Small Proline-rich Protein 1A Research Articles

Abstract P2099: Small Proline-rich Protein 1a Is A Key Downstream Effector Of Microrna-150 During Both Maladaptive Cardiac Remodeling In Mice And Human Cardiac Fibroblast Activation

Background & Objectives: MicroRNA-150 (miR-150) is conserved between rodents and humans, is significantly downregulated during heart failure (HF), and correlates with patient outcomes. We reported that miR-150 is protective during myocardial infarction (MI) in part by decreasing cardiomyocyte (CM) apoptosis and that proapoptotic small proline-rich protein 1a ( Sprr1a ) is a direct CM target of miR-150. We also showed that Sprr1a knockdown in mice improves cardiac dysfunction and fibrosis post-MI and that Sprr1a is upregulated in pathological mouse cardiac fibroblasts (CFs) from ischemic myocardium. However, the direct functional relationship between miR-150 and SPRR1A during both post-MI remodeling in mice and human CF (HCF) activation was not established. Methods & Results: Here, using a novel miR-150 knockout (KO); Sprr1a -hypomorphic ( Sprr1a hypo/hypo ) mouse model, we demonstrate that Sprr1a knockdown blunts adverse post-MI effects caused by miR-150 loss. MiR-150 KO; Sprr1a hypo/hypo mice also exhibit decreased profibrotic markers in the infarct areas of hearts as compared to miR-150 KO mice. Moreover, primary adult HCF studies reveal that SPRR1A is upregulated in hypoxia/reoxygenation-treated HCFs and is downregulated in HCFs exposed to the cardioprotective β-blocker carvedilol, which is inversely associated with miR-150 expression. Significantly, we show that the protective roles of miR-150 in HCFs are directly mediated by functional repression of profibrotic SPRR1A (See Figure). Conclusions: These findings delineate a pivotal functional interaction between miR-150 and SPRR1A as a novel regulatory mechanism pertinent to CF activation and ischemic HF.

SPRR1A is a key downstream effector of MiR-150 during both maladaptive cardiac remodeling in mice and human cardiac fibroblast activation

MicroRNA-150 (miR-150) is conserved between rodents and humans, is significantly downregulated during heart failure (HF), and correlates with patient outcomes. We previously reported that miR-150 is protective during myocardial infarction (MI) in part by decreasing cardiomyocyte (CM) apoptosis and that proapoptotic small proline-rich protein 1a (Sprr1a) is a direct CM target of miR-150. We also showed that Sprr1a knockdown in mice improves cardiac dysfunction and fibrosis post-MI and that Sprr1a is upregulated in pathological mouse cardiac fibroblasts (CFs) from ischemic myocardium. However, the direct functional relationship between miR-150 and SPRR1A during both post-MI remodeling in mice and human CF (HCF) activation was not established. Here, using a novel miR-150 knockout;Sprr1a-hypomorphic (Sprr1ahypo/hypo) mouse model, we demonstrate that Sprr1a knockdown blunts adverse post-MI effects caused by miR-150 loss. Moreover, HCF studies reveal that SPRR1A is upregulated in hypoxia/reoxygenation-treated HCFs and is downregulated in HCFs exposed to the cardioprotective β-blocker carvedilol, which is inversely associated with miR-150 expression. Significantly, we show that the protective roles of miR-150 in HCFs are directly mediated by functional repression of profibrotic SPRR1A. These findings delineate a pivotal functional interaction between miR-150 and SPRR1A as a novel regulatory mechanism pertinent to CF activation and ischemic HF.

Abstract 12156: Small Proline-Rich Repeat Protein 1a is a Significant Functional Target of Microrna-150 in Mouse Hearts and Human Cardiac Fibroblasts

