A novel porcine gene,alpha-1-antichymotrypsin 2 (SERPINA3-2): sequence,genomic organization,polymorphism and mapping

A novel porcine gene, alpha-1-antichymotrypsin 2 (SERPINA3-2), a member of the serpin superfamily, was isolated from a porcine genomic library and sequenced. The genomic organization of the approximately 9.0 kb gene was determined on the basis of the porcine liver cDNA of SERPINA3-1 and SERPINA3-2, and comprises five exons and four introns. The coding sequence of SERPINA3-2 shares 86% identity with the paralogue, SERPINA3-1. Porcine SERPINA3-2 was found to be an orthologue of human SERPINA3 (71% identity of the coding sequences) and both genes have a similar genomic organization. Polymorphisms were found in intron 4 of the porcine gene using polymerase chain reaction-restriction fragment length polymorphism. The gene was mapped by linkage analysis and radiation hybrid mapping to the distal end of chromosome 7q, to the gene cluster of the protease inhibitors including PI1 (SERPINA1), PI2, PI3, PI4 (apparently paralogues of SERPINA3), and PO1A and PO1B. SERPINA3-2 is the first porcine serpin gene whose genomic organization has been determined.     

Anti-angiogenic and anti-inflammatory effects of SERPINA3K on corneal injury

SERPINA3K is a member of the serine proteinase inhibitor (SERPIN) family. Here we evaluated the therapeutic effects of SERPINA3K on neovascularization and inflammation in a rat cornea alkali burn model that is commonly employed to study corneal wounding. Topical treatment of the injured rat cornea with SERPINA3K (20 μg/eye/day) for 7 days significantly decreased the neovascular area, compared with the groups treated with BSA or PBS. The SERPINA3K treatment also ameliorated the corneal inflammation as evaluated by the inflammatory index. Furthermore, SERPINA3K enhanced the recovery of corneal epithelium after the alkali injury. Toward the mechanism of action, SERPINA3K down-regulated the expression of the pro-angiogenic and pro-inflammatory factors, vascular endothelial growth factor and tumor necrosis factor-α and up-regulated the expression of the anti-angiogenic factor, pigment epithelium-derived factor. SERPINA3K specifically inhibited growth of vascular endothelial cells. Meanwhile, SERPINA3K significantly up-regulated the expression of EGFR in the corneal epithelium. These findings suggest that SERPINA3K has therapeutic potential for corneal inflammation and NV.     

Sequence diversity at the proximal 14q32.1 SERPIN subcluster: evidence for natural selection favoring the pseudogenization of SERPINA2

The superfamily of serine protease inhibitors (SERPINs) plays a key role in controlling the activity of proteinases in diverse biological processes. alpha1-antitrypsin (SERPINA1), the most studied member of this family, is encoded by a gene located within the proximal 14q32.1 SERPIN subcluster, together with the highly homologous alpha1-antitrypsin-like sequence (SERPINA2), which was previously proposed to be a pseudogene. Here, we performed a resequencing study encompassing both SERPINA1 and SERPINA2 as well as the adjacent gene coding for corticosteroid-binding globulin (SERPINA6) in samples from Europe and West Africa. In the African sample, we found that a common haplotype carrying a 2-kb deletion in the SERPINA2 gene is associated with remarkable long-range homozygozity as if it was quickly driven to high frequency by natural selection acting on an advantageous variant. An analysis of the HapMap Phase I data for the Yoruba sample confirmed that variation in this subcluster carries a strong signal of positive selection. We also show that the SERPINA2 gene is expressed and probably encodes a functional SERPIN. Finally, comparisons with orthologous sequences in nonhuman primates showed that SERPINA2 is present in some great apes, but in chimpanzees it was lost by a deletion event independent from that observed in humans. In agreement with the "less is more" hypothesis, we propose that loss of SERPINA2 is an ongoing process associated with a selective advantage during recent primate evolution, possibly because of a role in fertility or in host-pathogen interactions.     

Identification of TWIST-interacting genes in prostate cancer

Prostate cancer is one of the most common cancers in men worldwide, and the number of diagnosed patients has dramatically increased in recent years. Currently, the clinical parameters used to diagnose prostate cancer, such as Gleason score, pathological tumor staging, and prostate-specific antigen(PSA) expression level, are considered insufficient to inform recommendation to guide clinical practice. Thus, identification of a novel biomarker is necessary. TWIST is one of the well-studied targets and is correlated with cancer invasion and metastasis in several human cancers. We have investigated two largest prostate cancer patient cohorts available in GEO database and found that TWIST expression is positive correlated with Gleason score and associated with poorer survival. By using a prostate cancer cohort and a prostate cancer cell line dataset, we have identified three potential downstream targets of TWIST, PPM1 A, SRP72 and TBCB. TWIST's prognostic capacity is lost when the gene is mutated. Further investigation in the prostate cancer cohort revealed that gene expression of SERPINA, STX7, PDIA2, FMP5, GP1 BB, VGLL4,KCNMA1, SHMT2, SAA4 and DIDO1 influence the prognostic significance of TWIST and vice versa. Importantly, eight out of these ten genes are prognostic indicator by itself. In conclusion, our study has further confirmed that TWIST is a prognostic marker in prostate cancer, identified its potential downstream targets and genes that could possibly give additional prognostic value to predict TWIST-mediated prostate cancer progression.     

