Transmembrane protein 176B regulates amino acid metabolism through the PI3K-Akt-mTOR signaling pathway and promotes gastric cancer progression.

BACKGROUND: The present study aimed to investigate the expression level, biological function, and underlying mechanism of transmembrane protein 176B (TMEM176B) in gastric cancer (GC). METHODS: TMEM176B expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB). The function of TMEM176B was determined by various in vitro assays including colony formation, 5-ethynyl-2'-deoxyuridine (EdU), Transwell, and flow cytometry. Bioinformatics techniques were then used to elucidate the signaling pathways associated with TMEM176B activity. Tumor formation experiments were conducted on nude mice for in vivo validation of the preceding findings. TMEM176B expression was cross-referenced to clinicopathological parameters and survival outcomes. RESULTS: It was observed that TMEM176B was overexpressed in GC cells and tissues. Targeted TMEM176B abrogation inhibited colony formation, proliferation, migration, and invasion but promoted apoptosis in GC cell lines while TMEM176B overexpression had the opposite effects. Subsequent experimental validation disclosed an association between TMEM176B and the phosphatidylinositol 3-carboxykinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling axis. Moreover, TMEM176B affects GC cancer progression by regulating asparagine synthetase (ASNS). The in vivo assays confirmed that TMEM176B is oncogenic and the clinical data revealed a connection between TMEM176B expression and the clinicopathological determinants of GC. CONCLUSION: The foregoing results suggest that TMEM176B significantly promotes the development of gastric cancer and is an independent prognostic factor of it.

GRK2 Mediates Macrophage Polarization by Regulating EP4-cAMP-pCREB Signaling in Ulcerative Colitis and the Therapeutic Effect of Paroxetine on Mice with DSS-Induced Colitis.

G protein-coupled receptor kinase 2 (GRK2) is one of the cytosolic enzymes, and GRK2 translocation induces prostaglandin E2 receptor 4 (EP4) over-desensitization and reduces the level of cyclic adenosine monophosphate (cAMP) to regulate macrophage polarization. However, the role of GRK2 in the pathophysiology of ulcerative colitis (UC) remains unclear. In this study, we investigated the role of GRK2 in macrophage polarization in UC, using biopsies from patients, a GRK2 heterozygous mouse model with dextran sulfate sodium (DSS)-induced colitis, and THP-1 cells. The results showed that a high level of prostaglandin E2 (PGE2) stimulated the receptor EP4 and enhanced the transmembrane activity of GRK2 in colonic lamina propria mononuclear cells (LPMCs), resulting in a down-regulation of membrane EP4 expression. Then, the suppression of cAMP-cyclic AMP responsive element-binding (CREB) signal inhibited M2 polarization in UC. Paroxetine is acknowledged as one of the selective serotonin reuptake inhibitors (SSRI), which is also considered as a potent GRK2 inhibitor with a high selectivity for GRK2. We found that paroxetine could alleviate symptoms of DSS-induced colitis in mice by regulating GPCR signaling to affect macrophage polarization. Taken together, the current results show that GRK2 may act as a novel therapeutic target in UC by regulating macrophage polarization, and paroxetine as a GRK2 inhibitor may have therapeutic effect on mice with DSS-induced colitis.

PHB2 promotes colorectal cancer cell proliferation and tumorigenesis through NDUFS1-mediated oxidative phosphorylation.

