Osteopontin promotes gastric cancer progression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.

Osteopontin研究进展

Osteopontin (OPN)是一种分泌型钙结合磷蛋白 ,它能与矿化骨组织中的羟磷灰石紧密结合 ,参与调节骨钙的沉积。OPN通过其RGD序列与整合素αvβ3受体的相互作用 ,在成骨细胞和破骨细胞与骨基质结合的过程中起重要作用。此外 ,OPN与Ca2 +、NO等细胞的第二信使 ,肿瘤细胞的转化、浸润和转移 ,平滑肌细胞增生及表型转换 ,动脉粥样硬化 ,感染等的发生发展等多种生理、病理现象密切相关 ,是一种新的细胞信号分子。

Osteopontin在蛛网膜下腔出血后脑血管痉挛发生中的作用机制研究

目的研究Osteopontin(骨桥蛋白)在蛛网膜下腔出血后脑血管痉挛发生中的作用机制。方法收集2015年2月1—10日SD大鼠60只作为研究对象,采用电脑随机的方式进行分组,分为20只假手术加安慰剂组、20只SAH(蛛网膜下腔出血)加Osteopontin组、20只SAH加安慰剂组三组,对三组大鼠研究结果进行分析。结果在首次注血72 h后,三组大鼠的死亡率相比,具有显著差异(P<0.05); SAH加Osteopontin组神经功能评分与其他两组神经功能评分具有显著差异(P<0.05);与假手术加安慰剂组进行比较,SAH加安慰剂组基底动脉横截面积显著缩小,管壁厚度增加(P<0.05);与SAH加安慰剂组相比较,SAH加Osteopontin组的管壁厚度缩小、基底动脉横截面积显著增加(P<0.05); SAH加Osteopontin组ET-1浓度均低于其他两组(P<0.05)。结论蛛网膜下腔出血后采用Osteopontin,能有效预防脑血管痉挛发生,从而减少大鼠血管内皮细胞凋亡,对改善大鼠神经功能十分重要。

培哚普利对心肌梗死大鼠左室重构和心肌组织osteopontin蛋白表达的影响

目的观察血管紧张素转换酶抑制剂培哚普利对心肌梗死大鼠左室重构和心功能的影响,并探讨osteopontin蛋白在其中的作用。方法将成年雄性SD大鼠随机分为3组:假手术组,心肌梗死盐水组和心肌梗死培哚普利组。后两组动物在冠状动脉左前降支结扎第2天起给予生理盐水或者培哚普利灌胃,1次/d。4周后测定有创左室血流动力学,超声心动图测量左室内径和射血分数,组织学方法检测非梗死区心肌细胞直径和胶原纤维沉积,Westernblot检测心肌组织osteopontin蛋白表达水平。结果与假手术组相比,所有心肌梗死大鼠(盐水组和培哚普利组)均出现显著的左室收缩和舒张功能障碍,表现为LVEF、LVSP和±dp/dtmax下降,LVEDP显著升高。同时左室重量与体质量比值升高,LVEEDD和LVESD增大,非梗死区心肌细胞横径增加,心肌间质胶原纤维沉积增加,提示显著的左室重构。与盐水组相比,培哚普利治疗显著改善心肌梗死大鼠心功能,预防左室扩大,减轻非梗死区心肌细胞肥大和心肌间质胶原纤维沉积。Westernblot未检测到osteopontin蛋白在假手术大鼠心肌组织表达,在心肌梗死大鼠心肌组织有大量osteopontin蛋白表达,该上调的蛋白能被培哚普利治疗显著抑制。结论培哚普利抑制心肌梗死大鼠左室重构,改善心功能,可能与其抑制osteopontin蛋白表达有关。