Background and Aims: Cardiac injury induces dynamic changes in the expression of microRNAs (miRs). MiR-150 is conserved between rodents and humans, and its downregulation is markedly associated with heart failure (HF) severity and outcome in humans. Using systemic and cardiomyocyte (CM)-specific knockout (KO) mouse models, we reported that miR-150 plays a cardioprotective role in part by decreasing CM apoptosis, and that pro-apoptotic small proline-rich protein 1a ( Sprr1a ) is a direct and functional target of miR-150 in CMs. We also showed that Sprr1a knockdown in mice improves cardiac dysfunction and fibrosis post-myocardial infarction (MI), and that Sprr1a is upregulated in mouse cardiac fibroblasts (CFs) isolated from ischemic myocardium. However, the direct functional relationship between miR-150 and SPRR1A in MI and human CF (HCF) activation has never been established. Methods & Results: Here, we demonstrate using a novel miR-150 KO; Sprr1a -hypomorphic ( Sprr1a hypo/hypo ) mouse model that Sprr1a knockdown blunts cardiac dysfunction, damage, inflammation, apoptosis and fibrosis post-MI caused by miR-150 loss. MiR-150 KO; Sprr1a hypo/hypo mice also exhibit decreased pro-fibrotic markers in the infarct areas of hearts as compared to miR-150 KO mice. Our primary adult HCF studies further reveal that SPRR1A is upregulated in HCFs subjected to simulated ischemia/reperfusion, while its expression is downregulated in HCFs by a cardioprotective β-blocker carvedilol, which is inversely associated with miR-150 expression. MiR-150 knockdown in HCFs also increases pro-fibrotic SPRR1A . Lastly, protective roles of miR-150 in HCFs are in part mediated by the functional repression of SPRR1A (See Figure). Conclusions: Our findings delineate a pivotal functional interaction between miR-150 and Sprr1a as a novel regulatory mechanism pertinent to CF activation post-MI. This axis could be a therapeutic target for intervention in ischemic HF and cardiac fibrosis.

Small proline-rich protein 1A promotes lung adenocarcinoma progression and indicates unfavorable clinical outcomes.

Small proline-rich protein 1A (SPRR1A) plays a critical role in regulating squamous cell differentiation. SPRR1A overexpression was reported to be closely related to the progression of some tumors, such as gastric cancer and colon cancer. However, the function of SPRR1A in lung adenocarcinoma (LUAD) has not been elucidated. Here, we first examined the expression pattern of SPRR1A in LUAD tissues, which indicated that the SPRR1A expression level was significantly elevated in LUAD tissues compared with normal lung tissues. High expression of SPRR1A was closely related to larger tumor size. LUAD patients with higher SPRR1A expression had poorer overall survival and SPRR1A was identified as an independent unfavorable prognosis factor. In addition, the effects of SPRR1A on lung cancer cells were tested through cellular experiments and the result demonstrated that knockdown of SPRR1A can suppress the proliferation and invasion capacities of tumor cells, while overexpressing SPRR1A exerted opposite effects. Finally, our findings were substantiated by the data obtained from in vivo xenografts using a mice model. In conclusion, LUAD patients with higher SPRR1A expression were more predisposed to poorer clinical outcomes and unfavorable prognoses, indicating the potential role of SPRR1A as a novel clinical biomarker and therapeutic target.

Small proline-rich protein 2A is a gut bactericidal protein deployed during helminth infection.

A diverse group of antimicrobial proteins (AMPs) helps protect the mammalian intestine from varied microbial challenges. We show that small proline-rich protein 2A (SPRR2A) is an intestinal antibacterial protein that is phylogenetically unrelated to previously discovered mammalian AMPs. In this study, SPRR2A was expressed in Paneth cells and goblet cells and selectively killed Gram-positive bacteria by disrupting their membranes. SPRR2A shaped intestinal microbiota composition, restricted bacterial association with the intestinal surface, and protected against Listeria monocytogenes infection. SPRR2A differed from other intestinal AMPs in that it was induced by type 2 cytokines produced during helminth infection. Moreover, SPRR2A protected against helminth-induced bacterial invasion of intestinal tissue. Thus, SPRR2A is a distinctive AMP triggered by type 2 immunity that protects the intestinal barrier during helminth infection.

Cardiomyocyte microRNA-150 confers cardiac protection and directly represses proapoptotic small proline-rich protein 1A.

MicroRNA-150 (miR-150) is downregulated in patients with multiple cardiovascular diseases and in diverse mouse models of heart failure (HF). miR-150 is significantly associated with HF severity and outcome in humans. We previously reported that miR-150 is activated by β-blocker carvedilol (Carv) and plays a protective role in the heart using a systemic miR-150 KO mouse model. However, mechanisms that regulate cell-specific miR-150 expression and function in HF are unknown. Here, we demonstrate that potentially novel conditional cardiomyocyte–specific (CM-specific) miR-150 KO (miR-150 cKO) in mice worsens maladaptive cardiac remodeling after myocardial infarction (MI). Genome-wide transcriptomic analysis in miR-150 cKO mouse hearts identifies small proline–rich protein 1a (Sprr1a) as a potentially novel target of miR-150. Our studies further reveal that Sprr1a expression is upregulated in CMs isolated from ischemic myocardium and subjected to simulated ischemia/reperfusion, while its expression is downregulated in hearts and CMs by Carv. We also show that left ventricular SPRR1A is upregulated in patients with HF and that Sprr1a knockdown in mice prevents maladaptive post-MI remodeling. Lastly, protective roles of CM miR-150 are, in part, attributed to the direct and functional repression of proapoptotic Sprr1a. Our findings suggest a crucial role for the miR-150/SPRR1A axis in regulating CM function post-MI.

