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BackgroundImmune related interferon regulatory factor 4 (IRF4) is a member of the IRF family, whereas the clinical significance and possible role of IRF4 in lung adenocarcinoma (LUAD) remains unclear. We aimed to investigate the role of IRF4 in predicting the prognosis of LUAD patients.MethodsUsing The Cancer Genome Atlas (TCGA) database and our immunohistochemical (IHC) cohort, we analyzed the correlation between IRF4 expression and clinical characteristics, and the prognostic value of IRF4 was also evaluated in LUAD. The potential biological functions of IRF4 in LUAD were analyzed by Gene Set Enrichment Analysis (GSEA). The relationship between IRF4 and immune cell infiltration were evaluated by TISIDB database and our own IHC cohort. In addition, an immune checkpoint inhibitor (ICI) treated cohort from Gene Expression Omnibus database was used to determine the role of IRF4 in LUAD patients with immunotherapy.ResultsWe found that either mRNA or protein expression level of IRF4 was significantly higher in LUAD than in normal tissues (P < 0.001). The elevate in IRF4 expression in LUAD was significantly associated with the earlier clinical stage (P = 0.002). Patients with LUAD and IRF4 high expression correlated with significant longer overall survival in both TCGA database (P < 0.05) and our IHC-cohort (P = 0.001). Our results also demonstrated that IRF4 could serve as an independent favorable prognostic factor in patients with LUAD. GSEA analysis indicated that high IRF4 expression group enriched with several immune-related pathways, such as B cell receptor signaling pathway, T cell receptor signaling pathway and cytokine-cytokine receptor interaction signaling pathway. In LUAD, IRF4 positively correlated with several different immune infiltrations including various B cells, CD8+ T cells and CD4+ T cells both in mRNA and protein levels. Additionally, we found that the expression of IRF4 was positively associated with PD-1 and PD-L1 mRNA expression levels, and IRF4 high expression predicted moderate better survival in LUAD with immunotherapy (P = 0.071).ConclusionsOur results suggested that IRF4 was associated with higher B cells and T cells infiltration levels and might be a favorable prognostic biomarker in LUAD patients, whereas the potential prognostic role of IRF4 in ICI-treated patients needed further exploration.

Objective Currently, many studies have found that RFC4 was up‐regulated in various cancers, and related to the progression and development. While the effects of RFC4 in oral tongue squamous cell carcinoma remain unclear, the main purpose of this research is to explore the role of RFC4 in oral tongue squamous cell carcinoma. Methods The expression of RFC4 in various cancers was analyzed in GEPIA database, and the results were further verified by IHC assay. The relationship between RFC4 and several clinical parameters was analyzed; the proliferation was further observed by knockdown RFC4 in vitro. Finally, we constructed related nude mouse models by planting cells subcutaneous of nude mice, and the discrepancy was observed. Results Based on GEPIA database, RFC4 was up‐regulated in various cancers, including colorectal cancer, breast cancer, prostate cancer, lung cancer, and liver cancer. RFC4 was up‐regulated in oral tongue squamous cell carcinoma compared with the normal tissue from GEPIA online database; we further found that the expression of RFC4 was tightly associated with TNM stage (p = 0.005), but not with age, gender, and differentiation (p > 0.05). We further found that the proliferation of oral tongue squamous cell carcinoma was obviously restrained in vitro, and the carcinogenesis was also inhibited in vivo. Conclusions We found that RFC4 was up‐regulated and related to the progression of oral tongue squamous cell carcinoma, and knockdown RFC4 could restrain the proliferation and progression. RFC4 might serve a potential biomarker and provide a new treatment strategy for lots of patients with oral tongue squamous cell carcinoma., Figure 1. RFC4 was up‐regulated in various cancers. The expression of RFC4 in several cancers, including colorectal cancer, breast cancer, prostate cancer, lung cancer and liver cancer.Figure 2. The expression of RFC4 in oral tongue squamous cell carcinoma. Left panel: The expression of RFC4 in oral tongue squamous cell carcinoma (IHC), tissues were classified as low expression and high expression. Right panel: The expression of RFC4in normal tissues was used as normal control group. The expression of rfc4 in normal tissue is low and stain is weakly or negative.Table 1. Relationships of RFC4 and clinicopathological characteristics in 64 patients with tongue squamous cell carcinomaFigure 3. Knockdown RFC4 could restrain the proliferation of oral tongue squamous cell carcinoma CAL27 and TCA8113 cells. A. The mRNA expression of RFC4 in CAL27 and TCA8113 cells transfected with RFC4 shRNA. B. The protein expression of RFC4 in CAL27 and TCA8113 cells transfected with RFC4 shRNA. C. The CCK‐8 assay of CAL27 and TCA8113 cells transfected with RFC4 shRNA. D. The colony forming assay of CAL27 and TCA8113 cells transfected with RFC4 shRNA.Figure 4. Knockdown RFC4 inhibited the growth of oral tongue squamous cell carcinoma in vivo. A, B. CAL27 cell transfected with RFC4 shRNA was transplanted subcutaneously in nude mice, the tumor volume was measured with a caliper. C. IHC staining with RFC4 was performed in these tumors from nude mice.