Osteopontin基因SNP rs2728127和rs34527305与鼻咽癌易感性的相关性

目的研究鼻咽癌相关基因osteopontin SNP rs2728127和rs34527305对鼻咽癌易感性的影响。方法采用多重单碱基延伸PCR技术,对264例广西百色地区鼻咽癌患者和264例健康对照者的osteopontin基因SNP rs2728127和rs34527305进行基因分型,比较广西百色地区人群rs2728127和rs34527305基因型和等位基因型频率与其他地区人群的差异,分析rs2728127和rs34527305对鼻咽癌易感性的影响。结果广西百色地区人群osteopontin基因SNP rs2728127和rs34527305基因型和等位基因型频率与云南西双版纳地区人群差异无统计学意义(P>0.05),但是与北京、越南胡志明、日本东京[除rs34527305等位基因型频率与北京相比较差异无统计学意义(P>0.05)外]、秘鲁利马和芬兰地区人群相比差异有统计学意义(P<0.05)。对照组和病例组rs2728127和rs34527305基因型和等位基因型频率分布差异无统计学意义(P>0.05)。但是携带GA单倍型的个体相比携带AA单倍型个体罹患鼻咽癌的风险更低(OR=0.69,95%CI=0.48~0.96,P=0.038)。结论在广西百色地区人群中,osteopontin基因SNP rs2728127和rs34527305基因型和等位基因型可能与鼻咽癌的易感性无关,但是其GA单倍型可能降低鼻咽癌的风险。

Osteopontin特异性siRNA真核表达载体的构建及其在GC9811胃癌细胞中沉默效应的鉴定

目的:构建骨桥蛋白(OPN)特异性siRNA(smallin terfereRNA)真核表达载体,体外观察对OPN基因的沉默作 用.方法:采用基因克隆技术,将合成的特异性OPNRNA干 扰寡核苷酸序列插入真核表达载体mU6pro,构建OPNsiRNA 真核表达载体.以脂质体法将mU6pro空载体和2个 (mU6pro/OPN siRNA1和mU6pro/OPN siRNA2)重组质粒分 别导入具有高转移特性的GC9811胃癌细胞系.72h后用 RT PCR和Westernblotting技术检测各实验组胃癌细胞内 OPNmRNA及蛋白水平的表达情况.结果:成功构建OPN siRNA真核表达载体.转染OPN siRNA的胃癌细胞,72h后 OPNmRNA及蛋白表达下调,而以mU6pro/OPN siRNA1的抑 制效果更为明显.结论:构建的RNA干扰真核表达载体能明 显干扰OPNmRNA及蛋白的表达,为将其进一步应用于胃癌 的治疗研究奠定了基础.

Osteopontin: A non-invasive parameter of portal hypertension and prognostic marker of cirrhosis

AIM: To investigate the relationship between osteopontin plasma concentrations and the severity of portal hypertension and to assess osteopontin prognostic value.METHODS: A cohort of 154 patients with confirmed liver cirrhosis(112 ethylic, 108 men, age 34-72 years)were enrolled in the study. Hepatic venous pressure gradient(HVPG) measurement and laboratory and ultrasound examinations were carried out for all patients. HVPG was measured using a standard catheterization method with the balloon wedge technique. Osteopontin was measured using the enzyme-linked immunosorbent assay(ELISA) method in plasma. Patients were followed up with a specific focus on mortality. The control group consisted of 137 healthy age- and sex- matched individuals.RESULTS: The mean value of HVPG was 16.18 ± 5.6 mm Hg. Compared to controls, the plasma levels of osteopontin in cirrhotic patients were significantly higher(P < 0.001). The plasma levels of osteopontin were positively related to HVPG(P = 0.0022, r = 0.25) and differed among the individual Child-Pugh groups of patients. The cut-off value of 80 ng/m L osteopontin distinguished patients with significant portal hypertension(HVPG above 10 mm Hg) at 75% sensitivity and 63% specificity. The mean follow-up of patients was 3.7 ± 2.6 years. The probability of cumulative survival was 39% for patients with HVPG > 10 mm Hg and 65% for those with HVPG ≤ 10 mm Hg(P = 0.0086, odds ratio(OR), 2.92, 95% confidence interval(CI): 1.09-7.76). Osteopontin showed a similar prognostic value to HVPG. Patients with osteopontin values above 80 ng/m L had significantly lower cumulative survival compared to those with osteopontin ≤ 80 ng/m L(37% vs 56%, P = 0.00035; OR = 2.23, 95%CI: 1.06-4.68).CONCLUSION: Osteopontin is a non-invasive parameter of portal hypertension that distinguishes patients with clinically significant portal hypertension. It is a strong prognostic factor for survival.

