of SATB1 in 30 colorectal cancer (CRC) tissue samples and pair-matched adjacent nontumor samples. Cell growth was investigated after enhancing expression of SATB1. Wound-healing assay and Transwell assay were used to investigate the impact of SATB1 on migratory and invasive abilities of SW480 cells in vitro . Nude mice that received subcutaneous implantation or lateral tail vein were used to study the effects of SATB1 on tumor growth or metastasis in vivo . RESULTS: SATB1 was over-expressed in CRC tissues and CRC cell lines. SATB1 promotes cell proliferation and cell cycle progression in CRC SW480 cells. SATB1 over-expression could promote cell growth in vivo . In addition, SATB1 could significantly raise the ability of cell migration and invasion in vitro and promote the ability of tumor metastasis in vivo . SATB1 could up-regulate matrix metalloproteases 2, 9, cyclin D1 and vimentin, meanwhile SATB1 could down-regulate E-cadherin in CRC. CONCLUSION: SATB1 acts as a potential growth and metastasis promoter in CRC. SATB1 may be useful as a therapeutic target for CRC.

Transplantation of X-box-binding protein-1 gene-modified neural stem cells in the lateral ventricle of brain ischemia rats

X-box-binding protein-1 （XBP-1） is an essential transcription factor in endoplasmic reticulum stress In this study, XBP-1 gene-transfected neural stem cells （NSCs） were transplanted into lesion sites to ensure stability and persistent expression of XBP-1, resulting in the exertion of anti-apoptotic effects. Simultaneously, XBP-1 gene transfection promotes the survival and differentiation of transplanted NSCs. Results from this study demonstrated that survival, proliferation and differentiation of XBP-1 g^ne-modified NSCs were enhanced when compared to unmodified NSCs at 28 days post-transplantation （P 〈 0.05）. A diminished number of apoptotic neural cells increased Bcl-2 expression and reduced Bax expression, and were observed in the ischemic region of the XBP-1-NSCs group （P 〈 0.05）. These results indicated that modification of the XBP-1 gene enhances the survival and migration of NSCs in vivo and decreases the occurrence of apoptosis.

X-box-binding protein 1-modified neural stem cells for treatment of Parkinson's disease

X-box-binding protein 1-transfected neural stem cells were transplanted into the right lateral ventricles of rats with rotenone-induced Parkinson＇s disease. The survival capacities and differentiation rates of cells expressing the dopaminergic marker tyrosine hydroxylase were higher in X-box-binding protein 1-transfected neural stem cells compared to non-transfected cells. Moreover, dopamine and 3,4-dihydroxyphenylacetic acid levels in the substantia nigra were significantly increased, α-synuclein expression was decreased, and neurological behaviors were significantly ameliorated in rats following transplantation of X-box-binding protein 1-transfected neural stem cells. These results indicate that transplantation of X-box-binding protein 1-transfected neural stem cells can promote stem cell survival and differentiation into dopaminergic neurons, increase dopamine and 3,4-dihydroxyphenylacetic acid levels, reduce α-synuclein aggregation in the substantia nigra, and improve the symptoms of Parkinson＇s disease in rats.

Insulin-like growth factor 2 mRNA-binding protein 1 promotes cell proliferation via activation of AKT and is directly targeted by microRNA-494 in pancreatic cancer

BACKGROUND Studies have shown that insulin-like growth factor 2 mRNA-binding protein 1(IGF2BP1)plays critical roles in the genesis and development of human cancers.AIM To investigate the clinical significance and role of IGF2BP1 in pancreatic cancer.METHODS Expression levels of IGF2BP1 and microRNA-494(miR-494)were mined based on Gene Expression Omnibus datasets and validated in both clinical samples and cell lines by quantitative real-time polymerase chain reaction and Western blot.The relationship between IGF2BP1 expression and clinicopathological factors of pancreatic cancer patients was analyzed.The effect and mechanism of IGF2BP1 on pancreatic cancer cell proliferation were investigated in vitro and in vivo.Analyses were performed to explore underlying mechanisms of IGF2BP1 upregulation in pancreatic cancer and assays were carried out to verify the posttranscriptional regulation of IGF2BP1 by miR-494.RESULTS We found that IGF2BP1 was upregulated and associated with a poor prognosis in pancreatic cancer patients.We showed that downregulation of IGF2BP1 inhibited pancreatic cancer cell growth in vitro and in vivo via the AKT signaling pathway.Mechanistically,we showed that the frequent upregulation of IGF2BP1 was attributed to the downregulation of miR-494 expression in pancreatic cancer.Furthermore,we discovered that reexpression of miR-494 could partially abrogate the oncogenic role of IGF2BP1.CONCLUSION Our results revealed that upregulated IGF2BP1 promotes the proliferation of pancreatic cancer cells via the AKT signaling pathway and confirmed that the activation of IGF2BP1 is partly due to the silencing of miR-494.

