关键词： asporin   growth   differentiation factor-5   osteoarthritis   polymorphism  

摘要： Aim: Osteoarthritis (OA) is the most common chronic joint disorder, resulting from the breakdown of joint cartilage. It occurs in the knees, hands, and hips, leading to pain, stiffness, inflammation, and swelling. Methods: In this study, 100 hand and knee OA patients, meeting the American College of Rheumatology criteria were included in the case group, and 100 healthy individuals were allocated to the control group. Blood samples were collected from the participants. After DNA extraction, genotyping was carried out for GDF5 rs143383 C/T polymorphism by allele-specific polymerase chain reaction (ASPCR) and for D-repeat alleles of asporin (ASPN) by conventional PCR assay. Results: The results showed that the frequency of the D14 allele of ASPN was significantly higher than other alleles in the case group (P = .0001). Also, the frequency of the D14 allele among women was significantly higher than in men (P = .004). Moreover, the frequency of the TT allele in GDF5 rs143383 C/T polymorphism was significantly higher than the CC and CT alleles in the case group, compared with the control group (P = .001). A significant difference was found between the TT allele and other alleles in female and male patients compared with the control group (P = .02). Conclusions: The D14 allele of the ASPN gene and TT allele of the GDF5 gene (rs143383 + 104T/C) are associated with hand and knee OA in the Kurdish population, indicating that these alleles could be risk factors for OA, at least in our populations.

关键词： 椎间盘退变   基因治疗   锌指蛋白A20   NF-κB通路   炎症反应   兔  

摘要： 目的探讨锌指蛋白A20对兔腰椎间盘退变的影响。方法取3月龄新西兰大白兔26只,体质量2.0~2.5 kg,经腹细针穿刺法制备L3、4、L4、5、L5、6椎间盘退变模型,其中24只术后4周MRI检查明确造模成功,随机分为4组(n=6),于目标椎间盘中分别注射锌指蛋白A20过表达腺病毒(过表达A20组)、空载体腺病毒(空载体组)、PBS液(对照组)、锌指蛋白A20干扰腺病毒(干扰A20组)。于注射前1 d及注射后1、2、3、6 d行生物反应综合评分;注射后2、4、8周,各组行MRI检查并测量T2弛豫时间(T2信号值)后,取材行阿利辛蓝染色观察椎间盘髓核细胞退变情况,免疫组织化学染色检测锌指蛋白A20以及椎间盘退变相关指标(Ⅱ型胶原、蛋白聚糖)的表达,Western blot检测锌指蛋白A20、NF-κB结合蛋白[P65、磷酸化P65(phosphate P65,P-P65)、Ⅱ型胶原、蛋白聚糖]、自噬相关蛋白[LC3(LC3Ⅱ/LC3Ⅰ)、P62]以及炎症因子(TNF-α、IL-1β)的表达。结果各组注射后各时间点生物反应综合评分均明显低于注射前1 d(P<0.05);注射后6 d干扰A20组评分明显低于其他组(P<0.05),其他组间比较差异均无统计学意义(P>0.05)。MRI检测提示,注射后2、4、8周过表达A20组T2信号值均最高(P<0.05),2、4周时干扰A20组最低(P<0.05),其余组间差异均无统计学意义(P>0.05)。阿利辛蓝染色显示,注射后4周过表达A20组蛋白聚糖含量最高(P<0.05)、干扰A20组最低(P<0.05);8周时过表达A20组蛋白聚糖含量显著高于其他组(P<0.05),其他组间比较差异无统计学意义(P>0.05)。免疫组织化学染色示,锌指蛋白A20、Ⅱ型胶原、蛋白聚糖表达过表达A20组最高(P<0.05),干扰A20组上述蛋白表达最低(P<0.05)。Western blot检测示锌指蛋白A20、蛋白聚糖、Ⅱ型胶原、LC3(LC3Ⅱ/LC3Ⅰ)蛋白相对表达量过表达A20组最高、干扰A20组最低,而P-P65、TNF-α、IL-1β、P62蛋白相对表达量过表达A20组最低、干扰A20组最高,与其他组比较差异均有统计学意义(P<0.05);各组P65蛋白相对表达量差异均无统计学意义(P>0.05)。结论锌指蛋白A20能通过抑制炎症反应,有效延缓兔腰椎间盘退变的进程。