Osteopontin knockdown suppresses the growth and angiogenesis of colon cancer cells

AIM:To investigate the effects of osteopontin(OPN)gene expression knockdown on colon cancer Lovo cells in vitro.METHODS:Four candidate small interfering RNA(siRNA)constructs targeting the OPN gene and a scrambled control sequence(NC-siRNA)were synthesized and inserted into a pGPU6/GFP/Neo expression vector.After confirmation by restriction enzyme digestion and DNA sequencing,the recombinant plasmids were subsequently transfected into a human colon cancer cell line(Lovo)using a liposome transfection method.Stably transfected cells were maintained with G418 selection and referred to as Lovo-OPN-1,-2,-3,-4,and Lovo-NC cells.Knockdown efficiency of each of the four siRNA constructs was determined by realtime reverse transcription polymerase chain reaction assays and western blotting,and the construct with the most effective silencing was used for subsequent experiments.Cell proliferation,adhesion,and Matrigel invasion assays were performed to analyze the effects of OPN knockdown in stably transfected Lovo cells.The levels of four angiogenic factors,namely vascular endothelial growth factor(VEGF),matrix metalloproteinase(MMP)-2,MMP-9 and urokinase plasminogen activator were detected by enzyme-linked immunosorbent assays(ELISA).RESULTS:Recombinant vectors containing OPNspecific and scrambled siRNA sequences were successfully constructed and stably transfected into Lovo cells.Compared with the control Lovo and Lovo-NC cells,the levels of OPN mRNA and protein expression in LovoOPN-1,-2,-3,and-4 were significantly reduced(all P<0.05),with the most efficient reduction observed in Lovo-OPN-4 cells(P<0.05).Relative to untransfected Lovo cells,OPN mRNA expression levels in Lovo-NC and Lovo-OPN-4 cells were 1.008±0.067 and 0.160±0.023,respectively.The relative OPN protein expression levels in Lovo,Lovo-NC,and Lovo-OPN-4 cells were 3.024±0.211,2.974±0.630,and 0.121±0.008,respectively.Moreover,transfection with the scrambled sequence had no effect on the expression of OPN.After24,48,72,and 96 h of cultivation,absorption values at 450 nm to assess proliferation of Lovo-OPN-4 cells were 0.210±0.017,0.247±0.024,0.314±0.037,and 0.359±0.043,respectively,which were significantly lower than those of Lovo(0.244±0.031,0.313±0.024,0.513±0.048 and 0.783±0.051)and LovoNC cells(0.241±0.029,0.309±0.022,0.563±0.023,and 0.735±0.067)(all P<0.05).The absorption values at 595 nm,which were measured in a cell adhesion assay,showed that adhesion of Lovo-OPN-4 cells(0.215±0.036)was significantly decreased compared to Lovo(0.490±0.037)and Lovo-NC cells(0.462±0.043)(P<0.05).The number of invasive Lovo-OPN-4 cells(16.1±1.9)was also significantly decreased compared to Lovo(49.9±5.4)and Lovo-NC cells(48.8±4.5)(P<0.05).ELISA assays showed significant reductions in Lovo-OPN-4 cells compared to Lovo and Lovo-NC cells with regard to the expression of VEGF(1687.85±167.84 ng/L vs 2348.54±143.80 ng/L and 2284.39±138.62 ng/L,respectively),MMP-2(2966.07±177.36μg/L vs 4084.74±349.54μg/L and 4011.41±424.48μg/L,respectively),MMP-9(3782.89±300.64μg/L vs5062.90±303.02μg/L and 4986.38±300.75μg/L,respectively)and uPA(1152.69±120.79μg/L vs1380.90±147.25μg/L and 1449.80±189.92μg/L,respectively)(all P<0.05).CONCLUSION:Knockdown of OPN gene expression suppresses colon cancer cell growth,adherence,invasion,and expression of angiogenic factors.