The alteration of cellular energy metabolism is a hallmark of colorectal cancer (CRC). Accumulating evidence has suggested oxidative phosphorylation (OXPHOS) is upregulated to meet the demand for energy in tumor initiation and development. However, the role of OXPHOS and its regulatory mechanism in CRC tumorigenesis and progression remain unclear. Here, we reveal that Prohibitin 2 (PHB2) expression is elevated in precancerous adenomas and CRC, which promotes cell proliferation and tumorigenesis of CRC. Additionally, knockdown of PHB2 significantly reduces mitochondrial OXPHOS levels in CRC cells. Meanwhile, NADH:ubiquinone oxidoreductase core subunit S1 (NDUFS1), as a PHB2 binding partner, is screened and identified by co-immunoprecipitation and mass spectrometry. Furthermore, PHB2 directly interacts with NDUFS1 and they co-localize in mitochondria, which facilitates NDUFS1 binding to NADH:ubiquinone oxidoreductase core subunit V1 (NDUFV1), regulating the activity of complex I. Consistently, partial inhibition of complex I activity also abrogates the increased cell proliferation induced by overexpression of PHB2 in normal human intestinal epithelial cells and CRC cells. Collectively, these results indicate that increased PHB2 directly interacts with NDUFS1 to stabilize mitochondrial complex I and enhance its activity, leading to upregulated OXPHOS levels, thereby promoting cell proliferation and tumorigenesis of CRC. Our findings provide a new perspective for understanding CRC energy metabolism, as well as novel intervention strategies for CRC therapeutics.

Long intergenic non-protein coding RNA 467 inhibition elevates microRNA-27b-3p to repress malignant behaviors of gastric cancer cells via reducing STAT3.

Emerging evidence indicated that long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) exert critical effects on tumorigenesis of multiple malignancies, including gastric cancer (GC). We aim to explore the effects of long intergenic non-protein coding RNA 467 (LINC00467) and miR-27b-3p on GC. GC cells were initially cultured. LINC00467, miR-27b-3p, and signal transducer and activator of transcription 3 (STAT3) expression in GC were detected. The altered LINC00467 and/or miR-27b-3p were transfected into screened cells. Then, the biological activities of GC cells and the tumor growth in vivo were examined. The binding relationships among LINC00467, miR-27b-3p, and STAT3 were confirmed. It was indicated that LINC00467 was increased while miR-27b-3p was decreased in GC tissues and cells. Inhibition of LINC00467 hindered GC cell malignancy and blocked tumor development by upregulating miR-27b-3p. LINC00467 sponged miR-27b-3p and STAT3 was targeted by miR-27b-3p. It was discovered that LINC00467 reduction upregulates miR-27b-3p to repress malignant GC cell growth via inhibiting STAT3. This research may deepen the insight of molecular mechanisms on GC.

LINC01436 Promotes the Progression of Gastric Cancer via Regulating miR-513a-5p/APE1 Axis.

BACKGROUND: Gastric cancer (GC) is one of the deadliest cancer worldwide. Multiple long non-coding RNAs (lncRNAs) are recently identified as crucial oncogenic factors or tumor suppressors in GC. In this study, we aimed to probe into the effect of LINC01436 on GC progression. METHODS: LINC01436 and miR-513a-5p expressions in GC tissue samples were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was used to detect the expression of apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) expression. Human GC cell lines AGS and BGC-823 were employed to investigate the function and mechanism of LINC01436 in GC. Cell counting kit-8 (CCK-8) assay was used to assess the effect of LINC01436 on proliferation. Flow cytometry was utilized to explore the effect of LINC01436 on apoptosis, and Transwell assay was conducted to detect the effect of LINC01436 on the migration and invasion. Colony formation assay was performed to evaluate the effect of LINC01436 on radioresistance of GC cells. Furthermore, luciferase reporter assay and RNA immunoprecipitation assay were conducted to confirm the binding relationship between miR-513a-5p and LINC01436. Additionally, Western blot was used to study the regulatory function of LINC01436 and miR-513a-5p on APE1. RESULTS: LINC01436 expression of GC clinical samples was remarkably increased and LINC01436 was correlated with unfavorable pathological indexes. LINC01436 high expression was associated with shorter overall survival time. Its overexpression observably promoted the proliferation, metastasis and radioresistance of GC cells, and its knockdown suppressed the malignant phenotypes of GC cells. LINC01436 overexpression markedly reduced the miR-513a-5p expression via sponging it and enhanced the APE1 expression. MiR-513a-5p overexpression or APE1 knockdown reversed the effects of LINC01436 on GC cells. CONCLUSION: LINC01436 is a molecular sponge of tumor suppressor miR-513a-5p, which indirectly enhances the APE1 expression and functions as the oncogenic lncRNA in GC.