Ubiquitin ligase SYVN1/HRD1 facilitates degradation of the SERPINA1 Z variant/α-1-antitrypsin Z variant via SQSTM1/p62-dependent selective autophagy

SERPINA1/AAT/α-1-antitrypsin (serpin family A member 1) deficiency (SERPINA1/ AAT-D) is an autosomal recessive disorder characterized by the retention of misfolded SERPINA1/AAT in the endoplasmic reticulum (ER) of hepatocytes and a significant reduction of serum SERPINA1/AAT level. The Z variant of SERPINA1/AAT, containing a Glu342Lys (E342K) mutation (SERPINA1^E342K/ATZ), the most common form of SERPINA1/AAT-D, is prone to misfolding and polymerization, which retains it in the ER of hepatocytes and leads to liver injury. Both proteasome and macroautophagy/autophagy pathways are responsible for disposal of SERPINA1^E342K/ATZ after it accumulates in the ER. However, the mechanisms by which SERPINA1^E342K/ATZ is selectively degraded by autophagy remain unknown. Here, we showed that ER membrane-spanning ubiquitin ligase (E3) SYVN1/HRD1 enhances the degradation of SERPINA1^E342K/ATZ through the autophagy-lysosome pathway. We found that SYVN1 promoted SERPINA1^E342K/ATZ, especially Triton X 100-insoluble SERPINA1^E342K/ATZ clearance. However, the effect of SYVN1 in SERPINA1^E342K/ATZ clearance was impaired after autophagy inhibition, as well as in autophagy-related 5 (atg5) knockout cells. On the contrary, autophagy induction enhanced SYVN1-mediated SERPINA1^E342K/ATZ degradation. Further study showed that SYVN1 mediated SERPINA1^E342K/ATZ ubiquitination, which is required for autophagic degradation of SERPINA1^E342K/ATZ by promoting the interaction between SERPINA1^E342K/ATZ and SQSTM1/p62 for formation of the autophagy complex. Interestingly, SYVN1-mediated lysine 48 (K48)-linked polyubiquitin chains that conjugated onto SERPINA1^E342K/ATZ might predominantly bind to the ubiquitin-associated (UBA) domain of SQSTM1 and couple the ubiquitinated SERPINA1^E342K/ATZ to the lysosome for degradation. In addition, autophagy inhibition attenuated the suppressive effect of SYVN1 on SERPINA1^E342K/ATZ cytotoxicity, and the autophagy inducer rapamycin enhanced the suppressive effect of SYVN1 on SERPINA1^E342K/ATZ-induced cell apoptosis. Therefore, this study proved that SYVN1 enhances SERPINA1^E342K/ATZ degradation through SQSTM1-dependent autophagy and attenuates SERPINA1^E342K/ATZ cytotoxicity.     

Proteomics analysis of human serum of patients with non-small-cell lung cancer reveals proteins as diagnostic biomarker candidates

Non-small-cell lung carcinomas (NSCLC) is the most common type of lung cancer and it has a poor prognosis, because overall survival after 5 years is 20–25% for all stages. Thus, it is extremely important to increase the survival rate in the early stages NSCLC by focusing on novel screening tests of cancer identifying specific biomarkers expression associated with a more accurate tumor staging and patient prognosis. In this study, we focused our attention on quantitative proteomics of three heavily glycosylated serum proteins: AMBP, α2 macroglobulin, and SERPINA1. In particular, we analyzed serum samples from 20 NSCLC lung adenocarcinoma cancer patients in early and advanced stages, and 10 healthy donors to obtain a relative quantification through the MRM analysis of these proteins that have shown to be markers of cancer development and progression. AMBP, α2 macroglobulin, and SERPINA1 were chosen because all of them possess endopeptidase inhibitor activity and play key roles in cancer. We observe a variation in the expression of these proteins linked to the stage of the disease. Therefore, we believe that proteins like α2 macroglobulin, αmicroglobulin/bikunin, and SERPINA1 could be useful biomarkers for early detection of lung cancer and in monitoring its evolution.     