Increased osteopontin and liver stiffness measurement by transient elastography in biliary atresia

AIM: To analyze plasma osteopontin levels and liver stiffness using transient elastography in postoperative biliary atresia (BA) children compared with healthy controls. METHODS: Thirty children with postoperative BA and 10 normal controls were enrolled. The patients were categorized into two groups according to their jaundicestatus. Plasma levels of osteopontin were determined using commercially available enzyme-linked immunosorbent assay. Liver stiffness was measured by using transient elastography (Fibroscan). Ten validated Fibroscan measurements were performed in each patient and control with the result expressed in kilopascals (kPa). RESULTS: Plasma osteopontin was significantly elevated in BA children compared with that of healthy controls (47.0 ± 56.4 ng/mL vs 15.1 ± 15.0 ng/mL, P = 0.01). The liver stiffness measurement was markedly elevated in the patients with BA compared with that of controls (26.9 ± 24.6 kPa vs 3.9 ± 0.7 kPa, P = 0.001). Subgroup analysis showed that the BA patients with jaundice had more pronounced plasma osteopontin levels than those without jaundice (87.1 ± 61.6 ng/mL vs 11.9 ± 6.1 ng/mL, P = 0.001). Furthermore, the mean liver stiffness was significantly greater in the jaundiced BA patients compared with non-jaundiced patients (47.7 ± 21.8 kPa vs 8.7 ± 3.0 kPa, P = 0.001). Additionally, plasma osteopontin was positively related to serum total bilirubin (r = 0.64, P < 0.001). There was also a correlation between plasma osteopontin and liver stiffness values (r = 0.60, P < 0.001). CONCLUSION: High plasma osteopontin positively correlated with degree of hepatic fibrosis and could be used as a biochemical parameter reflecting disease severity in postoperative BA children.

FGF-2和osteopontin在非小细胞肺癌中的表达及其相关性

目的:探讨成纤维细胞生长因子-2(fibroblast growth factor-2,FGF-2)和骨桥蛋白(osteopon-tin,OPN)在非小细胞肺癌(non-small cell lung cancer,NSCLC)组织中的表达及其相关性。方法:应用免疫组织化学SP法分析76例NSCLC组织及15例正常肺组织中FGF-2和OPN蛋白的表达水平。采用RT-PCR和Western印迹检测FGF-2对人肺腺癌细胞株A549中OPN mRNA及蛋白表达水平的影响。结果:NSCLC组织中FGF-2和OPN的阳性率分别为65.8%(50/76)和60.5%(46/76),显著高于正常肺组织13.3%(2/15)和0(0/15)(P<0.01),FGF-2和OPN的阳性表达与淋巴结转移、临床分期密切相关(均P<0.01),与年龄、性别和病理分型无明显相关(均P>0.05)。两者表达在NSCLC中呈正相关(r=0.552,P<0.01)。FGF-2可以明显上调A549细胞中OPN mRNA和蛋白的表达(P<0.05)。结论:FGF-2和OPN的高表达与NSCLC的发生及侵袭转移有关,FGF-2的促癌机制可能部分通过上调OPN的表达。

Expression of NF-κ B and osteopontin of synovial fluid of patients with knee osteoarthritis

Objective:To explore the significance of osteopontin and nuclear factorκB(NF-κB) expression in patients with knee osteoarthritis.Methods:RT-PCR and enzyme-linked immunosorbent assay were used to measure the Osteopontin(OPN) and NF-κB concentration of knee joint synovial fluid of patients with knee osteoarthritis and trauma fractures,and analyze the relationship between the expressiones of them.Results:OPN and NF-κB expression at the mRNA and protein levels of patients with knee osteoarthritis were significantly higher than the control group, the result showed statistical significance(P【0.05).There was a positive correlation between the OPN levels in synovial fluid of patients with knee osteoarthritis and NF-κB expression levels (P【0.05).Conclusions:The high expression of OPN and NF-κB are closely related to occurrence and development of knee osteoarthritis.

