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Objective To observe the expression of insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) in human pancreatic tumor tissues and investigate its significance and relationship with clinic pathological characteristics and tumor microenvironment of the pancreatic neoplasms.Methods A total of 236 patients with surgically resected pancreatic tissue from January 2007 to December 2017 were selected from the First Hospital of Shanxi Medical University.Totally 236 patients were divided into paracancer control group (normal pancreatic tissue adjacent to the tumor,n =111),benign group (benign or low-grade malignant tumor such as solid pseudopapillary tumor,n =37),and malignant group (malignant tumors such as pancreatic ductal adenocarcinoma,n =88).The histomorphology and collagen deposition were observed using hematoxylin-eosin (H-E) staining and Sirius red staining in the three groups.The expressions of IGFBPrP1,transforming growth factor β1 (TGFβ1),α-smooth muscle actin (α-SMA)and collagen type Ⅰ of pancreatic tissues in the three groups were detected by immunohistochemical staining.The relationship between IGFBPrP1 and TGFβ1,α-SMA or collagen type Ⅰ,and the relathionship between IGFBPrP1 and clinicopathological features of the pancreatic neoplasms were analyzed.T test and one-way analysis of variance were used for statistical analysis,and Spearman rank correlation test was used for correlation analysis.Results In benign group and malignant group,there were obvious cell atypia,and the cell atypia of malignant group was more significant than benign group.The contents of collagen fibers in benign group and malignant group were significantly higher than that in paracancer control group.IGFBPrP1,TGFβ1,α-SMA and collagen type Ⅰ were highly expressed in the endochylema of the tumor cells and (or) the myofibroblast.The expression level of IGFBPrP1 in highly differentiated ductal adenocarcinoma was significantly higher than that in moderately and poorly differentiated ductal adenocarcinoma ((9.46 ± 2.10) × 104 vs.(6.48 ± 1.38) × 104 and (6.07 ± 1.29) × 104);t =7.430 and 6.767,both P ＜ 0.05).The expression of IGFBPrP1 in human pancreatic neoplasms was positively correlated with TGFβ1,α-SMA and collagen type Ⅰ (r=0.530,0.619,0.625;all P ＜0.05).Conclusions IGFBPrP1 is highly expressed in pancreatic tumor tissue and its expression level may correlate with the histological grade of pancreatic neoplasms.The expression of IGFBPrP1 in human pancreatic tumor tissues may be accompanied by the activation of pancreatic stellate cells and the generation of cancer-related fibroblasts,and IGFBPrP1 may involve in the formation of tumor by changing the tumor microenvironment.
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Objective To identify the effect of IGFBPrP1 on the secretion of extracellular matrix in hepatic stellate cells through the Smad3 signaling pathway. Methods (1)Two pairs of chemically synthesized siRNAs (siRNA1, siRNA2) targeting Smad3 were transfected into HSC-T6 cells,real-time PCR and Western blot were used to evaluate the silence efficiency, and the better siRNA was used. (2)HSC-T6 cells were divided into four groups: Negative control group, siRNA-Smad3 transfection group, siRNA-Smad3+IGFBPrP1 group and IGFBPrP1 group. The better siRNA was chosen to transfect into HSC-T6 cells. The protein expressions of Smad3, fibronectin and Collagen Ⅰ were evaluated by Western blot. Results (1)siRNA2-Smad3 inhibited Smad3 gene expression stronger than another siRNA. (2)After transfection of siRNA2-Smad3, the protein expression of Smad3 was significantly decreased compared to the negative control group(P＜0.01). The protein expression of fibronectin and Collagen Ⅰ in IGFBPrP1 stimulating HSCs treated with siRNA2-Smad3 were significantly decreased compared to that in IGFBPrP1 stimulating HSC without siRNA2-Smad3 (P ＜0. 01 ).Conclusion IGFBPrP1 induces the secretion of extracellular matrix in hepatic stellate cells through the Smad3 signaling pathway.
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Objective To investigate the effect and mechanism of exogenous IGFBPrP1 on collagen content in liver tissue. Methods Twenty-four male C57BL/6 wild-type mice were randomly divided into three groups: Control group (n=8), rmIGFBPrP1 2 weeks group (n=8) and rmIGFBPrP1 4weeks group (n=8). Both hematoxylin-eosin (HE) staining and picric acid-Sirius red staining were performed. The protein expression of IGFBPrP1, Collagen Ⅰ , Collagen Ⅲ, FN, TGF-β1, Smad3 andp-Smad2/3 was evaluated by immunohistochemistry or Western blot. Results Exogenous IGFBPrP1 can cause pathological changes in liver tissue. Collagen content was significantly increased by hematoxylin-eosin (HE) staining and picric acid-Sirius red staining (P＜0.05). The protein expression of IGFBPrP1, FN and Collagen Ⅰ was gradually increased after rmIGFBPrP1 injection for 2 weeks and 4 weeks by Western blot (P＜0.01). The protein expression of Collagen Ⅲ was obviously increased in the rmIGFBPrP1 2 weeks group and rmIGFBPrP1 4 weeks group by immunohistochemistry, and the level in the 4 weeks group was higher than that in the 2 weeks group (P＜0. 01). The protein expression of TGF-β1, Smad3 and p-Smad2/3 in liver tissue was significantly increased after rmIGFBPrP1 injection in a time-dependent manner by both immunohistochemistry and Western blot (P＜0. 01).Conclusion Exogenous IGFBPrP1 can cause a marked increase in collagen content and the excessive deposition of ECM through the TGF-β1/Smad3 pathway in liver tissue.
