TRPV4 channel is involved in HSV-2 infection in human vaginal epithelial cells through triggering Ca2+ oscillation.

Herpes simplex virus (HSV) infection induces a rapid and transient increase in intracellular calcium concentration ([Ca2+]i), which plays a critical role in facilitating viral entry. T-type calcium channel blockers and EGTA, a chelate of extracellular Ca2+, suppress HSV-2 infection. But the cellular mechanisms mediating HSV infection-activated Ca2+ signaling have not been completely defined. In this study we investigated whether the TRPV4 channel was involved in HSV-2 infection in human vaginal epithelial cells. We showed that the TRPV4 channel was expressed in human vaginal epithelial cells (VK2/E6E7). Using distinct pharmacological tools, we demonstrated that activation of the TRPV4 channel induced Ca2+ influx, and the TRPV4 channel worked as a Ca2+-permeable channel in VK2/E6E7 cells. We detected a direct interaction between the TRPV4 channel protein and HSV-2 glycoprotein D in the plasma membrane of VK2/E6E7 cells and the vaginal tissues of HSV-2-infected mice as well as in phallic biopsies from genital herpes patients. Pretreatment with specific TRPV4 channel inhibitors, GSK2193874 (1-4 µM) and HC067047 (100 nM), or gene silence of the TRPV4 channel not only suppressed HSV-2 infectivity but also reduced HSV-2-induced cytokine and chemokine generation in VK2/E6E7 cells by blocking Ca2+ influx through TRPV4 channel. These results reveal that the TRPV4 channel works as a Ca2+-permeable channel to facilitate HSV-2 infection in host epithelial cells and suggest that the design and development of novel TRPV4 channel inhibitors may help to treat HSV-2 infections.

Use of the alkaline phosphatase to prealbumin ratio as an independent predictive factor for the prognosis of gastric cancer.

BACKGROUND: Gastric cancer (GC) is characterized by a low 5-year survival rate. The prognosis is still not satisfactory although it has significantly improved due to developments in medicine. Thus, the identification of more efficient indices for the evaluation of GC prognosis is required. We propose, for the first time, that the alkaline phosphatase (ALP) to prealbumin (PA) ratio (APR) can be used as an independent prognostic factor in GC. AIM: To evaluate the prognostic value the APR in GC. METHODS: According to the exclusion strategy, we collected the preoperative serologic examination results and clinical information of 409 GC patients treated in Shandong Provincial Hospital from January to December, 2016. By calculating the APR, the neutrophil and lymphocyte ratio (NLR), C-reactive protein (CRP) and albumin (ALB) ratio, platelet and lymphocyte ratio, lymphocyte and monocyte ratio, and the relationship with clinical information, we verified the role of preoperative APR ratio in the prognosis of GC. In addition, we used a Cox model combined with the APR and tumor stage to demonstrate its efficacy in assessing the prognosis of GC patients. RESULTS: Preoperative APR was an independent prognostic factor for GC. The median age of patients in the APR-high group was greater compared with that in the APR-low group. Patients with a higher APR had a more advanced clinical stage, higher neutrophil to lymphocyte, CRP to ALB, and platelet to lymphocyte ratios, but a lower lymphocyte to monocyte ratio (P < 0.05). The APR-high group also had higher glycoprotein antigen 199 and carbohydrate antigen 125 levels than the APR-low group (P < 0.05). Median overall survival and disease-free survival were significantly longer in the APR-low group than in the APR-high group. In addition, a Cox model based on the APR and tumor stage was more effective in evaluating the prognosis of patients than models based on stage alone or stage plus the NLR. CONCLUSION: A higher APR is an independent and negative prognostic factor for GC. The prognosis of GC can be better evaluated using a Cox model based on the APR and stage.

Identification of New Therapeutic Targets for Gastric Cancer With Bioinformatics.