关键词： asporin   gastric cancer   HIF1 alpha   oxidative stress   ROS  

摘要： Asporin (ASPN), a small leucine-rich proteoglycan expressed predominantly by cancer associated fibroblasts (CAFs), plays a pivotal role in tumor progression. ASPN is also expressed by some cancer cells, but its biological significance is unclear. Here, we investigated the effects of ASPN expression in gastric cancer cells. Overexpression of ASPN in 2 gastric cancer cell lines, HSC-43 and 44As3, led to increased migration and invasion capacity, accompanied by induction of CD44 expression and activation of Rac1 and MMP9. ASPN expression increased resistance of HSC-43 cells to oxidative stress by reducing the amount of mitochondrial reactive oxygen species. ASPN induced expression of the transcription factor HIF1 alpha and upregulated lactate dehydrogenase A (LDHA) and PDH-E1 alpha, suggesting that ASPN reprograms HSC-43 cells to undergo anaerobic glycolysis and suppresses ROS generation in mitochondria, which has been observed in another cell line HSC-44PE. By contrast, 44As3 cells expressed high levels of HIF1 alpha in response to oxidant stress and escaped apoptosis regardless of ASPN expression. Examination of xenografts in the gastric wall of ASPN(-/-) mice revealed that growth of HSC-43 tumors with increased micro blood vessel density was significantly accelerated by ASPN;however, ASPN increased the invasion depth of both HSC-43 and 44As3 tumors. These results suggest that ASPN has 2 distinct effects on cancer cells: HIF1 alpha-mediated resistance to oxidative stress via reprogramming of glucose metabolism, and activation of CD44-Rac1 and MMP9 to promote cell migration and invasion. Therefore, ASPN may be a new therapeutic target in tumor fibroblasts and cancer cells in some gastric carcinomas.

关键词： asporin   CXCR4   DDR2   gut-microbiome   leptin   osteoarthritis   SDF-1  

摘要： Osteoarthritis (OA), a disorder of joints, is prevalent in older age. The contemporary cure for OA is aimed to confer symptomatic relief, consisting of temporary pain and swelling relief. In this paper, we discuss various modalities responsible for the onset of OA and associated with its severity. Inhibition of chondrocytes receptors such as DDR2, SDF-1, Asporin, and CXCR4 by specific pharmacological inhibitors attenuates OA, a critical step for finding potential disease modifying drugs. We critically analyzed recent OA studies with an emphasis on intermediate target molecules for OA intervention. We also explored some novel and safe treatments for OA by considering disease prognosis crosstalk with cellular signaling pathways.

关键词： Lung cancer   Fibroblasts   SCRIB   Invasion   Asporin  

摘要： Aims: Cancer associated fibroblasts (CAFs) play a crucial role in lung tumor development, but the underlying mechanism is still not fully understood. Main methods: SCRIB expression in the CAFs of human lung cancer tissues was examined by immunohistochemistry (IHC). A coculture of mouse Lewis lung cancer cells (LLC) and fibroblasts was used to investigate SCRIB expression in cocultured fibroblasts. Proliferation, scratch wound, and transwell assays were used to examine the proliferation, migration and invasion ability of SCRIB knockdown fibroblasts and their effects on LLC. A 3D-coculture system and co-injection xenograft model were used to examine LLC invasion. RNA sequencing and transwell experiments were used to explore the molecules that may participate in LLC invasion. Key findings: Herein, we found that the low expression of SCRIB in CAFs is correlated with advanced tumor stages and poor survival for human lung squamous cell carcinoma. SCRIB expression in fibroblasts is drastically downregulated by LLC cells. SCRIB knockdown fibroblasts not only enhance invasion but also facilitate LLC invasion in a 3D-coculture system and in an in vivo subcutaneous transplantation model. The upregulation of asporin in SCRIB knockdown fibroblasts is involved in LLC invasion in vitro. Significance: Collectively, the results indicate that fibroblasts with low SCRIB expression promote lung cancer cell invasion, which suggests that the downregulated expression of SCRIB may represent one of the important characteristics of tumor-promoting CAFs in lung squamous cell cancer.