SERPINA3K Plays Antioxidant Roles in Cultured Pterygial Epithelial Cells through Regulating ROS System

We recently demonstrated that SERPINA3K, a serine proteinase inhibitor, has antioxidant activity in the cornea. Here we investigated the antioxidant effects of SERPINA3K on the pterygial, which is partially caused by oxidative stress in pathogenesis. The head part of primary pterygial tissue was dissected and then cultured in keratinocyte serum-free defined medium (KSFM). The cultured pterygial epithelial cells (PECs) were treated with SERPINA3K. The cell proliferation and migration of PECs were measured and analyzed. Western blot and quantitative real-time polymerase chain reaction (PCR) assay were performed. It showed that SERPINA3K significantly suppressed the cell proliferation of PECs in a concentration-dependent manner, compared with cultured human conjunctival epithelial cells. SERPINA3K also inhibited the cell migration of PECs. Towards its underlying mechanism, SERPINA3K had antioxidant activities on the PECs by significantly inhibiting NADPH oxidase 4 (NOX4), which is an important enzyme of ROS generation, and by elevating the levels of key antioxidant factors of ROS: such as NAD(P)H dehydrogenase (quinone 1) (NQO1), NF-E2–related factor-2 (NRF2) and superoxide dismutases (SOD2). Meanwhile, SERPINA3K down-regulated the key effectors of Wnt signaling pathway: β-catenin, nonphospho-β-catenin, and low-density lipoprotein receptor-related protein 6 (LRP6). We provided novel evidence that SERPINA3K had inhibitory effects on pterygium and SERPINA3K played antioxidant role via regulating the ROS system and antioxidants.     

SERPINA2 Is a Novel Gene with a Divergent Function from SERPINA1

Serine protease inhibitors (SERPINs) are a superfamily of highly conserved proteins that play a key role in controlling the activity of proteases in diverse biological processes. The SERPIN cluster located at the 14q32.1 region includes the gene coding for SERPINA1, and a highly homologous sequence, SERPINA2, which was originally thought to be a pseudogene. We have previously shown that SERPINA2 is expressed in different tissues, namely leukocytes and testes, suggesting that it is a functional SERPIN. To investigate the function of SERPINA2, we used HeLa cells stably transduced with the different variants of SERPINA2 and SERPINA1 (M1, S and Z) and leukocytes as the in vivo model. We identified SERPINA2 as a 52 kDa intracellular glycoprotein, which is localized at the endoplasmic reticulum (ER), independently of the variant analyzed. SERPINA2 is not significantly regulated by proteasome, proposing that ER localization is not due to misfolding. Specific features of SERPINA2 include the absence of insoluble aggregates and the insignificant response to cell stress, suggesting that it is a non-polymerogenic protein with divergent activity of SERPINA1. Using phylogenetic analysis, we propose an origin of SERPINA2 in the crown of primates, and we unveiled the overall conservation of SERPINA2 and A1. Nonetheless, few SERPINA2 residues seem to have evolved faster, contributing to the emergence of a new advantageous function, possibly as a chymotrypsin-like SERPIN. Herein, we present evidences that SERPINA2 is an active gene, coding for an ER-resident protein, which may act as substrate or adjuvant of ER-chaperones.     

ING1 inhibits Twist1 expression to block EMT and is antagonized by the HDAC inhibitor vorinostat

ING1 is a chromatin targeting subunit of the Sin3a histone deacetylase (HDAC) complex that alters chromatin structure to subsequently regulate gene expression. We find that ING1 knockdown increases expression of Twist1, Zeb 1&2, Snai1, Bmi1 and TSHZ1 drivers of EMT, promoting EMT and cell motility. ING1 expression had the opposite effect, promoting epithelial cell morphology and inhibiting basal and TGF-β-induced motility in 3D organoid cultures. ING1 binds the Twist1 promoter and Twist1 was largely responsible for the ability of ING1 to reduce cell migration. Consistent with ING1 inhibiting Twist1 expression in vivo, an inverse relationship between ING1 and Twist1 levels was seen in breast cancer samples from The Cancer Genome Atlas (TCGA). The HDAC inhibitor vorinostat is approved for treatment of multiple myeloma and cutaneous T cell lymphoma and is in clinical trials for solid tumours as adjuvant therapy. One molecular target of vorinostat is INhibitor of Growth 2 (ING2), that together with ING1 serve as targeting subunits of the Sin3a HDAC complex. Treatment with sublethal (LD25-LD50) levels of vorinostat promoted breast cancer cell migration several-fold, which increased further upon ING1 knockout. These observations indicate that correct targeting of the Sin3a HDAC complex, and HDAC activity in general decreases luminal and basal breast cancer cell motility, suggesting that use of HDAC inhibitors as adjuvant therapies in breast cancers that are prone to metastasize may not be optimal and requires further investigation.     