We aimed to identify new targets affecting gastric cancer (GC) prognosis. Six target genes were identified from hub genes based on their relationship with important factors affecting tumor progression, like immune infiltration, purity, tumor mutation burden (TMB), and tumor microenvironment (TME) score. The effect of target genes' somatic mutations and copy number alteration (CNA) was examined to determine their effect on GC prognosis. Six target genes (FBN1, FN1, HGF, MMP9, THBS1, and VCAN) were identified. Reduced expression of each target gene, except MMP9, indicated better prognosis and lower grade in GC. FBN1, THBS1, and VCAN showed lower expression in stage I GC. Non-silencing mutations of the six genes played a role in significantly higher TMB and TME scores. THBS1 mutation was associated with earlier stage GC, and VCAN mutation was associated with lower grade GC. However, patients with target gene CNA displayed higher tumor purity. MMP9, THBS1, and VCAN CNA was associated with lower grade GC, while FBN1 CNA reflected earlier T stage. Additionally, the target genes may affect GC prognosis by influencing multiple oncogenic signaling pathways. FBN1, FN1, HGF, MMP9, THBS1, and VCAN may be new GC prognostic targets by affecting tumor purity, TMB, TME score, and multiple oncogenic signaling pathways.

Chemokine receptor 4 expression is correlated with the occurrence and prognosis of gastric cancer.

Gastric cancer (GC) is a common tumor with a low 5-year survival rate. The chemokine receptor 4 (CXCR4) protein contributes to the progression and prognosis of GC, but the relationship between CXCR4 and immune infiltration, somatic copy number alteration (SCNA), tumor purity, tumor mutation burden (TMB), cytolytic activity (CYT), and drug sensitivity in GC is poorly understood. This study aimed to systematically explore the role of CXCR4 in GC. Microarray and RNA-seq data were collected from the Gene Expression Omnibus and The Cancer Genome Atlas. Our analysis shows that CXCR4 is correlated with various types of immune cells. Patients with high CXCR4 expression had a higher fraction of B cells and CD8+ T cells, and a lower fraction of CD4+ T cells. In addition, high CXCR4 expression was associated with more advanced tumor stage, worse prognosis and higher stromal score, immune score, and cytolytic activity (P < 0.05). High CXCR4 expression also correlated with lower tumor purity and TMB. In summary, our analyses suggest that CXCR4 may affect the progression and prognosis of GC by influencing immune infiltration, TMB, CYT, tumor purity, and drug sensitivity.

Function of fibroblast growth factor 2 in gastric cancer occurrence and prognosis.

The present study aimed to explore the role of fibroblast growth factor 2 (FGF2) in the development and prognosis of gastric cancer (GC). The relationship between FGF2 mRNA expression levels and the clinical characteristics of GC was investigated using microarray data from four GC cohorts involving 726 patients obtained from the Gene Expression Omnibus. The results of the present study indicated that FGF2 expression levels were an independent factor affecting the prognosis of GC. The primary functions of FGF2 were related to cell adhesion and angiogenesis, and patients with high levels of FGF2 expression had poorer TNM staging and prognosis; these differences were statistically significant. In terms of immune infiltration, a higher extent of M2 macrophage intrusion was observed in patients with higher levels of FGF2. However, the degree of infiltration by dendritic and CD4+ T cells was lower, and this difference was statistically significant. Multivariate Cox proportional hazards model analysis revealed that age, TNM staging and FGF2 expression levels were independent prognostic factors for GC. In summary, FGF2 expression was demonstrated to be an independent prognostic factor in GC, and higher levels of FGF2 may promote the progression of this malignancy.

Usefulness of large-section cytokeratin 20 in the detection of intestinal wall infiltration and mesangial metastasis in patients with middle and lower rectal cancer.

