Expression Levels In Gastric Cancer Tissues Research Articles

Granzyme B Promotes Proliferation, Migration and EMT Process in Gastric Cancer.

Granzyme B (GZMB), a critical member of the Gr gene family, is known to play an essential role in diverse physiological and pathological processes such as inflammation, acute and chronic inflammatory diseases, and cancer progression. In this study, we delve deeper into the role of GZMB within the context of gastric cancer (GC) to examine its expression patterns and functional implications. To accomplish this, we applied a combination of quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry techniques. These methodologies allowed us to accurately gauge GZMB expression levels in GC tissues and investigate their correlation with various clinical-pathological variables. Our secondary focus was to discern the regulatory influence of GZMB on GC cell biology. We used an array of assays including cell counting kit-8 (CCK-8), colony formation, 5-ethynyl-2'-deoxyuridine, and migration assays. The effect of GZMB on gastric cancer progression was further validated through a subcutaneous xenograft mouse model. Our findings underscored that GZMB mRNA and protein levels were upregulated in GC tissues, a feature that showed a significant correlation with GC staging. We also discovered that a decrease in GZMB expression via knockdown experiments suppressed the proliferation and migration capabilities of GC cells. This effect was manifested through diminished expression levels of epithelial-mesenchymal transition (EMT) markers. In stark contrast, the overexpression of GZMB through plasmid transfection appeared to enhance the proliferation and migration abilities of GC cells. This was coupled with an upregulation in EMT expression. Our study concludes by emphasizing that GZMB promotes the growth, migration, and EMT processes in gastric cancer. In vitro, cell-based experiments and in vivo xenograft mouse models confirm this. Our findings provide a more comprehensive understanding of GZMB's role in gastric cancer pathogenesis, potentially opening doors for novel therapeutic strategies targeting this molecular pathway.

JAK3/STAT5 signaling-triggered upregulation of PIK3CD contributes to gastric carcinoma development.

Gastric cancer (GC) is one of the most common solid cancers with high incidence and mortality worldwide. Chronic gastritis and consequent inflammatory microenvironment is known as a major cause leading to gastric carcinogenesis. Here we report that PIK3CD that encodes p110δ, a catalytic subunit of the class IA PI3Ks, is overexpressed and tumorigenic in GC and associated with tumor inflammatory microenvironment. By investigating the data from TCGA database and our immunohistochemical staining and quantitative real-time PCR results from clinical samples, we found PIK3CD exhibits higher expression level in GC tissues compared with adjacent non-tumorous stomach tissues. Genetic silencing of PIK3CD in GC cells retards proliferation and migration in vitro and tumorigenicity and metastasis in vivo. In contrast, enhanced expression of PIK3CD promotes these phenotypes in vitro. Furthermore, pharmacological inhibition of PIK3CD could reduce GC cell viability and colony formation capacities. More importantly, we reveal a relevant mechanism that PIK3CD, but not PIK3CA and PIK3CB, is transcriptionally regulated by the pro-inflammatory IL2/JAK3/STAT5 axis and tumor-infiltrating immune cells such as lymphocytes. These observations may setup a new crosstalk between tumor inflammatory microenvironment, IL2/JAK3/STAT5 signaling and PI3K/AKT signaling. Targeting PIK3CD may be a promising therapy strategy for GC.

High expression of CPNE3 correlates with poor long-term prognosis of gastric cancer by inhibiting cell apoptosis via activating PI3K/AKT signaling

To explore the correlation of CPNE3 expression with long-term prognosis of patients with gastric cancer (GC) and the possible mechanism. We retrospectively collected the data of 104 GC patients undergoing radical surgery in our hospital from February, 2013 to October, 2017. TCGA database and immunohistochemistry were used to analyze CPNE3 expression level in GC tissues and its effects on tumor progression and long-term prognosis of the patients. GO analysis was performed to predict the biological role of CPNE3 in GC. We also conducted cell experiments with MGC803 cells and observed the effects of CPNE3 knockdown, CPNE3 overexpression and LY294002 (a PI3K/AKT inhibitor) treatment on cell apoptosis and cellular expressions of apoptotic proteins using flow cytometry and Western blotting. TCGA analysis and immunohistochemistry both showed high expressions of CPNE3 in GC (P < 0.05). The patients with high CPNE3 expressions had a reduced 5-year survival (P < 0.01), and a high CPNE3 expression, CEA level≥5 μg/L, CA19-9 level ≥37 kU/L, T3-T4 stage, and N2-N3 stage were all independent risk factors for a lowered 5-year survival rate after surgery. The sensitivity and specificity of CPNE3 for predicting 5-year mortality was 79.59% and 74.55%, respectively (P < 0.05). GO analysis predicted that CPNE3 negatively regulated GC cell apoptosis. In MGC803 cells, CPNE3 knockdown significantly increased cell apoptosis, enhanced Bax and Cleaved Caspase-3 expressions and decreased Bcl-2 expression, while CPNE3 overexpression produced the opposite results (P < 0.05). The cellular expressions of p-PI3K and p-AKT were significantly decreased following CPNE3 knockdown and increased following CPNE3 overexpression (P < 0.05). Treatment with LY294002 obviously attenuated the inhibitory effect of CPNE3 overexpression on apoptosis of MGC803 cells (P < 0.05). CPNE3 is highly expressed in GC tissues and affects the long-term prognosis of the patients possibly by inhibiting GC cell apoptosis through activation of PI3K/AKT signaling.

