Intracellular co-localization of SPLUNC1 protein with nanobacteria in nasopharyngeal carcinoma epithelia HNE1 cells depended on the bactericidal permeability increasing protein domain.

Epithelial surfaces constitute natural immunobarriers against environmental threats. These barriers are brimming with fluids that bind, transport, cleave or degrade bacterial cells and their endotoxic by-products. Saliva and the airway surface-lining fluid (ASL) comprise the important fluid constituents. Short palate, lung and nasal epithelium clone 1 (SPLUNC1) is a potential host defensive protein that is secreted from the submucosal gland to the saliva and nasal lavage fluid. However, its antimicrobial spectrum and antimicrobial mechanism is not clear. Through green fluorescence protein (GFP) mediated subcellular localization experiments in nasopharyngeal carcinoma (NPC) HNE1 cell line, we determined that the intracellular GFP-tagged SPLUNC1 protein binds to a miniscule microorganisms, approximately 50-400nm in size, after the bactericidal permeability increasing protein (BPI) domain was deleted, GFP-tagged truncated SPLUNC1 protein lost its function of binding to the miniscule microorganisms. We verified that these microorganisms are nanobacteria (NB) with a negative staining using transmitted electronic microscope (TEM) and immunofluorescent analysis using an NB-specific antibody. We isolated and cultured the NB from the cultured nasopharyngeal carcinoma epithelia HNE1 cell supernatant. We found that the NB did not absorb the Hoechst stain, even when we extended the staining time to 35min. However, with the time extension the larger sized NB (larger than 300nm) did stain positively. From the biopsy specimen of NPC, we also detected the NB, which can lead to the swelling of mitochondria in the infected host cells. We hypothesize that SPLUNC1 and NB co-localization is due to the GFP-tagged SPLUNC1 protein binding to the lipopolysaccharide (LPS) of the Gram-negative NB, which can play an important role in the host defense of nasopharyngeal epithelium. This research sheds new light on the mechanism of SPLUNC1 involvement in the host upper respiratory tract defense system.

[Effect of growth inhibition of the secretory protein SPLUNC1 on Pseudomonas aeruginosa].

OBJECTIVE: To express the recombinant SPLUNC1 protein in HNE1 cells and to study its function of bactericidal and binding to lipopolysaccharide (LPS). METHODS: Full length of SPLUNC1 gene was cloned into pCMV-tag4A vector and stably transfected into HNE1 cell lines, the supernatant of cell cultures was collected. After being treated with the supernatant, the Pseudomonas aeruginosa was seeded to LB soft agar plate, and the bacteria clones were counted and analyzed. For in vitro LPS binding assay, LPS was coated to 96-well plates. We incubated in the plate with SPLUNC1 protein, and detected the binded SPLUNC1 protein by ELISA. Incubating the FITC-LPS with the SPLUNC1 stably transfected or control cells, the intracellular intensity of fluorescence was observed under the fluorescence microscope. RESULTS: SPLUNC1 inhibited the bacteria clone formation obviously. Although the binding efficiency of LPS and SPLUNC1 in vitro was very low, more FITC-LPS entered into the SPLUNC1 stably transfected cells. CONCLUSION: SPLUNC1 can inhibit the growth of Pseudomonas aeruginosa and bind LPS, and play an important defensive role in innate immunity of the upper airway.

Expression of tumor related genes NGX6, NAG-7, BRD7 in gastric and colorectal cancer.