Histone modification of pain-related gene expression in spinal cord neurons under a persistent postsurgical pain-like state by electrocautery

Chronic postsurgical pain (CPSP) is a serious problem. We developed a mouse model of CPSP induced by electrocautery and examined the mechanism of CPSP. In this mouse model, while both incision and electrocautery each produced acute allodynia, persistent allodynia was only observed after electrocautery. Under these conditions, we found that the mRNA levels of Small proline rich protein 1A (Sprr1a) and Annexin A10 (Anxa10), which are the key modulators of neuropathic pain, in the spinal cord were more potently and persistently increased by electrocautery than by incision. Furthermore, these genes were overexpressed almost exclusively in chronic postsurgical pain-activated neurons. This event was associated with decreased levels of tri-methylated histone H3 at Lys27 and increased levels of acetylated histone H3 at Lys27 at their promoter regions. On the other hand, persistent allodynia and overexpression of Sprr1a and Anxa10 after electrocautery were dramatically suppressed by systemic administration of GSK-J4, which is a selective H3K27 demethylase inhibitor. These results suggest that the effects of electrocautery contribute to CPSP along with synaptic plasticity and epigenetic modification.

Small Proline-Rich Protein 2A and 2D Are Regulated by the RBM38-p73 Axis and Associated with p73-Dependent Suppression of Chronic Inflammation.

Simple SummarySmall proline-rich protein 2A and 2D (SPRR2A and SPRR2D) are structure proteins of cornified cell envelopes and function as a protective barrier against diverse external insults. However, the role of SPRR2A/2D in chronic inflammation remains unclear. Here, we showed that SPRR2A/2D expression is controlled by a regulatory loop formed by RNA-binding protein RBM38 and tumor suppressor p73. We also found that RBM38-mediated expression of SPRR2A/2D was p73-dependent and that induction of SPRR2A/2D during keratinocyte differentiation was dependent on both p73 and Rbm38. Furthermore, We found that Rbm38−/−;Trp73+/− mice exhibited weak expression of SPRR2A/2D in multiple tissues and were susceptible to systemic chronic inflammation. Together, our data reveal that SPRR2A/2D are novel targets of the RBM38-p73 loop and contribute to p73-dependent suppression of chronic inflammation.Small proline-rich protein 2A and 2D (SPRR2A and SPRR2D) provide barrier function in terminally differentiated stratified squamous epithelia through the epidermal differentiation complex. However, little is known how SPRR2A/2D expression is controlled and their role in chronic inflammation. Here, we showed that that SPRR2A/2D expression is controlled by a regulatory loop formed by RNA-binding protein RBM38 and tumor suppressor p73. Specifically, we found that SPRR2A/2D expression was induced by ectopic expression of RBM38 or p73 but suppressed by knockout of Rbm38 or p73. We also found that RBM38-mediated expression of SPRR2A/2D was p73-dependent and that induction of SPRR2A/2D during keratinocyte differentiation was dependent on both p73 and Rbm38. Additionally, we found that SPRR2A/2D expression was closely associated with p73 expression in normal and cancerous tissues. To determine the biological function of the RBM38-p73 loop potentially via SPRR2A/2D, we generated a cohort of wild-type, Rbm38−/−, Trp73+/−, and Rbm38−/−;Trp73+/− mice. We found that Rbm38−/−;Trp73+/− mice had a much shorter lifespan than that for Rbm38−/−—and to a lesser extent for Trp73+/− mice—but were less prone to spontaneous tumors than Trp73+/− or Rbm38−/− mice. We also found that Rbm38−/−;Trp73+/− mice exhibited weak expression of SPRR2A/2D in multiple tissues and were susceptible to systemic chronic inflammation, suggesting that decreased SPRR2A/2D expression is likely responsible for chronic inflammation in Rbm38−/−;Trp73+/− mice, leading to a shortened lifespan. Together, our data reveal that SPRR2A/2D are novel targets of the RBM38-p73 loop and contribute to p73-dependent suppression of chronic inflammation.