OBJECTIVE: The objective of the study was to evaluate the usefulness of large-section cytokeratin 20 (CK20) staining technique in the detection of infiltration on the distal wall and mesangial metastasis in patients with middle and lower rectal cancer. MATERIALS AND METHODS: A total of 62 patients with rectal cancer in the middle and lower segment were studied on large slices stained with CK20. Logistic regression was used to analyze the clinicopathologic factors related to distal low and middle rectal cancer metastasis to the mesorectum and rectal wall. RESULTS: Two types of distal metastasis of the tumor were observed in the rectal wall in 18% (11/62) of the patients: submucosal invasion and muscularis propria invasion. The extent of distal metastasis to the rectal wall was around 0.5-1.0 cm. Four types of distal metastasis occurred in the mesorectum: lymph node invasion, blood and lymphatic vessel invasion, perineural invasion, and isolated neoplastic microfoci. Distal metastasis to the mesorectum was observed in 24% (15/62) of the patients. The extent of metastasis to the mesorectum was around 0.5-4.0 cm. Another three patients with microcapillary invasion in the distal mesorectum were observed by immunohistochemistry, as it was difficult to determine the spread by conventional hematoxylin and eosin staining. CONCLUSION: The large-section CK20 staining technique is useful for the detection of infiltration on the distal wall and mesangial metastasis in patients with middle and lower rectal cancer.

Promotion of Sema4D expression by tumor-associated macrophages: Significance in gastric carcinoma.

AIM: To study the role of semaphorin 4D (Sema4D) expression promoted by tumor-associated macrophages (TAMs) in gastric carcinoma cells and its clinical significance in the invasion and metastasis of gastric carcinoma. METHODS: CD68 and Sema4D expression was analyzed in gastric carcinoma and adjacent normal tissues from 290 patients using the immunohistochemical streptavidin-peroxidase method, and their relationships with clinicopathological features were evaluated. Human M2 macrophages were induced in vitro and co-cultured in non-contact with gastric carcinoma SGC-7901 cells. Changes in the secretory Sema4D level in the SGC-7901 cell supernatant were measured using an enzyme-linked immunosorbent assay. The effects of TAMs on SGC-7901 cell invasion and migration were assessed with invasion and migration assays, respectively. RESULTS: CD68 and Sema4D protein expression was significantly higher in gastric carcinoma tissues than in adjacent normal tissues (71.7% vs 33.8% and 74.5% vs 42.8%, respectively; P < 0.01). CD68 and Sema4D protein expression was significantly associated with histological differentiation, TNM stage, and lymph node metastasis (P < 0.05), and their expression levels were positively correlated with one another (r = 0.467, P < 0.01). In the in vitro experiment, secretory Sema4D protein expression was significantly increased in the supernatant of SGC-7901 cells co-cultured with TAMs compared with the blank control (1224.13 ± 29.43 vs 637.15 ± 33.84, P < 0.01). Cell invasion and metastasis were enhanced in the Transwell invasion and migration assays (P < 0.01). CONCLUSION: TAMs promote the invasion and metastasis of gastric carcinoma cells possibly through upregulated secretory Sema4D protein expression. Combined detection of TAM markers, CD68 and Sema4D, in gastric carcinoma tissue shows potential to predict the trend of gastric carcinoma progression.

Activation of HIFa pathway in mature osteoblasts disrupts the integrity of the osteocyte/canalicular network.

The hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, are the central mediators of the homeostatic response that enables cells to survive and differentiate in low-oxygen conditions. Previous studies indicated that disruption of the von Hippel-Lindau gene (Vhl) coincides with the activation of HIFα signaling. Here we show that inactivation of Vhl in mature osteoblasts/osteocytes induces their apoptosis and disrupts the cell/canalicular network. VHL-deficient (ΔVHL) mice exhibited a significantly increased cortical bone area resulting from enhanced proliferation and osteogenic differentiation of the bone marrow stromal cells (BMSCs) by inducing the expression of ß-catenin in the BMSC. Our data suggest that the VHL/HIFα pathway in mature osteoblasts/osteocytes plays a critical role in the bone cell/canalicular network and that the changes of osteocyte morphology/function and cell/canalicular network may unleash the bone formation, The underlying mechanism of which was the accumulation of ß-catenin in the osteoblasts/osteoprogenitors of the bone marrow.

Semaphorin 4D and hypoxia-inducible factor-1α overexpression is related to prognosis in colorectal carcinoma.