The correlation between NEDD4L and HIF-1α levels as a gastric cancer prognostic marker.

NEDD4L (neural precursor cell expressed developmentally down-regulated 4-like) protein is a member of ubiquitin ligases Nedd4 family. Although studies have shown that Nedd4L may act as a tumor suppressor in various cancers, including gastric cancer (GC), its clinical significance and the diagnostic value in GC is not well defined. HIF-1α (hypoxia inducible factor family of transcription factors) is actively involved in the metabolism of many tumors, although the relationship between its expression levels and clinical significance in GC still need to be established. In this study, the level of HIF-1α and NEDD4L mRNA and protein in 25 freshly frozen GC- and matched normal-tissues were determined by western blot and quantitative PCR (qPCR). Additionally, immunohistochemistry assay was performed to measure the protein level of NEDD4L and HIF-1α in 124 GC and 25 normal control tissues. We observed that the NEDD4L mRNA and protein levels decreased significantly (P < 0.001) in GC tissues, while that of HIF-1α increased (P < 0.001), and they both were associated with a poor prognosis, as was the case in patients with lower NEDD4L and higher HIF-1α expression (P < 0.001). On correlation analysis, a significantly negative relationship (r = 0.288, P < 0.01) was revealed between NEDD4L and HIF-1α expressions. Multivariate analysis revealed that co-expression of NEDD4L (P < 0.05) and HIF-1α (P < 0.001) were independent predictors of GC prognosis. Thus, the correlation of NEDD4L and HIF-1α levels may act as a prognostic marker of GC.

Upregulation of KIF20A correlates with poor prognosis in gastric cancer.

BACKGROUND: KIF20A is well known as one of the key proteins in mitosis. Recently, a number of studies illustrated that KIF20A might function as an oncogene in some carcinomas. However, its expression levels and clinical value remained unclear in gastric cancer (GC). PATIENTS AND METHODS: In this study, we investigated the expression of KIF20A in samples from GC patients and cell lines by quantitative real-time PCR and Western blot. The function of KIF20A in cell proliferation of GC cell lines was examined via cell viability and colony formation assays. Immunohistochemistry assay based on a tissue microarray consisting of 146 cases was performed to evaluate the prognostic value of KIF20A. The overall survival rate of 122 GC patients based on KIF20A expression was analyzed as well. Finally, using KIF20A inhibitor, genistein, and combining it with cisplatin or fluorouracil, the antitumor effects were studied. RESULTS: Most GC samples (56.76%) showed higher KIF20A expression level compared to their corresponding normal specimens, which demonstrated the potential oncogenic role of KIF20A in GC. The functional studies elucidated the essential role of KIF20A in GC cell proliferation. Besides, tissue microarray result showed that the expression level of KIF20A was significantly related to the histological grades (P=0.036). Furthermore, we found the expression of KIF20A was related to poor overall survival rate, which is coincident with the results from Kaplan-Meier plotter database. In addition, we found that a KIF20A inhibitor, genistein, could enhance the antitumor activity of cisplatin and fluorouracil, which might be considered as a chemosensitive agent in GC. CONCLUSION: KIF20A can promote cell proliferation in GC, which might be used as an independent prognostic factor and a potential therapeutic target.

NDRG3 facilitates colorectal cancer metastasis through activating Src phosphorylation.

BACKGROUND: NDRG3 is an N-myc downregulated gene (NDRG). The aim of this article was to identify the role of NDRG3 in colorectal cancer (CRC) and to determine the mechanism underlying its function. METHODS: Using immunohistochemical staining, expression and clinicopathological variables of NDRG3 were analyzed in 170 CRC samples. Overexpression of NDRG3 was employed in SW1116 cells, downregulation of NDRG3 was achieved in RKO cells, then migration and invasion assays were performed in vitro, and a mouse model was constructed in vivo. RESULTS: Increased expression of NDRG3 was observed in primary CRC tissues, and this expression was correlated with distant metastasis. Consistently, ectopic expression of NDRG3 in SW1116 cells enhanced cell migration and invasion, while knockdown of NDRG3 in RKO cells significantly suppressed CRC cell metastasis. The portal vein injection models suggested that NDRG3 overexpression facilitates liver metastasis. These events were associated with the phosphorylation of Src (c-Src) at Tyr 419 site. CONCLUSION: Our results showed that NDRG3 facilitates CRC migration and invasion by activating Src phosphorylation, suggesting the role of NDRG3 as a candidate oncogene.