Abstract 12788: Cardiac MicroRNA-150 Confers Cardioprotection by Directly Repressing Pro-apoptotic Small Proline-rich Protein 1a, Sprr1a in Cardiomyocytes

Background and Aims: Cardiac injury induces dynamic changes in the expression of microRNAs (miRs). For example, the evolutionarily conserved miR-150 is downregulated in patients with multiple cardiovascular diseases such as myocardial infarction (MI) and cardiomyopathies, as well as in various mouse models of heart failure (HF). MiR-150 is significantly associated with HF severity and outcome in humans. Using a systemic miR-150 knockout (KO) mouse model, we previously showed that carvedilol (Carv)/β 1 -adrenergic receptor/β-arrestin1-responsive miR-150 confers cardiac protection against MI (Left side in Figure). However, the extent to which expression of miR-150 in cardiomyocytes (CMs) regulates MI is unknown and there is a lack of mechanistic insight by which CM miR-150 modulates cardiac protection. Methods and Results: Here, we demonstrate using a novel mouse model that conditional CM-specific miR-150 KO (miR-150 cKO) in mice worsens cardiac dysfunction, stress, fibrosis and apoptosis post-MI, without affecting mortality or inflammation. Genome-wide transcriptomic analysis in miR-150 cKO mouse hearts identifies small proline-rich protein 1a (sprr1a) as a novel regulatory target of miR-150. Our mouse and CM studies further reveal that sprr1a expression is upregulated in CMs isolated from ischemic myocardium and subjected to simulated ischemia/reperfusion. In contrast, its expression is downregulated in hearts and CMs by Carv. Our human heart data also show that left ventricular sprr1a is upregulated in patients with HF with reduced ejection fraction. Mechanistically, the cardioprotective roles of CM miR-150 during MI are in part attributed to the direct and functional repression of pro-apoptotic gene sprr1a in CMs (Right side in Figure). Conclusions: These findings reveal a pivotal role for the miR-150/sprr1a axis in regulating CM function post-MI, and this novel axis could be a therapeutic target for intervention in ischemic heart disease.

Serum Small Proline-Rich Protein 2A (SPRR2A) Is a Noninvasive Biomarker in Gastric Cancer.

Objective Since early diagnosis is very important for treating gastric cancer (GC), we aimed to detect serum small proline-rich protein2A (SPRR2A) to verify its diagnostic value for GC patients. Methods Serum samples were collected from 200 patients with GC, 100 patients with gastritis, 40 patients with rectal cancer (RC), 50 patients with colon cancer (CC), and 100 healthy controls. An enzyme-linked immunosorbent assay (ELISA) detection kit was applied to measure serum SPRR2A concentration. The correlations between serum SPRR2A and carcinoembryonic antigen (CEA), clinical pathological parameters of GC, and receiver operating characteristic (ROC) curve were also analyzed. Results The median serum SPRR2A concentration in GC patients was significantly higher than those in healthy controls and gastritis or colorectal cancer patients (P < 0.001). Serum SPRR2A concentration at a cut-off value of 80.7 pg/ml yielded an AUC of 0.851, with 75.7% sensitivity and 74.5% specificity for discriminating GC patients from healthy people. The AUC for the serum SPRR2A concentration combined with the CEA concentration was 0.876, with 79.7% sensitivity and 78.7% specificity. Similarly, serum SPRR2A discriminated GC patients from gastritis patients with an AUC of 0.820, with 90.5% sensitivity and 61.7% specificity. The AUC for the serum SPRR2A concentration combined with the CEA concentration was 0.848, with 87.8% sensitivity and 68.1% specificity. The serum SPRR2A levels in GC patients were associated with lymph node metastasis and the tumor-node-metastasis (TNM) stage (P < 0.05). There was an obvious difference in serum SPRR2A expression between GC patients before and after surgery (P < 0.0001). Conclusion These results suggest that serum SPRR2A can be used as an effective marker for GC.

High SPRR1A expression is associated with poor survival in patients with colon cancer.

High expression of small proline-rich protein 1A (SPRR1A) has been shown to be associated with tumor prognosis; however, the association between SPRR1A expression and colon cancer prognosis remains unclear. The present study sought to evaluate the association between SPRR1A expression and the clinicopathological characteristics of colon cancer, and to examine its potential prognostic value. A total of 114 patients with colon cancer were included. SPRR1A expression was evaluated by immunohistochemical staining, and the association between SPRR1A expression and clinicopathological parameters was analyzed. The prognostic value of SPRR1A was analyzed by Cox regression analysis, the Oncomine database and the R2 platform. SPRR1A expression was significantly increased in cancerous tissues compared with that in adjacent non-cancerous tissues. SPPRR1A expression was significantly associated with lymph node invasion. High SPRR1A expression was significantly associated with worse overall and disease-free survival rate. Cox regression analysis revealed that T stage, pathological N stage and high SPRR1A expression remained independent predictors for overall survival rate. The Oncomine database analysis demonstrated that SPRR1A mRNA expression levels were significantly increased in colorectal cancer tissues compared with those in adjacent non-cancerous tissues, and high SPRR1A expression was associated with a significantly worse event- and relapse-free survival time in the R2 platform. The data indicate that SPRR1A may serve as a potential biomarker for the prognosis of colon cancer.