AIM: To investigate semaphorin 4D (Sema4D) and hypoxia-inducible factor-1α (HIF-1α) expression in colorectal carcinoma and evaluate their clinicopathological and prognostic significance. METHODS: Eighty-six curatively resected colorectal carcinoma patients at different stages of disease were randomly selected from the group of patients who underwent surgery, and none of them received preoperative radiochemotherapy. Normal proximal adjacent bowel tissue, which served as an internal control, was obtained from 52 randomly selected patients. Immunohistochemistry was performed to analyze the expression of Sema4D and the tumor angiogenesis-related protein HIF-1α in normal colorectal tissues and colorectal carcinoma tissues. The relationships between the expression and clinical characters and prognosis were analyzed. RESULTS: HIF-1α and Sema4D were positively expressed in 58% and 60% of colorectal carcinoma tissues, respectively. Significantly lower expression levels were observed in normal mucosa (8% and 12%, respectively). HIF-1α and Sema4D expression was closely correlated with histological tumor type, tumor-node-metastasis (TNM) stage, and lymphatic metastasis (P<0.05), but not with age or tumor size (P>0.05). HIF-1α and Sema4D protein expression was significantly correlated with prognosis of colorectal carcinoma, as determined by Spearman rank correlation analysis (r=0.567; P<0.01). Multivariate Cox analysis revealed that only Sema4D expression played a significant role in predicting patient prognosis (P<0.05). CONCLUSION: These findings suggest that HIF-1α and Sema4D expression correlates with histological tumor type, TNM stage, and lymphatic metastasis in colorectal carcinoma and that Sema4D is a prognostic indicator of colorectal carcinoma.

Heme oxygenase-1 promotes Caco-2 cell proliferation and migration by targeting CTNND1.

BACKGROUND: Heme oxygenase-1 (HO-1) can be induced by inflammatory cytokines, oxidation, ischemia, hypoxia, and endotoxins. As a "graft survival protective gene," HO-1 is a hot spot in organ transplantation research. However, the role of HO-1 gene expression in the function of human colon adenocarcinoma cell line (Caco-2) cells has not been reported previously. METHODS: The role of HO-1 in the proliferation and migration of Caco-2 cells was analyzed using a stable HO-1 expression plasmid. We constructed a recombinant adeno-associated virus plasmid containing the HO-1 gene, heme oxygenase 1 (HMOX1), which was transfected into Caco-2 intestinal cells. We identified a number of target genes by global microarray analysis combined with real-time polymerase chain reaction (PCR) and chromatin immunoprecipitation assay. RESULTS: Our results showed that significant HO-1 upregulation was demonstrated in the Caco-2 cells after HO-1 transfection. Restoration of HO-1 expression promoted proliferation and invasion in vitro. The CTNND1 gene, a member of the armadillo protein family, was identified as a direct HO-1 target gene. CONCLUSION: Overexpression of HO-1 promotes Caco-2 cell proliferation and migration by targeting the CTNND1 gene.

Down-regulation of miR-622 in gastric cancer promotes cellular invasion and tumor metastasis by targeting ING1 gene.

AIM: To evaluate the biological and clinical characteristics of miR-622 in gastric cancer. METHODS: We analyzed the expression of miR-622 in 57 pair matched gastric neoplastic and adjacent non-neoplastic tissues by quantitative real-time polymerase chain reaction. Functional analysis of miR-622 expression was assessed in vitro in gastric cancer cell lines with miR-622 precursor and inhibitor. The roles of miR-622 in tumorigenesis and tumor metastasis were analyzed using a stable miR-622 expression plasmid in nude mice. A luciferase reporter assay was used to assess the effect of miR-622 on inhibitor of growth family, member 1 (ING1) expression. RESULTS: Expression of miR-622 was down-regulated in gastric cancer. MiR-622 was found involved in differentiation and lymphatic metastasis in human gastric cancer. Ectopic expression of miR-622 promoted invasion, tumorigenesis and metastasis of gastric cancer cells both in vitro and in vivo. ING1 is a direct target of miR-622. CONCLUSION: These findings help clarify the molecular mechanisms involved in gastric cancer metastasis and indicate that miR-622 modulation may be a bona fide treatment of gastric cancer.