Serum Y-Box Binding Protein 1 (YBX-1) and Interleukin 6 (IL-6) Are Associated with Metastasis in Breast Cancer Patients

Objectives: The aim of this study was to assess the levels of Y-box binding protein 1 (YBX-1) and interleukin 6 (IL-6) in the sera of metastatic and non-metastatic breast cancer patients (BC), investigate their clinicopathological significance and to analyze their potential use as biomarkers of breast cancer metastasis. Methods: The study included ninety subjects sub-grouped equally into metastatic BC, non-metastatic BC and healthy volunteers. Serum YBX-1 and IL-6 were quantified using ELISA technique while CA 15-3 was quantified using IRMA kit. Clinical data were collected from patients’ records. Results: YBX-1 (p < 0.001), IL-6 (p < 0.001) and CA15-3 (p = 0.017, 0.001) were significantly elevated in metastatic and non-metastatic BC patients compared to healthy controls, however, only YBX-1 (p 0.001) showed a significant difference with cancer metastasis. Generally, YBX-1 and IL-6 were correlated with worse histological grade and late clinical stage in breast cancer patients and they were also associated with axillary lymph nodes involvement and positive vascular invasion in metastatic BC patients. Serum YBX-1 and IL-6 levels were positively correlated to each other (rs = 0.615, p < 0.001) and they showed high sensitivity and specificity compared to CA 15-3 (p < 0.001 and p = 0.004 for YBX-1 and IL-6 respectively) for predicting cancer metastasis. Conclusions: Serum YBX-1 and IL-6 are potential biomarkers of breast cancer patients with significant correlation with bad clinicopathological characteristics. Serum YBX-1 and IL-6 have superior sensitivity and specificity compared to CA15-3 and can serve as potential follow up and prognostic markers.

Tribulus terrestris extracts alleviate muscle damage and promote anaerobic performance of trained male boxers and its mechanisms： Roles of androgen, IGF-1, and IGF binding protein-3

Purpose: To investigate the effects of Tribulus terrestris(TT) extracts on muscle mass, muscle damage, and anaerobic performances of trained male boxers and its mechanisms: roles of plasma androgen, insulin growth factor 1(IGF-1), and IGF-1 binding protein-3(IGFBP-3).Methods: Fifteen male boxers were divided into exercise group(E, n = 7) and exercise plus TT group(E + TT, n = 8). The 2 groups both undertook3-week high-intensity and 3-week high-volume trainings separated by a 4-week rest. TT extracts(1250 mg/day) were orally administered by boxers in E + TT group. TT extract compositions were detected by UHPLC–Q-TOF/MS. Before and at the end of the 2 trainings, muscle mass, anaerobic performance, and blood indicators were explored.Results: Compared with E group, decreases of plasma CK(1591.5 ± 909.6 U/L vs. 2719.9 ± 832.5 U/L) and IGFBP-3(3075.5 ± 1072.5 ng/m L vs. 3950.8 ± 479.3 ng/m L) as well as increases of mean power(MP, 459.4 ± 122.3 W vs. 434.6 ± 69.5 W) and MP/body weight(MP/BW, 7.5 ± 0.9 W/kg vs. 7.1 ± 1.1 W/kg) were detected in E + TT group after a high-intensity training. For high-volume training, reduction of IGFBP-3(2946.4 ± 974.1 ng/m L vs. 3632.7 ± 470.1 ng/m L) and increases of MP(508.7 ± 103.2 W vs. 477.8 ± 49.9 W) and MP/BW(8.2 ± 0.3 W/kg vs.7.5 ± 0.9 W/kg) were detected in E + TT group, compared with E group. Muscle mass, blood levels of testosterone, dihydrotestosterone(DHT),and IGF-1 were not signifiantly changed between the 2 groups.Conclusion: Taking 1250 mg capsules containing TT extracts did not change muscle mass and plasma levels of testosterone, DHT, and IGF-1 but significantly alleviated muscle damage and promoted anaerobic performance of trained male boxers, which may be related to the decrease of plasma IGFBP-3 rather than androgen in plasma.