Downregulation of Sprr1a Contributes to the Pathobiology of Sickle Cell Disease

Sickle cell disease (SCD) presents with multiple comorbidities including pain and organ damage. Transcriptomic analysis of dorsal root ganglion (DRG) revealed a significant decrease in the small proline rich protein 1a (Sprr1a) in HbSS-BERK sickle mice compared to control HbAA-BERK at an early age of ~2 months (Paul et al., Nature Sci Data 2017). Sprr1a is associated with axonal regeneration and cornification, which prevent nerve damage and evaporation from the skin, exposure to infections and mechanical stress, all of which occur in SCD. HbSS-BERK mice show thinner skin, nerve damage and increased sensitivity to mechanical and thermal stimuli (Kohli et al., Blood 2010). We hypothesized that Sprr1a downregulation in sickle mice leads to cutaneous alterations, thus increasing sensitivity to noxious stimuli leading to hyperalgesia and that restoring Sprr1a expression would reduce hyperalgesia. Utilizing Sprr1a-knockout (Sprr1a-KO) mice, we examined the gain and/or loss of skin and neuronal function with Sprr1a deletion. Compared to wild-type (WT) C57BL/6 mice, Sprr1a-KO mice showed a ~30% decrease in epidermal skin thickness (p&amp;lt;0.001); significant increase in Evan blue leakage in the skin in response to substance P and capsaicin (p&amp;lt;0.001 and 0.003, respectively); 30-40% increase in calcium release from DRG neurons in response to capsaicin and menthol (p&amp;lt;0.05 and 0.01, respectively); and increased phospho-p38 MAPK in the spinal cords (p&amp;lt;0.003). Complementary to these observations, Sprr1a-KO mice evinced heightened mechanical, heat and cold sensitivity, which increased with age from 1.2 to 7.0 months of age in both male and female mice compared to C57BL/6 WT. These data suggest that loss of Sprr1a leads to skin thinning, increased permeability and activation of peripheral and central nervous system leading to mechanical and thermal hyperalgesia (pain). To identify the molecular pathways involved in different functions of Sprr1a, we performed mass-spectrometry on the DRG of WT and Sprr1a-KO mice on a LTQ Orbitrap XL mass spectrometer (Thermo Scientific) coupled to a Prominence Nano LC (Shimadzu) using the Xcalibur version 2.7.0 (Thermo Scientific) and Proteome Discoverer 2.2 software. In parallel, we genetically overexpressed Sprr1a in HbSS-BERK mice (Sprr1a-OE-SS) using the Gateway cloning system combined with PiggyBac transposon: We obtained 1 cell embryo from HbSS mice, and carried out pronuclear injection with Sprr1a cDNA. The resulting HbSS mice harboring the Sprr1a transgene were screened by appropriate genotyping of tail snips. Proteomic analysis of DRG and subsequent network analysis using the STRING database tool of the significantly modified proteins (p&amp;lt;0.05) revealed that their interactions with Sprr1a have never been investigated. Many of the significantly altered proteins in Sprr1a-KO are associated with SCD pathobiology, including, but not limited to, SPIC (7-fold increase, p&amp;lt;0.0001), associated with red blood cell (RBC) recycling and VCAM1 regulation; CD9 (22-fold increase, p&amp;lt;0.0001), involved in platelet activation and cell adhesion; SPTBN4 (2-fold decrease, p&amp;lt;0.05), neuropathy, and kininogen (3-fold decrease, p&amp;lt;0.001). Decreased kininogen is observed in sickle cell patients, which is further reduced during crisis (Verma et al., Res Commun Chem Pathol Pharmacol 1983). Thus, downregulation of Sprr1a may contribute to SCD pathobiology, which may in turn lead to pain. This appears to be the case, because overexpression of Sprr1a in HbSS-BERK sickle mice (Sprr1a-OE-SS) led to amelioration of mechanical and cold hyperalgesia (p&amp;lt;0.01 &amp; 0.05, respectively) at 2.8 months of age compared to age/gender matched HbSS-BERK mice. This decrease was maintained up to 9.8 months of age (last period of observation) in Sprr1a-OE-SS, which was similar to the sensitivity in control HbAA-BERK mice and significantly lower than HbSS-BERK mice. Together, these data suggest a causal link between Sprr1a downregulation and pain in SCD. It is likely that Sprr1a loss contributes to complex sickle pathobiology leading to dehydration due to loss of water from thin and permeant skin, non-healing skin ulceration, neuropathy and pain. In conclusion, through genetic and proteomic approaches, we have identified Sprr1a as a novel treatable target for several consequences of SCD. Disclosures Gupta: 1910 Genetics: Research Funding.