SEC24A stimulates oncogenicity of human gastric cancer cells.

Gastric cancer (GC) is a worldwide health concern and is the second common malignancy. Despite rapid progression in diagnostic and therapeutic approaches over the past decades, the molecular mechanisms underlying the development and progression of GC remain unclear. SEC24 homolog A, COPII coat complex component (SEC24A) belongs to a protein family that are homologous to yeast SEC24, and is critical for neural tube closure. Thus, we focus on the relation of SEC24A and human GC cells. In our study, we found that SEC24A was highly expressed in tumor tissue compared to non-tumor tissue. Furthermore, less aggressive behavior was observed in the si-SEC24A transfected human GC cells (SGC-7901 and BGC-823). On the other hand, we have also found that over-expression of miR-101-3p down-regulated the expression of SEC24A. SEC24A played a role in promoting invasion and metastasis of human GC cells.

Anti-NY-ESO-1 autoantibody may be a tumor marker for intrahepatic cholangiocarcinoma.

Anti-NY-ESO-1 antibody is observed in a multitude of malignancies. This study was aimed to evaluate the expression of serum anti-NY-ESO-1 antibodies and its prognostic value in intrahepatic cholangiocarcinoma. A total of 103 patients with intrahepatic cholangiocarcinoma were enrolled in the study. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the serum level of anti-NY-ESO-1 antibody. Western blotting was performed to assess the NY-ESO-1 expression in tumor and adjacent tissues. The serum NY-ESO-1 antibody was detected in 18.4% of patients with intrahepatic cholangiocarcinoma, a value that was significantly higher than that in patients with chronic Hepatitis B. Serum NY-ESO-1 antibody was positively correlated with tumor differentiation, lymphatic metastasis, cTNM stage and abdominal pain. Finally, there was a higher cumulative survival rate in patients with serum NY-ESO-1 positivity than in those with serum NY-ESO-1 negativity among the patients with stage III + IV. Our data uncovered that NY-ESO-1 antibody might be a helpful tumor marker and prognostic predictor in intrahepatic cholangiocarcinoma.

Down regulation of RNA binding motif, single-stranded interacting protein 3, along with up regulation of nuclear HIF1A correlates with poor prognosis in patients with gastric cancer.

Frequent loss of multiple regions in short arm of chromosome 3 is found in various tumors including gastric cancer (GC). RNA binding motif, single-stranded interacting protein 3 (RBMS3) is a tumor suppressor gene located in this region and mediates cancer angiogenesis. However, the role of RBMS3 in GC remains unclear.To evaluate whether RBMS3, together with HIF1A, another key regulator of angiogenesis, predicts GC prognosis, the levels of RBMS3 and HIF1A were first examined by quantitative PCR (qPCR) and western blot from 27 fresh frozen GC and paired normal gastric tissues and then tested by immunohistochemistry (IHC) from 191 GC and 46 normal controls. Moreover, uni- and multivariate analysis were employed to assess the correlations between their levels and microvessel density (MVD) and clinical prognosis. To further identify RBMS3 function in vitro, cell proliferation assay, clonogenic assay, flow cytometry analysis and endothelial cell tube formation assay were employed.We found that RBMS3 level was decreased, whereas HIF1A was elevated in GC. Furthermore, we demonstrated that RBMS3 was an independent prognostic factor and the levels of RBMS3 and HIF1A were associated with GC angiogenesis and histopathological differentiation: patients with lower RBMS3 level and higher nuclear HIF1A expression had poorer prognosis. Besides, gain- and loss-of-function study revealed RBMS3 regulation on G1/S progression, cell proliferation and the tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. These findings implicated that RBMS3 and nuclear HIF1A could act as prognostic biomarkers and therapeutic targets for GC.