Integrin alpha v beta 6 mediates the potential for colon cancer cells to colonize in and metastasize to the liver.

Integrin alpha v beta 6 (alpha v beta 6) is correlated with colon cancer progression. To detect the effects of alpha v beta 6 on liver metastasis, the specificity of alpha v beta 6 against the monoclonal antibody (mAb) 2G2 was examined by immunoprecipitation. Integrin alpha v beta 6-immunoreactivity (IR) in liver metastasis tissues (63 cases) and colon carcinoma (358 cases) were examined. These results showed that alpha v beta 6 was specifically recognized by the mAb 2G2, and that rates of alpha v beta 6 positivity in liver metastatic tissues (71.4%, 45/63) were higher than that for primary colon cancer (34.0%, 122/358) (P < 0.01). Patients who were alpha v beta 6-positive had higher liver metastasis rates (17%, 21/122) than those who were alpha v beta 6-negative (only 3%, 7/236) (P < 0.01). To examine the underlying mechanisms associated with alpha v beta 6 regulating colonic metastasis in the liver, experimental liver metastasis (intrasplenic injection of HT29 transfectants) and liver colonization assays (direct injection of WiDr transfectants into the liver) in nude mice were performed; these demonstrated that alpha v beta 6 contributed to the promotion of the metastatic potential and the survival of cancer cells in the liver. Matrix metalloproteinase-9 (MMP-9) levels in the cultures of both HT29 and WiDr cells were detected by the Biotrak MMP-9 activity assay system and gelatin zymography assay, and showed that suppression of alpha v beta 6-IR inhibited MMP-9 activity and secretion. Transwell migration assay in vitro also showed that alpha v beta 6 promoted migration on fibronectin for HT29/WiDr mock compared with HT29/WiDr antisense beta 6 transfects (P < 0.01). We concluded that alpha v beta 6 may mediate the potential for colon cancer cells to colonize in and metastasize to the liver. The mechanisms that alpha v beta 6 may be involved in include the promotion of MMP-9 secretion, the enhancement of migration on fibronectin, and the survival of cancer cells in the liver.

[The regulation of hypoxia inducible factor-1alpha on osteoblast function in postmenopausal osteoporosis].

OBJECTIVE: To study the regulation of hypoxia inducible factor-1alpha (HIF-1alpha) on osteoblast function in postmenopausal osteoporosis. METHODS: From October 2004 to May 2006, Cre-Loxp recombinase was used to create mice which the HIF-1alpha gene in osteoblasts was conditional knock-out, 24 female wild-type (WT) mice and 24 female conditional knock-out (CKO) mice of 3 months old were operated on ovariotomy. At 0,4,8 weeks after operation, bone histomorphometry parameters were measured with computer image analysis in HE stain sections and in tetracycline bone double labeling fluorescence sections; Bone density and the trabecular bone architecture parameters were measured by Micro-CT; The mRNA expression of vascular endothelial growth factors (VEGF), RunX2, OC, ALP were detected with quantitative RT-PCR; The protein expression of VEGF and RunX2 were detected with Western-blotting. RESULTS: In CKO mice, the trabecular number, volume, thickness, bone density, mineral apposition rate (MAR), the expression of VEGF, RunX2, OC, ALP on mRNA level and the expression of VEGF, RunX2 on protein level decreased significantly compared with WT mice especially in 8 weeks after operation. CONCLUSIONS: The bone formation ability of osteoblasts in CKO mice was reduced compared with WT mice after ovariotomy. HIF-1alpha can regulate the bone formation ability of osteoblasts in postmenopausal osteoporosis.

[Effects of antisense 6 gene on colon cancer cells].