Antiviral Effect of Interferon-Induced Guanylate Binding Protein-1 against Coxsackie Virus and Hepatitis B Virus B3 in Vitro

Guanylate binding protein-1(GBP-1)is an interferon-induced protein.To observe its antiviral effect against Hepatitis B virus(HBV)and Coxsackie virus B3(CVB3),we constructed an eukaryotic expression vector of human GBP-1(hGBP-1).Full-length encoding sequence of hGBP-1 was amplified by long chain RT-PCR and inserted into a pCR2.1 vector,then subcloned into a pCDNA3.1(-)vector.Recombinant hGBP-1 plasmids and pHBV1.3 carrying 1.3-fold genome of HBV were contransfected into HepG2 cells,and inhibition effect of hGBP-1 against HBV replication was observed.Hela cells transfected with recombinant hGBP-1 plasmids were challenged with CVB3,and viral yield in cultures were detected.The results indicated that recombinant eukaryotic expression plasmid of hGBP-1 was constructed successfully and the hGBP-1 gene carried in this plasmid could be efficiently expressed in HepG2 cells and Hela cells.hGBP-1 inhibit CVB3 but not HBV replication in vitro.These results demonstrate that hGBP-1 mediates an antiviral effect against CVB3 but not HBV and perhaps plays an important role in the interferon-mediated antiviral response against CVB3.

The Evolution of Microtubule End-Binding Protein 1 (EB1) and Roles in Regulating Microtubule Behavior

All organisms must transmit genetic information to offspring through cell division, and mitotic spindle participates in the process. Spindle dynamics through depolymerization or polymerization of microtubules generates the driving force required for chromosome movements in mitosis. To date, studies have shown that microtubule arrays control the directions of cell division and diverse microtubule-associated proteins regulate cell division. But a clear picture of how microtubules and microtubule-associated proteins modulate cell division remains unknown. Depletion of end-binding protein 1 by RNA-mediated inhibition shows that one of the microtubule-associated proteins, end-binding protein 1, plays a crucial role in mitotic spindle formation and promotes microtubule dynamics and is needed for the proper segregation of mitotic chromosomes during anaphase in Drosophila cells. Here, we review the properties of end-binding protein 1 and the roles of end-binding protein 1 in regulating microtubule behavior and in cell cycle.

Insulin-like growth factor-binding protein-3 inhibits IGF-1-induced proliferation of human hepatocellular carcinoma cells

OBJECTIVE Basic fibroblast growth factor(b FGF)and platelet-derived growth factor(PDGF)produced by hepatocellular carcinoma(HCC)cells are responsible for the cell growth.Accumulating evidence shows that insulin-like growth factor-binding protein-3(IGFBP-3)suppresses HCC cell proliferation in both IGF-dependent and independent manners.The present study is to investigate whether treatment with exogenous IGFBP-3 inhibits bF GF and PDGF production and the cell proliferation of HCC cells.METHODS Cell Counting Kit 8 assay were designed to detect HCC cell proliferation,transcription factor early growth response-1(EGR1)involving in IGFBP-3 regulation of b FGF and PDGF were detected by RT-PCR and Western blot assays.Western blot assay was adopted to detect the IGFBP-3 regulating insulin-like growth factor 1 receptor(IGF-1R)signaling pathway.RESULTS The present study demonstrates that IGFBP-3 suppressed IGF-1-induced b FGF and PDGF expression while it does not affect their expression in the absence of IGF-1.To delineate the underlying mechanism,Western-blot and RT-PCR assays confirmed that the transcription factor early growth response protein 1(EGR1)is involved in IGFBP-3 regulation of b FGF and PDGF.IGFBP-3 inhibition of type 1 insulin-like growth factor receptor(IGF1R),ERK and AKT activation is IGF-1-dependent.Furthermore,transient transfection with constitutively activated AKT or MEK partially blocks the IGFBP-3 inhibition of EGR1,b FGF and PDGF expression.CONCLUSION In conclusion,these findings suggest that IGFBP-3suppresses transcription of EGR1 and its target genes b FGF and PDGF through inhibiting IGF-1-dependent ERK and AKT activation.It demonstrates the importance of IGFBP-3 in the regulation of HCC cell proliferation,suggesting that IGFBP-3 could be a target for the treatment of HCC.