Lymph node metastasis is one of the most important predictors of the prognosis for esophageal cancer (EC) patients. However, the mechanism underlying the lymph node metastasis is largely unknown. Progranulin (PGRN) is shown to be highly expressed in various types of cancers and could promote the angiogenesis and epithelial-mesenchymal transition of cancer cells in previous studies. However, the expression status of PGRN and its effects on the lymphangiogenesis in EC are largely unclear. In this study, we show for the first time that PGRN is expressed in EC tissue samples and cell lines and could promote the expression of VEGF-C in vitro, a well-known lymphangiogenesis inducer, through the putative signaling transducers p-ERK and p-AKT. Besides, increased levels of PGRN are correlated with lymph node metastasis, high levels of lymph microvessel density, and lymph vessel space invasion in tissue samples of EC patients. In addition, Cox proportional risk model shows that patients with high levels of PGRN would have 2-fold increases in 5-year mortality compared with patients with low levels of PGRN. Finally, we establish a clinically useful nomogram to predict the possibility of mortality for individual EC patients. In conclusion, PGRN may play an important role in the lymphangiogenesis through activation of VEGF-C in the EC patients.

Hsp70 is the most strongly induced in response to various cellular stresses and a good candidate for investigating its role in tissue injury. We firstly cloned full-length cDNA of hsp70 from Paramisgurnus dabryanus (PdHsp70) by RACE method (GenBank: KP402408.1). Then regeneration and inflammation of fin were established by amputation and scratch respectively. Quantitative RT-PCR detected the PdHsp70 began to increase rapidly its expression at 1 days post amputation (dpa) and reached the peak at 2 dpa during fin regeneration. Its expression was also up-regulated at 2 days post scratch (dps) of inflammation but still significant weaker in comparison with it in regenerated fin at 2 dpa. Next, immunohistochemistry analysis of PdHsp70 showed that PdHsp70 located mainly in the deeper epidermis of regenerated fin and was stronger than its expression in the scratched inflammatory fin which was involved in whole epidermal. SDS-PAGE and Western blotting confirmed that the PdHsp70 protein expressed efficiently in Escherichia coli BL21. These findings have implied that PdHsp70 are implicated in different regulation of regeneration and inflammation in response to injury stimulation. During the regeneration, it is involved in the formation of wound epidermis by mediating cellular protection whereas it can modulate inflammatory by activating the innate immune response.

Podoplanin and fibroblast growth factor (FGF) 1 have been detected more frequently in lung squamous cell carcinoma (SQCC) compared with lung adenocarcinoma. Furthermore, it has been previous demonstrated that FGF1 is located on the edge of tumor nests in certain lung SQCC sections, which resembles the characteristic expression pattern of podoplanin. Podoplanin and FGF1 have roles in lymphangiogenesis and angiogenesis. Based on their consistently specific expression in lung SQCC and similar localization patterns, the present study aimed to investigate whether the expression of podoplanin in tumor cells is correlated with FGF1 expression in lung SQCC and whether their co-expression has clinicopathological significance, particularly for lymphangiogenesis/angiogenesis. The correlation between podoplanin and FGF1 expression in tumor cells of 82 lung SQCC cases was investigated by immunohistochemical staining and the association between the co-expression of podoplanin and FGF1, and clinicopathological factors such as microvessel density (MVD), was examined in these samples. In addition, the prognostic value of co-expression of podoplanin and FGF1 in tumor cells was determined, and the regulation of FGF1 expression and angiogenesis by podoplanin was examined in vitro in a human lung SQCC cell line. Immunohistochemical analysis demonstrated that there was a significant correlation between podoplanin and FGF1 expression in lung SQCC tumor cells (R=0.591; P