Osteopontin特异性siRNA慢病毒表达载体的构建及其在U251胶质瘤细胞中OPN的表达

目的:观察慢病毒表达载体介导的RNA干扰(RNAinterference,RNAi)对人胶质瘤细胞U251骨桥蛋白(osteopon-tin,OPN)表达的影响,为后续的以OPN基因为靶点的神经胶质瘤基因治疗研究奠定基础.方法:应用基因工程技术,筛选出2条针对OPN基因的RNAi靶序列,分别与pGCL-GFP载体连接,构建2个重组慢病毒表达载体LV-OPNshRNA1,LV-OPNshRNA2;将连接产物转化到DH5α感受态细胞,经PCR筛选阳性克隆、测序鉴定.将LV-OPNshRNA,pHelper 1.0,pHelper 2.0共转染293T细胞,包装产生慢病毒颗粒并测定病毒滴度.将包装产生的2种重组慢病毒分别感染U251细胞,实时定量PCR和Western印迹检测U251细胞OPN mRNA和蛋白的表达,并与未转染及空转染细胞进行比较.结果:2个慢病毒载体PCR和测序结果与预期结果一致,经包装产生的病毒滴度为8×1010TU/L和5×1010TU/L.感染U251细胞后,OPN基因mRNA和蛋白的表达量与未感染慢病毒的细胞组及空载体感染组相比均明显下降.结论:成功构建针对OPN基因的2个慢病毒载体OPN shRNA,体外感染U251细胞后可有效抑制OPN基因和蛋白的表达.

Implication of Expression of Osteopontin and Its Receptor Integrin αvβ3 in the Placenta in the Development of Preeclampsia

To investigate the expression of osteopontin （OPN） and its receptor integrin αvβ3 in the placental tissue from pregnant women complicated with preeclampsia, the expression of OPN and αvβ3 in the placenta of the pregnant women with preeclampsia and healthy pregnant women was detected by immunohistochemistry, Western blotting and RT-PCR. Our results showed that OPN and αvβ3 protein were expressed in the placenta from normal pregnant woman and those with preeclampsia. OPN was located in the placental syncytiotrophoblasts and the cytoplasm of capillary endothelial cells and integrin αvβ3 was mainly expressed on the surface of trophoblast cells. Expression of OPN and integrin αvβ3 in the placental tissue from preeclampsia subjects was significantly lower than that from the control group （P〈0.05）. Compared with the control group, expression of OPN in the placental tissue from preeclampsia group was significantly lower （P〈0.05） but there was no significant difference in the expression of αv and β3 between the preeclampsia group and the controls. It is concluded that OPN and its receptor integrin αvβ3 may be involved in the pathogenesis of preeclampsia.

Influence of Osteopontin Short Hairpin RNA on the Proliferation and Activity of Rat Vascular Smooth Muscle Cells