Sema3A inhibits axonal regeneration of retinal ganglion cells via ROCK2

Successful regeneration of injured axons in the adult mammalian central nervous system (CNS) is mainly limited by lesion-induced neuronal apoptosis and the inhibitory environment consisting of numerous extrinsic and intrinsic factors. Semaphorin 3A (Sema3A), a classic axonal guidance cue, contributes to the failure of axonal regeneration and can be neutralized to enhance axonal regeneration. Previous studies have suggested that blockage of rho-associated protein kinase 2 (ROCK2) also exerts a protective effect on the survival and axonal regeneration of retinal ganglion cells (RGC, RGCs) after injury. Yet unresolved question is the interaction between the two factors. We thus evaluated the role of Sema3A and ROCK2 in RGC axonal regeneration. In this study, we first examined the expression of Sema3A and ROCK2 against optic nerve crush in vivo and oxygen-glucose deprivation insult to RGCs in vitro at different time points. Then Sema3A, ROCK2 inhibitor Y-27632, combination of both and phosphate-buffered saline (PBS) only were injected into the vitreous cavity after optic nerve crush at various times in different experiments. In order to assess axonal regeneration, we detected the mRNA levels of small proline-rich protein 1A (Sprr1A) and growth-associated protein 43 (GAP43) by quantitative real time-polymerase chain reaction (RT-qPCR), evaluated visual function by Flash Visual Evoked Potentials (F-VEPs), and checked the protein level of GAP43 by immunofluorescent staining. Our results demonstrated that Sema3A significantly suppressed optic nerve regeneration and this effect can be attenuated via blocking ROCK2. Moreover, Sema3A promoted the phosphorylation of myosin light chain 2 (MLC2) (specific downstream effector of ROCK2 concerning neurite growth). Collectively, Sema3A may negatively regulate axonal regeneration through ROCK2 in RGCs.

Elevation of S100 calcium-binding protein A7 in recurrent pterygium.

Recurrent pterygium, a common ophthalmic disease, is difficult to treat as its pathogenesis is unclear. To investigate the key genes responsible for the recurrence of pterygium, tissue samples were collected from six patients with primary pterygium (primary group), six patients with recurrent pterygium (recurrent group) and six patients with ocular trauma (control group) who underwent surgery between December 2014 and June 2017. The differentially expressed genes amongst these tissues were detected using expression profiling microarrays and verified by reverse transcription-quantitative PCR (RT-qPCR). Comparing the primary and control groups, 10 genes, including PP7080, small proline-rich protein 2A, keratin 24, small proline-rich protein 2F, defensin β4A, serpin family A member 3, S100 calcium-binding protein A7 (S100A7), Fc fragment of IgG binding protein and BPI Fold Containing Family A Member 1, were identified to be consistently upregulated in recurrent pterygium tissues, whilst two genes (H19 imprinted maternally expressed transcript and secretoglobin family 2A member 1) were consistently downregulated. Following RT-qPCR verification, it was identified that that S100A7 gene was significantly upregulated in recurrent pterygium tissues compared with the other groups. Protein-protein interaction and Gene Ontology analysis further revealed that all genes interacting with S100A7 were mainly involved in the regulation of defense mechanisms against bacteria, mitogen-activated protein kinase (MAPK) pathway activation and receptor for advanced glycation end-products receptor binding. The present findings confirmed that elevation of S100A7 expression in recurrent pterygium may be associated with the inflammatory response and activation of the MAPK signaling pathway.

Comprehensive Genomic Profiling of Patient-matched Head and Neck Cancer Cells: A Preclinical Pipeline for Metastatic and Recurrent Disease.

Metastases and tumor recurrence have a major prognostic impact in head and neck squamous cell carcinoma (HNSCC); however, cellular models that comprehensively characterize metastatic and recurrent HNSCC are lacking. To this end, we obtained genomic, transcriptomic, and copy number profiles of the UM-SCC cell line panel, encompassing patient-matched metastatic and recurrent cells. UM-SCC cells recapitulate the most prevalent genomic alterations described in HNSCC, featuring common TP53, PI3K, NOTCH, and Hippo pathway mutations. This analysis identified a novel F977Y kinase domain PIK3CA mutation exclusively present in a recurrent cell line (UM-SCC14B), potentially conferring resistance to PI3K inhibitors. Small proline-rich protein 2A (SPRR2A), a protein involved in epithelial homeostasis and invasion, was one of the most consistently downregulated transcripts in metastatic and recurrent UM-SCC cells. Assessment of SPRR2A protein expression in a clinical cohort of patients with HNSCC confirmed common SPRR2A downregulation in primary tumors (61.9% of cases) and lymph node metastases (31.3%), but not in normal tissue. High expression of SPRR2A in lymph node metastases was, along with nonoropharyngeal location of the primary tumor, an independent prognostic factor for regional disease recurrence after surgery and radiotherapy (HR 2.81; 95% CI, 1.16-6.79; P = 0.02). These results suggest that SPRR2A plays a dual role in invasion and therapeutic resistance in HNSCC, respectively through its downregulation and overexpression. IMPLICATIONS: The current study reveals translationally relevant mechanisms underlying metastasis and recurrence in HNSCC and represents an adjuvant tool for preclinical research in this disease setting. Underlining its discovery potential this approach identified a PIK3CA-resistant mutation as well as SPRR2A as possible theragnostic markers.