In this paper, we aimed to explore whether progranulin (PGRN) could induce epithelial ovarian cancer cells to undergo an epithelial mesenchymal transition (EMT) program directly and through its activation of cancer associated fibroblasts (CAFs) indirectly. Immunohistochemistry(IHC) staining of tissue samples of 78 cases of epithelial ovarian cancer (EOC) patients found that PGRN expression levels were negatively correlated with E-cadherin levels (r=-0.289, P=0.013) and positively correlated with Slug levels (r=0.332, P=0.003); Cell experiments showed that PGRN overexpression could increase the migratory and invasive abilities of A2780 cells significantly. Moreover, high doses (62ng/ml) of recombinant PGRN could induce 14.7 fold high expression of smooth muscle actin α (α-SMA) in human normal fibroblasts. In addition, patients with both high levels of PGRN and α-SMA in their tissue samples had the worst disease free survival (DFS) and overall survival (OS) than those with low levels of PGRN or α-SMA. All the results suggest that PGRN could promote invasiveness of EOC cells through an EMT program directly and through activation of CAFs indirectly. This may provide a new effective therapy target for EOC.

Fibroblast growth factor (FGF) 1 is identified as a candidate cancer biomarker in several kinds of cancer. However, little is known about its expression and function in non-small cell lung cancer (NSCLC). The aim of this study is to identify the expression of FGF1 in primary human NSCLC tissues and evaluate its clinical significance for NSCLC patients. Archived tissues from NSCLC (n = 113) and adjacent normal lung tissues (n = 71) were examined for the immunohistochemical expression of FGF1; then we analyzed the correlations of FGF1 expression with clinicopathological factors and overall survival of the patients. FGF1 expression was identified in the cytoplasm or both cytoplasm and nucleus of NSCLC cells. Immunoreactive scores of FGF1 were significantly higher in NSCLC specimens than in peritumoral normal tissues. High expression of FGF1 (immunoreactive score >3) was detected in 61.9% (70/113) of NSCLC specimens, and high FGF1 expression in cancer cells was significantly correlated with larger primary tumor size, squamous cell carcinoma (SQCC), and vascular invasion. In addition, FGF1 expression was correlated with intratumoral microvessel density in both SQCC and adenocarcinoma subgroups. Moreover, NSCLC patients with high FGF1 expression had a significantly lower overall survival rate, compared with those with low FGF1 expression. Furthermore, subgroup analyses showed that FGF1 expression was associated with poor prognosis in lung SQCC, but not in adenocarcinoma. These findings suggest that the presence of FGF1 in NSCLC cells may serve as a prognostic indicator and a potential therapeutic target for NSCLC patients, especially for lung SQCC.

Immunoglobulin-like transcript (ILT) 4 is critical for the inhibitory function of certain immune cells. We previously demonstrated that ILT4 is over-expressed in human non-small cell lung cancer (NSCLC) cells and is involved in tumour evasion via an unknown mechanism. In this report, we demonstrate that ILT4 increases the expression of the co-inhibitory molecule B7-H3 through PI3K/AKT/mTOR signalling. In primary human NSCLC tissues, a significant positive relationship is observed between ILT4 and B7-H3 expression. ILT4/B7-H3 co-expression is significantly associated with a reduction in T infiltrating lymphoid cells and lower overall survival. In summary, ILT4 increases B7-H3 expression and ILT4/B7-H3 co-expression may be involved in NSCLC progression.

Background Previous evidence have demonstrated that p21-activated kinase PAK4 was correlated with breast cancer. The aim of this paper is to study the expression and interaction of p21-activated kinase (pAK)-4 and P54 protein in breast cancer. Methods A total of 80 patients were enrolled in our study (breast fibroma n = 20, breast noninvasive cancer n = 20, early breast invasive cancer n = 20, and advanced breast invasive cancer). The expression of PAK4 was detected by immunohistochemical S-P method, and the relationship between them and the different pathological characteristics were compared. The subcellular localization of P54 and PAK4 in vitro was observed by immunofluorescence assay. Results The expression of both PAK4 and P54 in breast cancer was much higher than that in breast fibroma. Meanwhile, we found that both PAK4 and P54 increased gradually as breast cancer progressed (advanced invasive > early invasive > noninvasive). The positive staining of P54 were mainly located in the cytoplasm, especially around the nucleus. There was no significant stained region in the cell matrix. The P54 localization in the cytoplasm was verified by confocal experiment, and the PAK4 was co-localized. Conclusions PAK4 and P54 proteins may be used as molecular markers for diagnosis and treatment of breast cancer.