miR-429与肿瘤

miR-429是miR-200家族成员之一。研究表明,miR-429异常表达与肿瘤的发生、发展、转移、凋亡和耐药等密切相关。但miR-429在肿瘤中所起的作用一直有争议,可作为肿瘤抑制剂或促进剂,具肿瘤细胞/组织特异性。其在骨肉瘤、肾癌、卵巢癌、乳腺癌、宫颈癌、胶质瘤、口腔鳞状细胞癌、胃癌、食管癌、胰腺癌中起抑癌作用,而在肺癌、前列腺癌和子宫内膜癌中起促癌作用,但在结肠癌、肝癌、膀胱癌中的作用尚不明确。本文综述了近年来miR-429在肿瘤发生、发展中的作用及潜在的调控机制,为其作为肿瘤诊断、治疗及预后的潜在生物标记分子提供新的启示和参考。     

Lipocalin 2: A multifaceted modulator of human cancer

Lipocalin 2 (Lcn2), a member of the lipocalin family that carries small lipophilic ligands, has gained recent attention as both a potential biomarker and a modulator of human cancers. Here we describe recent findings of the functions of Lcn2 in breast cancer and the potential mechanisms that underlie its actions. Lcn2 has been shown to induce the epithelial to mesenchymal transition (EMT) in breast cancer cells and to promote breast tumor invasion. Estrogen receptor α may participate in the pathway that leads to Lcn2-induced EMT. Preliminary evidence also suggests that Lcn2 may be used as a potential non-invasive urinary biomarker of breast cancer. Elevated levels of Lcn2 have also been reported in other human cancers. The potential roles of Lcn2 in some other epithelial tumors as well as leukemia are also reviewed and discussed here.     

FHL2 蛋白在肿瘤中的转录调控及其分子生物学机制

FHL2是仅有四个半LIM结构域(FHL)蛋白家族的成员,目前在FHL蛋白家族中研究最为广泛。FHL2作为重要的衔接蛋白和支架蛋白,主要通过LIM结构域介导蛋白分子间的相互作用以实现其生物学功能。Fhl2基因在转录水平受多种肿瘤相关基因的调控,如p53,血清应答因子等。FHL2与恶性肿瘤的关系是近年来的研究热点,目前认为FHL2能够作为癌蛋白及抑癌蛋白通过不同机制广泛影响乳腺癌、胃肠道肿瘤、肝癌、前列腺癌等肿瘤的发生发展,并且在不同肿瘤中的表达具有组织特异性。本文就FHL2的结构特点、功能、转录调控及与肿瘤的关系几个方面展开综述,从而明确FHL2在不同肿瘤中所发挥的作用及其分子生物学机制将会为治疗相关肿瘤提供新的干预靶点。     

Src Regulates the Activity of the ING1 Tumor Suppressor

The INhibitor of Growth 1 (ING1) is stoichiometric member of histone deacetylase (HDAC) complexes and functions as an epigenetic regulator and a type II tumor suppressor. It impacts cell growth, aging, apoptosis, and DNA repair, by affecting chromatin conformation and gene expression. Down regulation and mislocalization of ING1 have been reported in diverse tumor types and Ser/Thr phosphorylation has been implicated in both of these processes. Here we demonstrate that both in vitro and in vivo, the tyrosine kinase Src is able to physically associate with, and phosphorylate ING1, which results in a nuclear to cytoplasmic relocalization of ING1 in cells and a decrease of ING1 stability. Functionally, Src antagonizes the ability of ING1 to induce apoptosis, most likely through relocalization of ING1 and down regulation of ING1 levels. These effects were due to both kinase-dependent and kinase-independent properties of Src, and were most apparent at elevated levels of Src expression. These findings suggest that Src may play a major role in regulating ING1 levels during tumorigenesis in those cancers in which high levels of Src expression or activity are present. These data represent the first report of tyrosine kinase-mediated regulation of ING1 levels and suggest that kinase activation can impact chromatin structure through the ING1 epigenetic regulator.     

A targeted proteomic strategy for the measurement of oral cancer candidate biomarkers in human saliva

Head and neck cancers, including oral squamous cell carcinoma (OSCC), are the sixth most common malignancy in the world and are characterized by poor prognosis and a low survival rate. Saliva is oral fluid with intimate contact with OSCC. Besides non‐invasive, simple, and rapid to collect, saliva is a potential source of biomarkers. In this study, we build an SRM assay that targets fourteen OSCC candidate biomarker proteins, which were evaluated in a set of clinically‐derived saliva samples. Using Skyline software package, we demonstrated a statistically significant higher abundance of the C1R, LCN2, SLPI, FAM49B, TAGLN2, CFB, C3, C4B, LRG1, SERPINA1 candidate biomarkers in the saliva of OSCC patients. Furthermore, our study also demonstrated that CFB, C3, C4B, SERPINA1 and LRG1 are associated with the risk of developing OSCC. Overall, this study successfully used targeted proteomics to measure in saliva a panel of biomarker candidates for OSCC.