To investigate the influence of osteopontin （OPN） short hairpin RNA （shRNA） on the proliferation and activity of rat vascular smooth muscle cells （VSMCs）, the expressing vector of shRNA targeting OPN was constructed and transferred into the rat VSMCs. After amplification and purification, pGenesil-1/OPNshRNA1 （PG1）, pGenesil-1/OPNshRNA2 （PG2） and pGenesil-1/OPNshRNAHK （PGH） were transfected into the cultured rat VSMC by LipofectamineTM 2000. Transfected cells were visualized by using an inverted fluorescent microscope. VSMCs transfected by optimal recombined plasmid was selected by culturing in G418 48 h later. Nude cells and cells transfected by PGH were used as control. The expression levels of OPN mRNA and protein were assayed by RT-PCR and Western blotting. The OPN of VSMCs was suppressed by transfection of optimal recombined plasmid, and the changes in cell proliferation, adhesion and motility were evaluated by MTT, adhesion test and transwell chamber test. Levels of type I and Ⅲ collagen were measured with ELISA kit. Our results showed that VSMCs stably transfected by OPN shRNA accounted for over 50% of total cells. OPN mRNA and protein were reduced by 81% and 67% （P〈0.01） by PG1, 73% and 52% （P〈0.01） by PG2, respectively while no change was found in PGH and non-treated VSMCs. PG1 significantly suppressed the proliferation, adhesion, mobility of VSMCs and reduced the amount of type Ⅰ and Ⅲ collagen. It is concluded that recombinant plasmid can be success-fully transfected into VSMCs by LipofectamineTM 2000 and inhibit the expression of OPN. The proliferation, adhesion and mobility of VSMCs can be inhibited by knocking down OPN expression. Moreover, the transferring capability of cells is attenuated, and the secretion of type Ⅰ and Ⅲ collagen is inhibited aftter knocking-down of OPN expression. The study provides experimental evidence for clinical prevention of restenosis after percutaneous coronary intervention （PCI） by RNA interference （RNAi） technology.

Osteopontin expression in vitreous and proliferative retinal membranes of patients with proliferative vitreous retinopathy

AIM: To analyze osteopontin (OPN) expression in vitreous and proliferative retinal membranes of patients with proliferative vitreous retinopathy (PVR). METHODS: A total of 54 vitreous fluid samples were obtained between 2009 and 2010, which contained 45 with PVR (group A) and 9 without PVR (group B). Enzyme-linked immunosorbent assay was applied to quantify the OPN concentrations in vitreous fluid. Four samples of proliferative retinal membrane were also obtained at the time of vitrectomy, and their contents of OPN were measured by Real-time RT-PCR. RESULTS: The OPN levels in the vitreous fluid were 778.48+/- 62.06ng/mL in group A and 452.99 32.52ng/mL in group B. The vitreous OPN levels in group A were significantly higher than those in group B and to rise by time in the early stages of PVR. The average OPN levels in the proliferative retinal membranes (F =0.14) were also higher than those in the retinal pigment cells (F =0) using Real-time RT-PCR. CONCLUSION: The high vitreous and proliferative retinal membrane OPN levels in PVR suggest that OPN might promote the development of PVR. The vitreous OPN concentrations are rising by the time in the early phases of PVR.

Osteopontin is a biomarker for early autoimmune uveoretinitis

Osteopontin(OPN)is an extracellular matrix protein with a diverse range of functions,including roles in cell adhesion,migration,and immunomodulation,which are associated with the modulation of neuroinflammation in the central nervous system.The present study was performed to evaluate the involvement of OPN in the eyes of an experimental autoimmune uveoretinitis(EAU)model.The EAU model was developed by immunization of Lewis rats with interphotoreceptor retinoid-binding protein.The results showed the OPN level was remarkably upregulated in the eye of EAU rats on day 9 post-immunization.The level of CD44,a ligand of OPN,was increased in the ciliary body of EAU rats.Furthermore,OPN was also detected in the ciliary body and activated microglia/macrophages in the EAU retina.The results suggest that OPN was significantly upregulated in the eyes of EAU rats,and that it may be useful as an early biomarker of ocular autoimmune diseases.All animal experiments were approved by the Institutional Animal Care and Use Committee of Jeju National University(approval No.2020-0012)on March 11,2020.

Influence of Osteopontin Short Hairpin RNA on the Proliferation and Invasion of Human Renal Cancer Cells

The influence of short hairpin RNA(shRNA)-mediated osteopontin(OPN)gene silencing on the proliferation and invasion of human renal cancer ACHN cells was investigated.Four types of OPN shRNA recombinant plasmids were constructed and RT-PCR assays were used to screen the most highly functional shRNA recombinant plasmids,which were transferred into the cultured ACHN cells by LipofectamineTM 2000.The cells transfected by shRNA expression vectors(ACHN/OPN)were visualized under an inverted microscope and screened...