Interaction of calcium- and integrin-binding protein 1 with integrin <i>α</i>11 and its possible involvement in pulmonary fibrosis

Integrin α11 (ITGA11) is one of the collagen-binding integrin α chains;however, its biological significance remains unknown. To determine the functions of ITGA11, we performed a yeast two-hybrid screen using the cytoplasmic domain of ITGA11 as bait and transformed an EGY48 yeast strain with the bait-containing plasmid using the plasmid from a human lung fibroblast cDNA library. This screen identified calcium- and integrin-binding protein 1 (CIB1) as prey. Recombinant ITGA11 and CIB1 were expressed in mammalian cells and used in coimmunoprecipitation experiments, which showed that full-length ITGA11 and CIB1 are also associated in vivo. Over-expression of CIB1 in the human lung myofibroblast MRC-5 cells decreased the expression of α-smooth muscle actin and fibronectin. Using a mouse model of pulmonary fibrosis (bleomycin-treatment), we detected elevated expression of CIB1 in lung tissues compared with controls. These data suggest that CIB1 may regulate pulmonary fibrosis in concert with IT-GA11.

抑制lncRNA TUG1下调核苷酸结合寡聚结构域样受体蛋白1炎症小体在延缓阿尔茨海默病进展的作用

目的探讨敲低长链非编码RNA(lncRNA)牛磺酸上调基因1(TUG1)抑制核苷酸结合寡聚结构域样受体蛋白1(NLRP1)炎症小体在缓解阿尔茨海默病进展中的作用。方法选取9~10周龄遗传背景为C57/BL6的野生型小鼠(WT组,10只)或淀粉样前体蛋白(APP)/早老素1(PS1)转基因小鼠(30只)。APP/PS1转基因小鼠随机分为模型(model)组,模型+敲低lncRNA TUG1组[model+lncRNA TUG1短发夹RNA(shRNA)组]和model+shRNA非靶标(NT)组,每组10只。分别采集12周龄第1天(3月龄)和32周龄第1天(8月龄)小鼠外周血和脑皮质组织,并分离皮质中的原代小胶质细胞和原代星形胶质细胞,每个时间点每组5只小鼠。Real-time PCR分别测定3月龄和8月龄上述4个分组小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和巨噬细胞移动抑制因子(MIF)mRNA的水平,以及原代星形胶质细胞中补体蛋白C1r和C1s mRNA的水平。ELISA法测定其外周血浆中MIF含量。对3月龄和8月龄小鼠脑皮质原代小胶质细胞和原代星形胶质细胞共培养。CCK-8法测定上述2种细胞的增殖能力。Western blotting分别测定3月龄和8月龄上述4个分组小鼠脑皮质组织中MIF、白细胞介素1β前体(pro-IL-1β)、凋亡相关斑点样蛋白(ASC)、Caspase-1(p20)、Caspase-1(full)、NLRP1及NLRP3蛋白的表达水平。采用免疫荧光染色法测定8月龄各分组小鼠脑皮质组织中β淀粉样蛋白(Aβ)表达。结果3月龄和8月龄时,与WT组小鼠相比,model组小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和MIF相对表达水平显著上调,原代小胶质细胞和原代星形胶质细胞增殖能力增强(P<0.05)。与model组相比,model+lncRNA TUG1 shRNA组小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和MIF的相对表达水平显著降低,原代小胶质细胞和原代星形胶质细胞增殖能力降低(P<0.05)。与WT组相比,model组小鼠外周血浆中MIF含量显著升高;小鼠脑皮质组织中pro-IL-1β、ASC、Caspase-1(p20)、Caspase-1(full)、NLRP1以及NLRP3的蛋白表达水平显著升高;Aβ免疫荧光强度明显增强(P<0.05)。与model组相比,model+lncRNA TUG1 shRNA组小鼠外周血浆中MIF含量显著降低;小鼠脑皮质组织中pro-IL-1β、ASC、Caspase-1(p20)、Caspase-1(full)和NLRP1的蛋白表达水平显著降低,Aβ免疫荧光强度明显降低(P<0.05),而NLRP3蛋白质的表达水平无明显变化(P>0.05)。与model组相比,model+shRNA NT组小鼠上述所有检测指标差异均无显著性(P>0.05)。结论APP/PS1转基因小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和MIF因子表达上调与脑皮质内NLRP1炎症小体激活成正相关,敲低lncRNA TUG1可缓解阿尔茨海默病的进展。