关键词： asporin   cardiomyocyte apoptosis   glycated low-density lipoproteins   grape seed procyanidin B2   transforming growth factor-beta 1  

摘要： The progression of diabetic cardiomyopathy is related to cardiomyocyte dysfunction and apoptosis. Our previous studies showed that asporin (ASPN) was significantly increased in the myocardium of db/db mice through proteomics, and grape seed procyanidin B2 (GSPB2) significantly inhibited the expression of ASPN in the heart of db/ db mice. We report here that ASPN played a critical role in glycated low-density lipoproteins (gly-LDL) induced-cardiomyocyte apoptosis. We found that gly-LDL upregulated ASPN expression. ASPN increased H9C2 cardiomyocyte apoptosis with down-regulation of Bcl-2, upregulation of transforming growth factor-beta 1, Bax, collagen III, fibronectin, and phosphorylation of smad2 and smad3. However, GSPB2 treatment reversed ASPN-induced impairments in H9C2 cardiomyocytes. These results provide evidence for the cardioprotective action of GSPB2 against ASPN injury, and thus suggest a new target for fighting against diabetic cardiomyopathy.

关键词： Periodontal   Mesenchymal stem cells   Asporin   Periostin   Cementum Protein-1   Bone marrow  

摘要： Background: Periodontal ligament stem cells are a source of mesenchymal stem cells, but it is unclear whether their phenotype is distinct from mesenchymal stem cells derived from different tissues, such as those derived from bone marrow. Objective: To investigate the expression of the putative PDL markers asporin, periostin, nestin and cementum protein 1, by periodontal ligament stem cells both constitutively and during osteogenic differentiation when compared to bone marrow-derived mesenchymal stem cells, and dental pulp stem cells. Methods: The primary human periodontal ligament, bone marrow, and dental pulp stem cells, and osteoblasts from different donors were cultured in vitro. The expression of periodontal marker associated genes during osteogenic induction was tested by qRT-PCR and immunofluorescence staining. Results: Asporin expression was detected in periodontal ligament stem cells and increased markedly during the time in culture (upregulated x53 fold at 21 days post-induction). During osteogenic differentiation, asporin expression significantly decreased in periodontal ligament cells whereas periostin significantly decreased in dental pulp cells. Periostin expression was absent in osteoblasts, but expression gradually increased in all other cells with time in culture. Nestin expression was mainly seen in the periodontal ligament and dental pulp cells and was largely absent in osteoblasts and bone marrow cells. Cementum protein-1 was most highly expressed in bone marrow cells and osteoblasts following osteogenic induction. Conclusions: The results provide further evidence that periodontal ligament-derived and bone marrow derived mesenchymal stem cells are phenotypically distinct. Periodontal markers are also expressed in dental pulp stem cells.

摘要： Worldwide, calcific aortic valve disease is one of the leading causes of morbidity and mortality among patients with cardiac abnormalities. Aortic valve mineralization and calcification are the key events of adult calcific aortic valve disease manifestation and functional insufficiency. Due to heavy mineralization and calcification, adult aortic valvular cusps show disorganized and dispersed stratification concomitant with deposition of calcific nodules with severely compromised adult valve function. Interestingly, shared gene regulatory pathways are identified between bone-forming cells and heart valve cells during development. Asporin, a small leucine-rich proteoglycan (43 kDa), acts to inhibit mineralization in periodontal ligament cells and is also detected in normal murine adult aortic valve leaflets with unknown function. Therefore, to understand the Asporin function in aortic cusp mineralization and calcification, adult avian aortic valvular interstitial cell culture system is established and osteogenesis has been induced in these cells successfully. Upon induction of osteogenesis, reduced expression of Asporin mRNA and increased expression of bone and osteogenesis markers are detected compared to cells maintained without osteogenic induction. Importantly, treatment with human recombinant Asporin protein reduces the mineralization level in osteogenic media-induced aortic valvular interstitial cells with the concomitant decreased level of Wnt/β-catenin signaling. Overall, all these data are highly indicative that Asporin might be a novel biomolecular target to treat patients of calcific aortic valve disease over current cusp replacement surgery. © 2020 Anisha Polley et al.