Analysis of differential gene expression caused by cervical intraepithelial neoplasia based on GEO database.

The aim of the present study was to identify the differentially expressed genes between cervical intraepithelial neoplasias (CIN) and adjacent normal tissue, and to construct a protein-protein interaction (PPI) network. A CIN dataset was obtained from Gene Expression Omnibus, and data of gene expression in CIN and adjacent normal tissue were extracted from GSE64217. The differentially expressed genes were selected using software package and heat map was drawn using the ‘pheatmap’ package. The selected differentially expressed genes were subjected to PPI, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis using Cytoscape, Database for Annotation, Visualization and Integrated Discovery, STRING and KOBAS. In the present study, 287 genes were differentially expressed between CIN and adjacent normal tissue, of which 170 were significantly upregulated and 118 genes were significantly downregulated (P<0.00001, fold-change >6). A differential gene expression network map was constructed to show the interactions of 30 protein products encoded by differentially expressed genes using STRING software. In particular, the key gene, EGR1, was identified using Cytoscape software. The KEGG pathway analysis revealed that the differential genes were mainly involved in several pathways, including ‘glutathione metabolism’, ‘arachidonic acid metabolism’, and ‘pentose phosphate pathway’. Results of the GO analysis showed that differential genes were enriched in different subsets. Specifically, small proline-rich protein 2E and 3, distal-less homeobox 5, epithelial membrane protein 1, cornifelin, periplakin, homeobox protein Hox-A13, estrogen receptor α, transglutaminase 1, small proline-rich protein 2A, Rh C glycoprotein, tumor protein p63, TGM3, homeobox B5 and small proline-rich protein 2D were enriched in ‘epithelial cell differentiation’, which affected the differentiation of epithelial cells. In conclusion, 287 differentially expressed genes were identified successfully. The key gene was identified based on the results of PPI, GO and KEGG analyses, and functional annotation and pathway analysis were also performed. Our study provides the basis for further studies on the interaction among differentially expressed genes.

Identification of cancer biomarkers of prognostic value using specific gene regulatory networks (GRN): a novel role of RAD51AP1 for ovarian and lung cancers.

To date, microarray analyses have led to the discovery of numerous individual 'molecular signatures' associated with specific cancers. However, there are serious limitations for the adoption of these multi-gene signatures in the clinical environment for diagnostic or prognostic testing as studies with more power need to be carried out. This may involve larger richer cohorts and more advanced analyses. In this study, we conduct analyses-based on gene regulatory network-to reveal distinct and common biomarkers across cancer types. Using microarray data of triple-negative and medullary breast, ovarian and lung cancers applied to a combination of glasso and Bayesian networks (BNs), we derived a unique network-containing genes that are uniquely involved: small proline-rich protein 1A (SPRR1A), follistatin like 1 (FSTL1), collagen type XII alpha 1 (COL12A1) and RAD51 associated protein 1 (RAD51AP1). RAD51AP1 and FSTL1 are significantly overexpressed in ovarian cancer patients but only RAD51AP1 is upregulated in lung cancer patients compared with healthy controls. The upregulation of RAD51AP1 was mirrored in the bloods of both ovarian and lung cancer patients, and Kaplan-Meier (KM) plots predicted poorer overall survival (OS) in patients with high expression of RAD51AP1. Suppression of RAD51AP1 by RNA interference reduced cell proliferation in vitro in ovarian (SKOV3) and lung (A549) cancer cells. This effect appears to be modulated by a decrease in the expression of mTOR-related genes and pro-metastatic candidate genes. Our data describe how an initial in silico approach can generate novel biomarkers that could potentially support current clinical practice and improve long-term outcomes.