LncRNA ZEB1-AS1和LncRNA SOX2OT在糖尿病肾病患者中的表达及与肾功能的相关性研究

目的探究长链非编码RNA锌指E盒结合同源盒蛋白1反义链1(LncRNA ZEB1-AS1)和长链非编码RNA性别决定相关基因簇2重叠转录本(LncRNA SOX2OT)在糖尿病肾病(DN)患者中的表达及与肾功能的相关性。方法选取于2021年11月—2023年12月在武汉大学人民医院肾内科收治的DN患者106例为DN组,并根据24 h尿蛋白定量(24 h Upro)水平分为正常蛋白尿亚组43例(<30 mg)、微量蛋白尿亚组39例(30~<300 mg)、大量蛋白尿亚组24例(≥300 mg),另选取同期医院单纯糖尿病患者106例作对照组,检测患者血清LncRNA ZEB1-AS1、LncRNA SOX2OT水平;Pearson法分析LncRNA ZEB1-AS1和LncRNA SOX2OT与肾功能指标的相关性;Logistic分析影响DN患者肾功能损伤的因素。结果DN组血清LncRNA ZEB1-AS1、LncRNA SOX2OT水平低于对照组(t=11.471、10.257,P均<0.001)。血清LncRNA ZEB1-AS1、LncRNA SOX2OT比较,正常尿蛋白亚组>微量尿蛋白亚组>大量尿蛋白亚组(F=58.720、117.722,P均<0.001),BUN、SCr、UA水平比较,正常尿蛋白亚组<微量尿蛋白亚组<大量尿蛋白亚组,差异均有统计学意义(F=122.493、595.589、53.178,P均<0.001);LncRNA ZEB1-AS1、LncRNA SOX2OT分别与BUN、SCr、UA呈负相关(r=-0.487、-0.498、-0.521,-0.527、-0.515、-0.534,P均<0.001);Logistic回归分析显示,糖尿病病程长及高BUN、SCr、UA水平是影响DN患者肾功能损伤的危险因素[OR(95%CI)=1.672(1.128~2.479)、2.839(1.534~5.253)、2.754(1.512~5.017)、2.693(1.464~4.954)],高LncRNA ZEB1-AS1、LncRNA SOX2OT是保护因素[OR(95%CI)=0.875(0.798~0.959)、0.898(0.832~0.969)]。结论血清LncRNA ZEB1-AS1、LncRNA SOX2OT水平与DN患者肾功能有关,可能是评估DN患者肾功能的潜在指标。

RNPS1通过Notch信号通路参与调控胰腺癌细胞的生存及进展

目的:探讨富含丝氨酸结构域1的RNA结合蛋白(RNA-binding protein with serine-rich domain 1,RNPS1)在胰腺癌进展中的作用及可能分子机制。方法:免疫组织化学与免疫荧光检测RNPS1与Notch3在胰腺癌组织及癌旁组织的表达;RTq PCR、免疫荧光检测RNPS1与Notch3在胰腺癌细胞中的表达情况;Hoechst与CCK-8实验检测胰腺癌细胞凋亡与增殖;划痕实验与transwell实验检测胰腺癌细胞迁移与侵袭能力;Western blot实验检测胰腺癌细胞中N-Cadherin和E-Cadherin的表达;Western blot与RT-q PCR实验检测胰腺癌细胞中Notch3与HEY1的表达。结果:与癌旁组织与正常细胞系相比较,RNPS1与Notch3在胰腺癌组织中及胰腺癌细胞的表达均增高(F=121.612、34.649,均P<0.05);与对照组相比较,敲低RNPS1抑制生物标志物N-Cadherin的表达(t=39.922,P<0.05),促进E-Cadherin的表达(t=8.281,P<0.05),敲低RNPS1可减弱癌细胞的生存、迁移侵袭的能力(t=2.017、4.874、19.747,均P<0.05,),促进了细胞凋亡(t=33.673,P<0.05);敲低RNPS1降低了癌细胞中Notch3与HEY1的表达(t=17.546、6.258,均P<0.05)。结论:RNPS1的表达与胰腺癌细胞生存、恶性表型有关,RNPS1可能通过调控Notch3/HEY1信号通路促进胰腺癌细胞的生存及肿瘤进展。