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Background Antagonists against vascular endothelial growth factor (VECF) play key roles in treating and preventing neovascular ophthalmopathy. As a novel anti-angiogenic factor, insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) might be an antagonist against VEGF in eye. Objective This study was to explore the inhibitory effect of IGFBP-rP1, a novel anti-angiogenic factor, on VEGF-induced retinal angiogenesis in vitro. Methods The retina-choroid endothelial cell line ( RF/6A ) was cultured in DMEM containing 10% fetal bovine serum. Culture cells were divided into control group(free-serum culture group) ,10mg/L VEGF culture group and different concentrations of IGFBP-rP1 (50,100,200 mg/L) +10 mg/L VEGF group. The expression of IGFBP-rP1 in the cells was detected by immunofluorescence assay. The proliferation and migration of RF/6A cells were evaluated using MTS colorimetric assay and the chemotactic motility assay, respectively. Flow cytometry was used to detect the apoptosis of RF/6A cells. Results The immunofluorescence assay RF/6A cells showed the green fluorescence in cytoplasm and red fluorescence in nuclei after cells were exposed to any concentration of IGFBP-rP1 ,but only red fluorescence was seen in nuclei in control cells. After stimulation of 10 mg/L VEGF,the proliferation value (A490) was elevated and the numbers of cell migration were increased in comparison with control group (t = -15. 191, P = 0. 000; t = -21. 274, P = 0. 000 ) , but the cellular apoptosis rate was lower than the control group (t - 10. 228, P = 0. 000 ) . After treated with various concentrations of IGFBP-rP +10% VEGF, the proliferation and migration of RF/6A cells were significantly decreased in comparison with only 10% VEGF group (F = 534. 158,P = 0. 000;F = 2742. 323,P = 0.000,respectively) ,and the inhibitory effects were gradually enhanced with the increase of IGFBP-rP1 levels (P<0. 05). The apoptosis rate of RF/6A cells in 50,100 and 200 mg/L + 10 mg/L VEGF groups increased by ( 1. 26±0. 04)% ,( 1. 50±0. 07)% and ( 1. 93±0. 27)% respectively,showing significant differences among different groups ( F = 274. 273, P = 0. 000). Conclusion IGFBP-rP1 inhibits the proliferation and activity of retina and choroid endothelial cells induced by VEGF at a concentration-independent manner. It appears to be as a novel endogenous inhibitory factor in retinal angiogenesis.
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Objective To investigate the effect of insulin-like growth factor binding protein-related protein 1 ( IGFBPrP1 ) in the formation and development of hepatic fibrosis. Methods Hepatic fibrosis model of mice was made by intraperitoneal injecting with thioacetamide, then the mice were sacrificed four, five and six weeks later (10 mice were sacrificed at each time point, model groups). Mice in the control groups were treated by normal saline (6 mice were sacrificed at each time point). Collagen accumulation in liver tissues was detected by Masson stain. Distribution and dynamic expressions of IGFBPrP1, alpha-smooth muscle actin ( α-SMA ),Collagen Ⅰ , fibronectin ( FN), TGF-β1 and Smad3 in different groups were detected by immunohistochemistry.The expressions of IGFBPrP1, α-SMA and Smad3 were detected by Western blot. All data were analyzed using the analysis of variance (ANOVA), Pearson rank correlation coefficient. Results The expressions of IGFBPrP1 in the liver tissues were increased from 0.21 ±0.03 to 5.03 ±0.09, α-SMA from 0. 11 ±0.04 to 10.09 ±0. 18,Collagen Ⅰ from 0.22 ±0.01 to 11.01 ±0. 16, FN from 0.31 ±0.09 to 19.81 ±1.62, TGF-β1 from 0.49 ±0.02 to 5.97 ± 0. 19, and Smad3 from 0.22 ± 0.03 to 2.03 ± 0.07. Compared with the control groups, the expressions of IGFBPrP1, α-SMA, Collagen Ⅰ , FN, TGF-β1 and Smad3 in the model groups were significantly increased as time passed by ( F = 783. 141,998. 200,886. 715,935. 242, 931. 241,697. 118, P < 0. 05 ). During the formation of hepatic fibrosis, the expression of IGFBPrP1 was positively correlated with the expressions of α-SMA,Collagen Ⅰ , FN, TGF-β1 and Smad3 ( r = 0. 906, 0. 927, 0. 988, 0. 947, 0. 977, P < 0.05 ). The results of Western blot showed that the protein expression of IGFBPrP1 was increased from 0. 23 ± 0.01 to 0.92 ± 0.07,α-SMA from 0.36 ± 0. 02 to 1.39 ± 0.03, FN from 0.03 ± 0.00 to 0.12 ± 0.02, and Smad3 from 0.09 ± 0. 01 to 0.56 ±0.04. The protein expressions of IGFBPrP1, α-SMA, FN and Smad3 were significantly increased in the model groups when compared with the control groups (F =57. 316, 201. 214, 103. 871, 72. 966, P <0.05).During the formation of hepatic fibrosis. IGFBPrP1 was positively correlated with the expressions of α-SMA, FN and Smad3 (r = 0. 982, 0. 924, 0. 965, P < 0.05 ). Conclusions The expression of IGFBPrP1 increases as the aggravation of the fibrosis. IGFBPrP1 promotes the formation and development of hepatic fibrosis by activating hepatic stellate cells, accelerating the synthesis and secretion of Collagen Ⅰ and FN which are the principal components of extracellular matrix, and affecting the TGF-β1/Smad3 pathway.