Defining a mechanistic link between pigment epithelium–derived factor, docosahexaenoic acid, and corneal nerve regeneration

The cornea is densely innervated to sustain the integrity of the ocular surface. Corneal nerve damage produced by aging, diabetes, refractive surgeries, and viral or bacterial infections impairs tear production, the blinking reflex, and epithelial wound healing, resulting in loss of transparency and vision. A combination of the known neuroprotective molecule, pigment epithelium–derived factor (PEDF) plus docosahexaenoic acid (DHA), has been shown to stimulate corneal nerve regeneration, but the mechanisms involved are unclear. Here, we sought to define the molecular events of this effect in an in vivo mouse injury model. We first confirmed that PEDF + DHA increased nerve regeneration in the mouse cornea. Treatment with PEDF activates the phospholipase A2 activity of the PEDF-receptor (PEDF-R) leading to the release of DHA; this free DHA led to enhanced docosanoid synthesis and induction of bdnf, ngf, and the axon growth promoter semaphorin 7a (sema7a), and as a consequence, their products appeared in the mouse tears. Surprisingly, corneal injury and treatment with PEDF + DHA induced transcription of neuropeptide y (npy), small proline-rich protein 1a (sprr1a), and vasoactive intestinal peptide (vip) in the trigeminal ganglia (TG). The PEDF-R inhibitor, atglistatin, blocked all of these changes in the cornea and TG. In conclusion, we uncovered here an active cornea–TG axis, driven by PEDF-R activation, that fosters axon outgrowth in the cornea.

Overexpression of tumor suppressor gene ZNF750 inhibits oral squamous cell carcinoma metastasis.

Zinc-finger protein 750 (ZNF750) encodes a putative C2H2 zinc finger protein and is typically mutated or deleted in squamous cell carcinoma. The role of ZNF750 in oral squamous cell carcinoma (OSCC) remains unknown. The aim of the present study was to investigate the effects of ZNF750 overexpression in CAL-27 cells. Cell viability, and the expression of genes associated with proliferation, differentiation and the epithelial-mesenchymal transition were investigated in CAL-27 cells following ZNF750 overexpression, using Cell Counting kit-8, reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. In addition, scratch wound, invasion and migration assays were performed. Cell viability, matrix metalloproteinase 28 expression, cyclin B1 expression and mesenchymal marker neural cadherin expression were decreased following ZNF750 overexpression compared with the control groups. ZNF750 overexpression induced the differentiation-associated genes late cornified envelope 3A and small proline-rich protein 1A and upregulated the expression of late epidermal differentiation factor Kruppel-like factor 4. Overexpression of ZNF750 in CAL-27 cells resulted in inhibition of cell invasion and migration. Taken together, these data suggest that ZNF750 may inhibit the metastasis of OSCC.

Clinical significance of ZNF750 gene expression, a novel tumor suppressor gene, in esophageal squamous cell carcinoma.

The present authors previously identified a novel candidate tumor suppressor gene, zinc finger protein 750 (ZNF750), in esophageal squamous cell carcinoma (ESCC) (1). The present study aimed to clarify the clinical significance of ZNF750 expression in ESCC. The association between ZNF750 DNA mutation status and the mRNA expression was examined by whole exome sequence analysis and quantitative reverse transcription polymerase chain reaction (RT-qPCR). The expression of ZNF750 in 76 patients with ESCC (Kyushu University Beppu Hospital) was measured using immunohistochemistry and RT-qPCR. Using this dataset, the association between ZNF750 mRNA expression and clinicopathological factors was examined. Additionally, survival analysis was performed using datasets from the Kyushu University Beppu Hospital and The Cancer Genome Atlas (TCGA). The biological effects of ZNF750 expression were explored using gene set enrichment analysis (GSEA) and were validated using datasets from the Cancer Cell Line Encyclopedia (CCLE) and the Kyushu University Beppu Hospital. ZNF750 expression analyses demonstrated that ZNF750 mRNA expression was lower in patients with the DNA mutations compared with those without the mutations (P<0.05), and ZNF750 expression was downregulated in tumor tissues compared with normal tissues (P<0.00005). In the clinicopathological analysis, the low ZNF750 expression group exhibited a higher incidence of undifferentiated histology (P<0.05) compared with the high expression group. The low ZNF750 expression group exhibited a poorer prognosis in the Kyushu and TCGA datasets (P<0.0005 and P<0.05, respectively). GSEA indicated that ZNF750 expression was significantly correlated with epithelial differentiation in ESCC. This was confirmed using the datasets from CCLE and the Kyushu University Beppu Hospital by analyzing the levels of small proline rich protein 1A mRNA, an epithelial differentiation-associated gene. In conclusion, the results of the present study suggested that ZNF750 serves a role as a tumor suppressor; potentially via regulating epithelial differentiation and that it may be a promising biomarker of poor outcomes in ESCC.