高糖通过调控miR-429/ZEB1轴对胰腺癌细胞免疫逃逸的影响

目的探究高糖干预对胰腺癌细胞免疫逃逸的影响及作用分子机制。方法采用不同浓度葡萄糖(0、7.5、15、30 mmol/L)处理PANC-1细胞24 h构建高糖干预的PANC-1细胞。将miR-429 mimics及其阴性对照(mimics NC)转染至PANC-1细胞,分为对照组、HG组、HG+mimics NC组、HG+mimics组、HG+mimics+oe-NC组和HG+mimics+oe-ZEB1组。流式细胞术检测细胞表面分子细胞程序性死亡-配体1(PD-L1)表达水平;qRT-PCR检测细胞miR-429和锌指E-盒结合同源盒蛋白1(ZEB1)mRNA表达水平;Western blot检测细胞ZEB1蛋白表达水平。将以上各组PANC-1细胞与CD8^(+)T细胞建立共培养体系,CCK-8检测细胞增殖活性;流式细胞术检测细胞凋亡水平;乳酸脱氢酶(LDH)释放法检测CD8^(+)T细胞对PANC-1细胞的杀伤作用;采用双荧光素酶报告系统验证miR-429和ZEB1的靶向调控关系。结果HG可促进PANC-1细胞表面分子PD-L1及ZEB1表达(P<0.05),抑制miR-429表达,且呈浓度依赖性。miR-429过表达可显著抑制HG诱导的PANC-1细胞表面分子PD-L1表达,而过表达ZEB1可逆转miR-429过表达对HG诱导PANC-1细胞表面分子PD-L1表达的抑制作用。建立与CD8^(+)T细胞共培养体系后,与对照组比较,HG组PANC-1细胞增殖活性明显增加,细胞凋亡率和杀伤活性明显降低(P<0.05);与HG+mimics NC组比较,HG+mimics组PANC-1细胞增殖活性明显降低,细胞凋亡水平和杀伤活性明显升高(P<0.05)。与HG+mimics+oe-NC组比较,HG+mimics+oe-ZEB1组PANC-1细胞增殖活性明显增加,细胞凋亡率和杀伤活性明显降低(P<0.05)。双荧光素酶报告基因实验证实,miR-429靶向负调控ZEB1。结论高糖通过下调miR-429表达水平,靶向负调控ZEB1 mRNA的表达,提高PANC-1细胞表面分子PD-L1表达水平,进而促进PANC-1细胞免疫逃逸。

血清TyG指数和nesfatin-1及RBP4联合预测糖尿病视网膜病变的价值

目的:探究血清甘油三酯-葡萄糖(TyG)指数、摄食抑制因子-1(nesfatin-1)、视黄醇结合蛋白4(RBP4)联合预测糖尿病视网膜病变(DR)的价值,为DR早期预测提供支持。方法:回顾性分析。收集2022-02/2023-12我院接诊的2型糖尿病(T2DM)患者164例的临床资料,按照眼底检查结果分为DR组43例(其中增殖性DR 19例,非增殖性DR 24例),不合并DR的T2DM组121例。入院后记录患者基本资料,检查血清TyG指数、nesfatin-1、RBP4水平。结果:DR组病程长于T2DM组,空腹血糖、糖化血红蛋白、甘油三酯、总胆固醇、低密度脂蛋白及TyG指数、RBP4水平高于T2DM组,高密度脂蛋白、nesfatin-1水平低于T2DM组(均P<0.001)。多因素Logistic回归分析可知,T2DM病程(OR=1.338,95%CI:1.059-1.690)、糖化血红蛋白(OR=5.065,95%CI:1.659-15.470)、低密度脂蛋白(OR=12.715,95%CI:2.385-67.790)、TyG指数(OR=23.057,95%CI:2.936-181.073)、RBP4(OR=1.319,95%CI:1.028-1.692)是T2DM患者发生DR的危险因素,nesfatin-1(OR=0.007,95%CI:0.003-0.016)为保护因素。绘制ROC曲线显示,TyG指数、nesfatin-1、RBP4均对T2DM患者并发DR具有一定预测价值,曲线下面积(areas under curve,AUC)分别为0.804、0.878、0.738,各指标联合预测时AUC为0.946,预测敏感度为83.72%、特异度为92.56%。增殖性DR患者TyG指数、RBP4水平高于非增殖性DR患者,nesfatin-1水平低于非增殖性DR患者(均P<0.05)。Spearman相关性分析显示,TyG指数、RBP4水平与DR病情程度呈正相关,nesfatin-1水平与DR病情程度呈负相关(r_(s)=0.557、0.392、-0.359,均P<0.05)。Pearson相关分析显示,T2DM并发DR患者TyG指数与nesfatin-1水平呈负相关,与RBP4水平呈正相关,nesfatin-1与RBP4水平呈负相关(r=-0.486、0.538、-0.592,均P<0.05)。结论:血清TyG指数、nesfatin-1、RBP4水平与DR发病风险及病情程度有关,可作为DR早期预测的标志物,且联合预测效能更好。