OBJECTIVE: To investigate the effect of antisense integrin beta6 gene on the growth of colon cancer cells. METHODS: Expressing vector of antisense alphavbeta6 was constructed. Human colon cancer cells of the line HT29 were cultured and divided into 3 groups: Group A, remaining wild type; Group B, transfected with antisense integrin beta6 gene; and Group C, transfected with blank vector. RT-PCR was used to detect the integrin beta6 mRNA expression of in the HT29 cells. The integrinbeta6 protein expression on the surface of the cells was detected by immunohistochemistry and flow cytometry. The binding between the cells and fibronectin was examined. (3)H-labeled thymidine (T) was added into the culture fluid of the cells, and then the radiation amount was detected every 6 days so as to determine the capacity to proliferation of the cells in vitro. Thirty female nude mice were divided into 3 groups to be injected subcutaneously with suspension of HT29 cells of Groups A, B, and C as mentioned above. Six weeks later the size of tumors was measured and part of the tumor nodules were resected 5 weeks after the inoculation to undergo pathological examination. RESULTS: Compared with Groups A and C, no corresponding band at 141 bp was found in Group B by RT-PCR. Flow cytometry showed that the expression level of beta6 protein had was (0.30 +/- 0.051, 30%), significantly lower than those of Groups A and C [(0.80 +/- 0.038, 80%) and (0.85 +/- 0.045, 85%), both P < 0.01]. The binding between the HT29 cells and fibronectin of Group B was significantly degraded after the further addition of anti-beta1 and anti-alphav in comparison of Groups A and C (both P < 0.01). The accumulation values of (3)H-labeled T of Group B 2, 4, and 6 days after addition were all significantly lower than those of Groups A and C (all P < 0.01). The tumors in 9 of the 10 mice injected with the HT29 cells of Group B disappeared and the tumor in the only one mice in Group B was only less than 1 mm(3), significantly smaller then those in Groups A and C (15 mm(3) on average, all P < 0.01). CONCLUSION: Antisense beta6 gene significantly inhibits the mRNA and protein expression of the beta6 gene, and then inhibits the growth and proliferation of colon cancer cells, thus proving that integrin beta6 plays an important role in the regulation of colon cancer cells.

[Regulation of hypoxia inducible factor-1alpha on osteoblast function in osteogenesis].

OBJECTIVE: To explore the regulation of hypoxia inducible factor-1alpha (HIF-1alpha) on osteoblast function in osteogenesis. METHODS: Skull-cap bone of HIF-1alpha Loxp/Loxp and VHL Loxp/Loxp C57/BL6 mice were taken out and cultured so as to obtain osteoblasts which were infected with the recombinant adenovirus Ad-Cre so as to conditionally knock out the HIF-1alpha gene and its up-stream gene for von Hippel-Lindau disease (VHL) using Cre-Loxp recombinase technique. Then the osteoblasts were cultured under 2% O2 for 48 hours. Real-time PCR and Western-blotting were used to detect the mRNA and protein expression of vascular endothelial growth factor (VEGF), core binding factor al (RunX2), alkaline phosphatase (ALP) and osteocalcin (OC). Transgenic FVB mice mated with C57/BL6 mice with both HIF-1alpha and VHL alleles to obtain the mice with the osteoblasts with the HIF-1alpha and VHL genes conditionally knocked-out. At the age of 3 months the distal femurs of HIF-1alpha/VHL conditionally knocked-out mice and wild type mice were obtained to undergo hematoxylin-eosin staining and micro-CT to evaluate the bone histomorphometry and bone mineral density (BMD). RESULTS: The mRNA and protein expressions of VEGF, RunX2, ALP, and OC of the HIF-1alpha conditionally knocked-out osteoblasts were all decreased and the mRNA and protein expressions of VEGF, RunX2, ALP, and OC of the VHL conditionally knocked-out osteoblasts were all increased. The values of bone histomorphometry and bone mineral density (BMD) of the HIF-1alpha conditionally knocked-out mice were both significantly lower than those of the wild-type mice, whereas the values of bone histomorphometry and BMD of the VHL conditionally knocked-out mice were both significantly higher than those of the wild-type mice (all P < 0.01). CONCLUSION: Under both the physiological and pathological hypoxia environment in bone tissues HIF-1alpha can promote the bone formation ability of osteoblast.