[Expression of KIF18A in gastric cancer and its association with prognosis].

OBJECTIVE: To explore the expression of KIF18A gene protein in gastric cancer tissues and its association with the prognosis of patients. METHODS: Twenty fresh paired gastric cancer specimens and adjacent normal mucosa(at least 5 cm from the edge of tumor) from 20 gastric cancer patients undergoing operation in Department of General Surgery at the First Affiliated Hosptial of Anhui Medical University between March 2015 and July 2015 were collected. Real-time PCR was used to examine KIF18A mRNA expression in above specimens. Meanwhile, paraffin embedded cancer tissue samples from 129 gastric cancer patients undergoing operation and 23 samples of randomly selected normal gastric tissue(adjacent non-cancer tissue) were collected to establish the microarray. Immunohistochemistry method was applied to detect the KIF18A protein expression in the microarray after confirmation by pathologists. Association of KIF18A expression with clinicopathological features in gastric cancer patients was evaluated. Cox proportional hazard model was used to identify prognostic risk factors. RESULTS: Among 20 fresh paired gastric cancer specimens, mRNA expression of KIF18A in 16 specimens was obviously lower than that in adjacent normal tissues. The positive rate of KIF18A protein expression in gastric cancer tissues was significantly lower than that in normal gastric tissues in microarray[45.0%(58/129) vs. 69.6%(16/23), P=0.041]. KIF18A protein expression was significantly associated with invasion depth (P=0.008) and TNM staging (P=0.032). The median overall survival of all the 129 patients was 44.0(95% CI: 39.78-49.24) months. The three-year survival rates of patients with high and low KIF18A expression were 67.2% and 36.6% respectively(P=0.020). Cox regression analysis showed that KIF18A expression was an independent protective factor of the prognosis of gastric cancer patients (HR=0.570, 95% CI:0.335 to 0.970). CONCLUSIONS: KIF18A expression is down-regulated in gastric cancer tissue, which may play a critical role in gastric cancer carcinogenesis. Lower expression of KIF18A is associated with poor prognosis of gastric cancer patients. KIF18A may be a potential prognostic marker of gastric cancer.

Overexpression of eukaryotic elongation factor 1 alpha-2 is associated with poorer prognosis in patients with gastric cancer.

PURPOSE: Eukaryotic elongation factor 1 alpha-2 (eEF1A2) is a protein translation factor involved in protein synthesis. It is overexpressed in various cancers, which indicates potential vital functions in tumorigenesis and progression. Our study aims to investigate the expression levels of eEF1A2 in gastric cancer and its roles in clinical practice. METHODS: A total of 129 patients with pathologically confirmed gastric cancer and 24 normal controls were recruited for this study. The expression levels of eEF1A2 in gastric cancer and normal tissues were evaluated by tissue microarrays, quantitative real-time PCR, and western blot analysis. Kaplan-Meier analysis and Cox's proportional hazards model were used in survival analysis. RESULTS: Compared with corresponding controls, gastric cancer specimens had significantly increased expressions of eEF1A2 at mRNA and protein levels (both P < 0.05). Moreover, multivariate analysis confirmed that overexpression of eEF1A2 was a significant and independent indicator for predicting poor prognosis of gastric cancer. CONCLUSIONS: Our results showed for the first time that overexpression of eEF1A2 was correlated with worse outcomes in gastric cancer patients, suggesting its critical roles in the carcinogenesis of gastric cancer.

Down regulation of Thrombospondin2 predicts poor prognosis in patients with gastric cancer.