AIM: NGX6, NAG-7 and BRD7 genes are tumor related genes, which have been newly cloned by positional candidate cloning strategy. This study was designed to investigate the expression levels of NGX6, NAG-7 and BRD7 genes in human gastric and colorectal cancer tissues, and their corresponding normal tissues, and to investigate whether these genes play a role in the pathogenesis of gastric and colorectal cancers. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), dot hybridization and Northern blot analysis were used to compare the expression levels of NGX6, NAG-7 and BRD7 genes in 34 gastric cancer tissues and 34 colorectal cancer tissues with their corresponding normal tissues of the same patients, respectively. RESULTS: Among the 34 colorectal cancer specimens and the 34 gastric cancer specimens, the expression of NGX6 in 25 colorectal cancer tissues was absent or very weak (73.5 %) by RT-PCR analysis. The down-regulation rate of NGX6 in colorectal cancer tissues was significantly higher than that in corresponding normal tissues (26.5 %,9/34) (P<0.005). Moreover, the down-regulation of NGX6 was significantly correlated with lymph node and/or distance metastases. Patients with lymph node and/or distance metastasis had much higher down-regulation rate of NGX6 than patients without metastases (93.8 % vs 55.6 %, P<0.05). However no correlation was found between the expression of NGX6 and pathologic type of colorectal cancer in this study, and also the expression of NGX6 did not display any difference between gastric cancer and corresponding normal tissues (58.8 % vs 70.6 %, P>0.25). Dot hybridization and Northern blot analysis confirmed the results of RT-PCR. Furthermore, NAG-7 and BRD7 mRNA was not up- or down-regulated in gastric and colorectal cancers compared with their corresponding normal tissues in our study. CONCLUSION: The down-regulation of NGX6 may be closely associated with tumorigenesis and metastasis of colorectal carcinoma. However, it may not contribute to the development and progression of gastric carcinoma. In addition, the expression levels of NAG-7, and BRD7 did not alter in gastric and colorectal cancers. This seems to suggest that NAG-7 and BRD7 genes may not play a role in gastric and colorectal carcinogenesis.

Expression of tumor related gene NAG6 in gastric cancer and restriction fragment length polymorphism analysis.

AIM: NAG6 gene is a novel tumor related gene identified recently. This study was designed to examine the expression of this gene in gastric cancer and corresponding normal tissues, and to investigate its role in the occurrence and development of gastric cancer, also to study if the genetic structure of NAG6 was altered in gastric cancer. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), Northern blot analysis and dot hybridization were used to compare the expression level of NAG6 gene in 42 cases of gastric cancer tissues with their corresponding normal tissues of the same patients respectively. In addition, restriction fragment length polymorphism (RFLP) analysis was adopted to study if the genetic structure of NAG6 was altered in gastric carcinomas. RESULTS: The expression of NAG6 in 57.1% gastric cancer tissues (25/42) was absent by RT-PCR analysis. The down-regulation rate of NAG6 in gastric cancer tissues was significantly higher than that in corresponding normal tissues (P<0.01). However no correlation between the down-regulation of NAG6 and lymph-node and/or distance metastasis was found in this study (P>0.05). Dot hybridization confirmed the results of RT-PCR. Furthermore, the results of EcoRI RFLP analysis of NAG6 gene demonstrated that 3 of 7 cases of gastric cancer showed loss of 5 kb fragment in comparison with their corresponding normal tissues. CONCLUSION: NAG6 gene is significantly down regulated in gastric cancer. The loss of genetic materials may be the cause of down-regulation of NAG6 expression. This seems to suggest that NAG6 may represent a candidate of putative tumor suppressor gene at 7q31-32 loci associated with gastric carcinoma. The down-regulation of this gene may play a role in occurrence and development of this disease, however it may not be associated with lymph node and/or distance metastasis.

Effect of SPLUNC1 protein on the Pseudomonas aeruginosa and Epstein-Barr virus.

Short palate, lung and nasal epithelium clone 1 (SPLUNC1) gene coded a secreted protein found at the surface of nasopharyngeal epithelium, which may be an innate immunity defensive molecular and a risk factor for nasopharyngeal carcinoma (NPC). Here, we observed the effects of SPLUNC1 on the Gram negative bacteria Pseudomonas aeruginosa, evaluated the ability of SPLUNC1 protein binding to lipopolysaccharide. To observe the effect of SPLUNC1 protein on Epstein-Barr virus (EBV), we raised three EBV-transformed B-lymphocyte lines and treated the cells by SPLUNC1 protein; cellular disruption, apoptosis, EBV DNA content, and viral oncogene expression were analyzed. We found that SPLUNC1 protein can bind to bacterial lipopolysaccharide, inhibit the growth of P. aeruginosa, enhance the disruption and apoptosis of EBV-infected B-lymphocytes, downregulate protein expression of EBV latent membrane protein 1, while upregulate protein expression of EBV envelope glycoprotein gp350/220. The total EBV DNA in the culture medium was decreased significantly after 7 days of treatment by SPLUNC1. This study shows that SPLUNC1 not only has the role of antibacteria and antivirus, but also inhibits the potential oncogenicity of EBV in respiratory epithelium.