Introduction Deficiency of Treg cells and hyperactivity of Th17 cells together are involved in the immunological pathogenesis of asthma. The adenosine A2A receptor (A2AR) plays a critical role in the increased Foxp3 expression of Treg cells and the decreased Th17 generation. Objective The study aimed to investigate A2AR expression in peripheral blood and its regulatory effect on balance of Treg/Th17 cells in asthma. Methods Thirty-one patients with chronic persistent asthma were recruited and divided into 18 intermittent to mild asthma patients, 13 moderate to severe asthma patients. A2AR, Foxp3, and ROR-γt mRNA expression levels in peripheral blood mononuclear cells (PBMCs) were measured by quantitative polymerase chain reaction (qPCR). TGF-β, IL-17, and IgE in plasma were detected with enzyme-linked immunosorbent assay (ELISA). Forty-two BALB/c mice were randomly, equally assigned to control group, ovalbumin (OVA) group and OVA + CGS (CGS21680, A2AR agonist) group. The infiltration of lung inflammation cells were evaluated by HE, A2AR, Foxp3, and ROR-γt mRNA in lung tissues measured by qPCR, TGF-β, IL-17, and IgE in plasma measured with ELISA, and IL-17 and TGF-β protein in lung tissues analyzed with immunohistochemical. Results Our results showed that expression A2AR mRNA in PBMCs was associated with asthma severity. Foxp3 mRNA, TGF-β, and FEV1%pred positively correlated with A2AR mRNA in asthma. ROR-γt mRNA and IL-17 negatively correlated with A2AR mRNA in asthma. CGS could promote Foxp3 mRNA expression, TGF-β, and improve lung function while inhibit ROR-γt mRNA expression, IL-17, and the infiltration of lung inflammation cells. Conclusion A2AR could regulate the balance of Treg/Th17 cells in asthma.

To investigate the expression of growth arrest-specific protein 6 (Gas6) in the placenta and decidua tissues and its relationship with the pathogenesis of preeclampsia.All the patients were recruited in Qilu Hospital of Shangdong University from October 2013 to June 2014. Among them, thirty-two women with early-onset severe preeclampsia who received cesarean section were assigned to the preeclampsia group, and thirty healthy pregnant women who received cesarean section were defined as the control group. Blood glucose, blood lipids, platelet count, D-dimer levels and other clinical indicators of the two groups were detected. Immunohistochemistry of SP was conducted to identify the localization of Gas6 protein in the placenta and decidua tissues. And reverse transcription (RT)-PCR was performed for quantitative analysis of Gas6 RNA expression in placentas. The correlations between placental Gas6 mRNA levels with clinical indicators were analyzed.(1) The gestational age at delivery, blood pressure, serum albumin, platelet count and birth weight of fetuses showed statistically significant differences between the two groups (P0.05). (2) The Gas6 protein expressed in the cytoplasm and nucleus of the syncytiotrophoblasts and decidual cells in the placenta and decidual tissues of the two groups. (3) The Gas6 mRNA expression elevated significantly in the placenta of preeclampsia group (0.60 ± 0.38) when compared to that of the control group (0.34 ± 0.22; P0.05). (4) The expression of Gas6 mRNA was positively related with body mass index, diastolic blood pressure, systolic blood pressure, free fatty acids and creatinine (P0.05), while it was negatively associated with serum albumin (P0.05).The high expression of Gas6 in the placenta and decidua tissues may be related to the pathogenesis of severe preeclampsia.