BACKGROUND: Thrombospondins (THBSs) are a family of multidomain and secreted matricellular Ca(2+)-binding glycoproteins which has at least five members encoded by independent genes. As a THBSs family member, Thrombospondin2 (THBS2) has been reported to regulate angiogenesis. Nevertheless, the functions and clinical significance of THBS2 still remains unclear in gastric cancer. METHODS: The mRNA and protein expression levels of THBS2 were assessed in 14 paired of gastric cancer specimens and corresponding normal mucosas using quantitative real-time PCR and western blot analysis. Immunohistochemistry of THBS2 and CD34 on population-based tissue microarrays consisting of 129 gastric cancer cases were used to evaluate the prognostic significance of THBS2 and microvessel density (MVD) of each sample. Survival analyses were performed by Kaplan-Meier method and Cox's proportional hazards model. Colony formation assay, endothelial cell tube formation assay, cell migration assay and apoptosis analysis in MKN-45 and SGC-7901 cell lines were carried out to evaluate the effects of THBS2 on gastric cancer in vitro. RESULTS: 85.71% (12 of 14) gastric cancer tissues expressed remarkably lower THBS2 in both mRNA and protein levels than the corresponding normal controls. Consistently, tissue microarray (TMA) results showed THBS2 levels were also inhibited in gastric cancer tissues compared with the normal controls. Moreover, we observed that patients with higher levels of THBS2 were significantly correlated with more favourable prognosis while decreased THBS2 expression were associated with poorer histological grades of gastric cancer. Additionally, our in vitro experiments further demonstrated that overexpression of THBS2 could impede both the proliferation rate and the tube formation of Human umbilical vein endothelial cells (HUVECs) in MKN-45 and SGC-7901 cell lines. CONCLUSION: Our study suggests THBS2 is aberrantly expressed in gastric cancer and plays a critical role in cancer progression, which can be a potential prognosis predictor of gastric cancer.

[Effects and mechanism of osthole on intestinal ischemia-reperfusion injury in mice].

OBJECTIVE: To explore the protective role of osthole in intestinal ischemia-reperfusion (I/R) injury in mice and examine its underlying mechanism. METHODS: A murine model of intestinal I/R injury was established with clamping of superior mesenteric artery for 120 min and then clamping was relieved for 60 min. Forty-five SD male mice weighing 27-31 g were randomly divided into 3 groups (n = 15 each): sham group (S), I/R injury group (I/R) and I/R plus osthole treatment group (Ost+). Intestinal wet/dry weight ratio, superoxide dismutase (SOD), malondialdehyde (MDA) in serum were examined by colorimetric assay and diamine oxidase (DAO) was examined by automatic biochemical analyzer, the levels of tumor necrosis factor (TNF) -α, interleukin (IL)-1ß and IL-2 were examined by enzyme-linked immunosorbent assay (ELISA). Intestinal barrier permeability was detected by Evans blue (EB) test. One-way ANOVA was used to analyze all experimental data variance. RESULTS: Intestinal tissues wet/dry weight ratios, Evans blue content and Chiu's score of I/R group mice were significantly higher than those of S and Ost+ groups (0.80% ± 0.03% vs 0.77% ± 0.02% & 0.78% ± 0.02%, (0.11 ± 0.04) vs (0.05 ± 0.02) & (0.06 ± 0.02) µg/mg, 3.42 ± 0.73 vs 0.87 ± 0.35 & 2.63 ± 0.58, P < 0.05 or P < 0.01) . Serum level of DAO, MDA, IL-1ß & TNF-α of I/R group mice were significantly higher than those of S and Ost+ groups ( (18.9 ± 4.0) vs (14.5 ± 2.3) & (16.0 ± 2.6) U/L, (8.4 ± 1.2) vs (6.9 ± 1.7) & (6.1 ± 2.4) µmol/L, (93 ± 6) vs (51 ± 4) & (67 ± 5) ng/L, (467 ± 31) vs (235 ± 21) & (323 ± 30) ng/L, P < 0.01 or P < 0.05) . Serum SOD activity and IL-2 level were significantly lower than those of S and Ost+ groups ( (75 ± 7) vs (93 ± 16) & (89 ± 5) U/ml, (95 ± 16) vs (198 ± 14) & (139 ± 11) ng/L, all P < 0.05) . All parameters showed no significant difference between S and Ost+ groups (all P > 0.05). CONCLUSIONS: Treatment of osthole may protect murine intestinal tissue against intestinal I/R injury. And the mechanisms may be due to its actions of preventing oxygen stress and inflammatory responses.