关键词： 乳头状甲状腺癌   TMT定量蛋白质组学   Asporin   HER2   肿瘤侵袭和转移  

摘要： 甲状腺癌是内分泌系统最常见的恶性肿瘤。过去二十年,甲状腺癌新发病例在全球呈现“潮涌”态势,已成为各个国家及地区发病增长速度最快的恶性肿瘤之一。84%的甲状腺癌是乳头状甲状腺癌(Papillary thyroid cancer,PTC),对小于45岁PTC病人,淋巴结转移是预后变差的独立危险因素尚存在争议;但对于大于45岁PTC病人,学术界普遍认为这类病人的淋巴结转移与其高死亡率、手术后复发及预后差正相关。特别是与无淋巴结转移(N0)或Ⅵ区淋巴结转移(N1a)相比,发生Ⅰ-Ⅵ区淋巴结转移(N1b)的PTC病人具有更高的术后复发率。遗憾的是,目前尚未有研究报道大于45岁PTC病人淋巴结转移的分子机制。为此,本课题从两方面展开研究:第一,分析PTC-N0、PTC-N1a和PTC-N1b癌组织之间蛋白水平和分子功能的差异;第二,分析PTC-N0、PTC-N1a和PTC-N1b癌与癌旁正常组织之间蛋白水平和分子功能的差异,并深入研究潜在的调控机制,寻找新的肿瘤标志物。第一部分研究结果:通过非监督的主成分和火山图分析发现,大于45岁的PTC病人,淋巴结转移程度不同,癌组织中蛋白的表达也明显不同,而癌旁正常组织中蛋白的表达差异却很小。通过热图、信号通路和基因本体富集分析PTC-N0、PTC-N1a和PTC-N1b 癌组织中的差异表达蛋白,发现这些差异表达蛋白主要富集于细胞外基质、能量代谢和细胞增殖等生物学过程,从宏观蛋白层面揭示PTC-N0、PTC-N1a和PTC-N1b预后不同的可能分子机制。第二部分研究结果:与癌旁正常组织相比,PTC-N0、PTC-N1a和PTC-N1b癌组织中蛋白的表达模式不甚相同。通过生物信息学工具筛选出PTC-N0、PTC-N1a和PTC-N1b癌组织中共同高表达的差异蛋白,并从中挑选出5个进行Western Blotting 验证,发现 Versican、PL3、SERP1NA1、CD55 和 Asporin 蛋白在 PTC-N0、PTC-N1a和PTC-N1b癌组织中均高表达。免疫组化实验发现,相比癌旁正常组织,Asporin在PTC癌组织中明显高表达,且主要表达于细胞外基质、细胞膜及胞浆。TCGA THCA数据库验证发现,PTC癌组织中高表达的Asporin mRNA与大直径肿瘤,淋巴结转移,双叶占位,高TNM分期和BRAFV600E突变,总生存期和无进展生存期缩短正相关。以上结果提示Asporin在PTC中可能是个促癌基因,可促进PTC增殖、侵袭和转移。CCK8实验发现,敲低Asporin后,BCPAP和KTC-1细胞活力明显降低。克隆形成实验发现,敲低Asporin后,BCPAP和KTC-1细胞克隆形成能力明显减弱,提示Asporin具有促进PTC癌细胞增殖和克隆形成的作用。Transwell实验发现,敲低Asporin后,BCPAP、KTC-1和BHT101细胞侵袭和转移能力都明显降低,提示Asporin具有促进甲状腺癌细胞侵袭和转移的作用。为了进一步研究Asporin致病的分子机制,Western Blotting发现,敲低BCPAP、KTC-1和BHT101细胞中Asporin后,MAPK信号通路中BRAF和p-ERK1/2蛋白表达降低,而t-ERK1/2蛋白表达不变,且MAPK信号通路下游的正向调控EMT相关蛋白β-catenin、SNAIL、SLUG、ZEB1 和 ZEB2 表达降低。提示 Asporin 可通过 MAPK 信号通路上调其下游EMT相关蛋白的表达水平。此外,敲低Asporin后,BCPAP和KTC-1细胞形态主要呈圆形,伪足减少,胞内微丝分布紊乱,提示Asporin可通过促进MAPK信号通路的激活,上调EMT相关蛋白的表达水平,进而引起细胞骨架的改变,最后促进PTC的侵袭和转移。免疫共沉淀实验发现,Asporin可结合MAPK信号通路上游蛋白HER2,而不结合EGFR和SRC。免疫荧光实验发现,Asporin和HER2在BCPAP和KTC-1细胞膜及胞浆中共定位,进一步提示Asproin和HER2存在相互作用。通过Western Blotting实验发现,相比对照细胞,Asporin敲低后,BCPAP和KTC-1细胞中HER2、p-HER2Y1248、p-SRCY418、p-EGFRY845 和 p-EGFRY1173 表达水平降低,而 SRC 和EGFR表达水平不变,提示Asporin可通过结合HER2,上调HER2表达水平,进而使p-EGFR和p-SRC表达升高,从而激活MAPK信号通路。体外rhAsporin刺激和PLX4032抑制实验发现,rhAsporin并不能在细胞外通过结合HER2激活MAPK信号通路,且MAPK信号通路并不能够正反馈调控Asporin蛋白表达水平