The Elevated Expression of Osteopontin and NF-κB in Human Aortic Aneurysms and Its Implication

The expression and significance of osteopontin （OPN） and NF-κB in patients with thoracic aortic aneurysm （TAA） and abdominal aortic aneurysm （AAA） were investigated. Thirteen TAA specimens, 20 AAA specimens and 6 normal aortic specimens were collected. The expression of OPN, nuclear factor-κB P65 （NF-κB P65）, urokinase plasminogen activator （uPA）, matrix metalloproteinase-2 （MMP-2） and matrix metalloproteinase-9 （MMP-9） were detected by using immunohisto-chemistry and Western blotting was employed to determine the expression of OPN and NF-κB P65. Immunohistochemical results showed that the expression of OPN, NF-κB P65, uPA, MMP-2 and MMP-9 was positive in all TAA and AAA specimens and negative in normal specimens, with the difference being statistically significant （P〈0.05）. There was no difference in the expression between TAA and AAA specimens （P〉0.05）. Correlation analysis revealed that there existed a positive correlation between the expression of OPN and that of NF-κB P65, uPA, MMP-2 and MMP-9 and between the expression of NF-κB P65 and that of uPA, MMP-2, MMP-9 （P〈0.05）. Western blotting demonstrated that OPN and NF-κB P65 were positive in AAA and TAA specimens, and negative in normal specimens with the differences being statistically significant （P〈0.05）. There were no statistically significant differences in the expression of OPN and NF-κB P65 between AAA and TAA specimens （P〉0.05）. It was concluded that OPN and NF-κB P65 were involved in the pathogenesis of TAA and AAA. OPN can up-regulate the expression of MMP and uPA via NF-κB signaling pathway thereby accelerating the degradation of extracellular matrix and playing an important role in the pathogenesis and development of TAA and AAA.

Expression of Phosphorylated-STAT3 and Osteopontin and Their Correlation in Melanoma

The expressions of p-STAT3 and osteopontin in 22 cases of normal nevi and 43 cases of malignant melanoma were immunohistochemically detected, and the correlation between p-STAT3 and osteopontin in malignant melanoma and the correlations of p-STAT3 （or osteopontin） with invasion, metastasis and thickness of malignant melanoma were examined. The results showed p-STAT3 was expressed in 2 of 22 cases of normal nevi and 30 of 43 cases of malignant melanoma, while osteopontin was expressed in 3 cases of normal nevi and 29 cases of malignant melanoma. The expressions of p-STAT3 and osteopontin in melanoma were significantly higher than that in benign nevi. There existed significant correlations between the expression of p-STAT3 and that of osteopontin in melanoma. Furthermore, the expression rates of p-STAT3 were significantly higher in invasive or metastatic melanomas than that their non-invasive or non-metastatic counterparts, and the expression rates of osteopontin were significantly higher in invasive melanomas than that in non-invasive ones. It is concluded that p-STAT3 and osteopontin may play important roles in the pathogenesis of malignant melanoma.

Rapid Purification and Identification of Osteopontin from Human Milk by HPLC Coupled to FT-ICR-MS

Milk as a key element for infant nutrition represents the only source of feeding for newborns and infants, breast-feeding milk normally contains several bioactive proteins or peptides useful for the development of the immune system that protects infants from diseases. Osteopontin（OPN） plays a distinct role during the processes of lactation, Some studies on OPN isolated and purified from the human milk via HPLC on SCX and Ca columns adopted biological mass spectrometry. After digesting the purified OPN sample with trypsin, the typical correlative polypeptide fragments GDSVVYGLR and QNLLAPQTLPSK are identified by FT-ICR-MS. The rapid identification of OPN is assumed to be reasonably well behaving in a standard bottom-up experiment. It shows that nano-spray HPLC combined with FT-ICR-MS and Mascot search can be used as a high efficient method for the identification of purified OPN and its polypeptide fragments. Further study will provide more academic theories of their different modifications and the bioactivity as well.