To investigate the expression of interleukin-37 (IL-37) in placenta tissue and its relationship with the pathogenesis of severe preeclampsia.All the patients were recruited in Qilu Hospital of Shangdong University from November 2012 to November 2013. Among them, thirty patients with severe preeclampsia were assigned to the preeclampsia group, and thirty-one healthy pregnant women were assigned to the control group. Immunohistochemistry of SP was used to detect the IL-37 protein expression in placenta tissue of the two groups. The expression level of IL-37 in placenta tissue of the two groups was detected by western blot. Besides, reverse transcription (RT)-PCR was used to detect the expression of IL-37 mRNA. The correlation between the expression of IL-37 mRNA and the delivery gestational age, body mass index (BMI) was analyzed.(1) IL-37 were detected in the placenta of both the preeclampsia group and the control group, and the expression site mainly located in the syncytiotrophoblast, with a small amount in cytotrophoblast. (2) The expression levels of IL-37 protein in the preeclampsia group and the control group were 0.59 ± 0.39 and 0.88 ± 0.22, respectively. The IL-37 mRNA levels in the preeclampsia group and the control group were 0.55 ± 0.17 and 1.11 ± 0.21, respectively. Both decreased significantly when compared to the control group (P0.05). (3) Significant correlation between the expression of IL-37 mRNA and the delivery gestational weeks (r = 0.209, P0.05) was seen neither in the preeclampsia group nor in the control group (r = -0.053, P0.05). In the severe preeclampsia group, the pregnant women's BMI had no significant correlation with IL-37 mRNA expression of placenta tissue (r = 0.102, P0.05), neither did the control group(r = -0.115, P0.05).IL-37 expression is significantly lower in severe preeclampsia placenta tissue than that in the normal pregnant women, which may play a protective role in the course of severe preeclampsia.

microRNAs (miRNAs) play an important role in development and are associated with birth defects. Data are scant on the role of miRNAs in birth defects arising from exposure to environmental factors such as alcohol.In this study, we determined the expression levels of 509 mature miRNAs in fetal mouse brains with or without prenatal ethanol exposure using a miRNA microarray technique, verified by northern blot and PCR. Mouse embryos in culture were used to examine the effect of ethanol treatment on expression of the putative target genes of miR-10a (Hoxa1 and other Hox members) at mRNA and protein level. Open field and Morris water maze tests were also performed at post-natal day 35.Ethanol treatment induced major fetal teratogenesis in mice and caused mental retardation in their offspring, namely lower locomotor activity (P0.01) and impaired task acquisition. Of the screened miRNAs, miR-10a, miR-10b, miR-9, miR-145, miR-30a-3p and miR-152 were up-regulated (fold change1.5) in fetal brains with prenatal ethanol exposure, whereas miR-200a, miR-496, miR-296, miR-30e-5p, miR-362, miR-339, miR-29c and miR-154 were down-regulated (fold change0.67). Both miR-10a and miR-10b were significantly up-regulated (P0.01) in brain after prenatal ethanol exposure. Ethanol treatment also caused major obstruction in the development of cultured embryos, with down-regulated Hoxa1. Co-incubation with folic acid blocked ethanol-induced teratogenesis, with up-regulated Hoxa1 and down-regulated miR-10a (P0.01).The study provided new insights into the role of miRNAs and their target genes in the pathogenesis of fetal alcohol syndrome.

Background. Cytomegalovirus (CMV) infection of the gastrointestinal (GI) tract is associated with high mortality in immunosuppressed patients. However, few studies have correlated blood CMV load with GI histopathological findings. Furthermore, there have been few studies determining the clinical significance of isolated CMV infection. Design. Cases were selected for the diagnosis of GI CMV infection by searching the information system of a tertiary hospital. The electronic medical record was reviewed for each case to determine blood viral load, clinicopathological features at the time of diagnosis and clinical outcomes after discharge. Results. In all, 30 patients with CMV-positive intestinal biopsies confirmed by immunohistochemistry (IHC) were identified. All were immunosuppressed. CMV inclusions were also recognized by hematoxylin and eosin stain in 27% of the cases, and the remaining cases were identified by IHC alone. CMV blood load was only positive in 17% of the cases; 8 cases had only isolated CMV-infected cells (0.1-0.5 IHC count/high-power field), with the following outcomes: worsening symptoms that responded to antiviral therapy (n = 5); clinical improvement without treatment (n = 1); death without treatment (n = 2). Conclusions. CMV infection of the intestines is clinically significant but will not always present with classic viral cytopathic changes. IHC should be considered in any case where there is a clinical suspicion for CMV infection. Identification of isolated CMV infection by IHC should be considered clinically significant. Current blood viral load tests have poor sensitivity in detecting CMV intestinal infection. Future studies will investigate the predictive value of positive peripheral blood viral load in patients with intestinal symptoms.