Correlation of mir-221-3p differential expression with clinical characteristics of gastric cancer patients.

Gastric cancer (GC) is one of the most common digestive malignancies. Although miR-221-3p was defined as a novel biomarker in many types of cancer, the relationship between its expression differences and the clinicopathological characteristics and prognosis of GC patients was yet to be fully understood. TCGA database was utilized to predict the potential biological function of miR-221-3p in GC. QRT-PCR and RNA FISH were performed to detect the expression levels of miR-221-3p in GC. The miR-221-3p expression levels in GC tissues and cells were significantly higher than those in paracancerous tissues (p < 0.001) and normal gastric mucosal cells (p < 0.05). Higher expression levels of miR-221-3p were associated with tumor diameter ≥ 4cm (χ2 = 5.519, p = 0.019), cTNM stage (III + IV) (χ2 = 28.013, p = 0.000), lymph node metastasis (χ2 = 23.272, p = 0.000) and distant metastasis (χ2 = 7.930, p = 0.005). Kaplan-Meier survival analysis showed a better prognosis for GC patients with miR-221-3p low expression(HR = 4.520, 95% CI: 1.844-11.075). miR-221-3p is highly expressed in GC tissues, which plays an important role in tumorigenesis, invasion and metastasis. miR-221-3p may become an important biomarker and potential molecular therapeutic target for patients with GC.

Regulation of the RB1 Gene through DNMT1 by SGI-1027 and its Impact on the Growth and Metastasis of Gastric Cancer Cells.

SGI-1027 is a recognized inhibitor of DNA methyltransferase 1 (DNMT1), and earlier investigations have indicated an inverse correlation between dysregulated DNMT1 expression in gastric cancer (GC) and retinoblastoma 1 (RB1) gene expression. Despite this knowledge, the precise mechanisms underlying the action of SGI-1027 in GC cells remain inadequately comprehended. The primary objective of this study is to elucidate the impact of SGI-1027 on the behavior of GC cells, encompassing aspects such as growth and metastatic potential, by intervening in DNMT1, thereby influencing the regulation of RB1 gene expression. The acquisition of the normal gastric mucosal cell line GES-1 and the human gastric cancer cell line MKN45 was followed by employing Western blot (WB) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) techniques to evaluate the expression levels of RB1 and DNMT1 in these two cell lines. Subsequently, the MKN45 cell line was cultured in medium containing varying concentrations of SGI-1027, and the impact of SGI-1027 on the regulation of RB1 and DNMT1 in GC cells was reassessed using WB and qRT-PCR techniques. To scrutinize the effect of SGI-1027 on GC cells, we utilized the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay to determine cell proliferation and performed Transwell experiments to assess cell migration and invasion capabilities. Throughout this process, we also employed WB to assess the levels of cell cycle-associated proteins (Cyclin D1, Cyclin E1, and Cyclin B1) and proteins related to apoptosis (BCL-2 associated protein X apoptosis regulator (BAX) and B-cell lymphoma 2 apoptosis regulator (BCL-2)). Furthermore, we injected the MKN45 cell line and MKN45 cell line cultured with the optimal concentration of SGI-1027 for 5 days and 10 days into mice subcutaneously and through the tail vein, dividing them into the Model group, Model+SGI-1027 5d group, and Model+SGI-1027 10d group. We monitored changes in tumor size and volume in mice, and tumor tissues as well as lung tissues were collected for hematoxylin and eosin (HE) staining. Finally, DNMT1 expression levels in GC tissues were detected using both WB and immunohistochemistry (IHC) techniques. Additionally, RB1 expression levels in GC tissues were assessed using WB. In contrast to GES-1 cells, MKN45 cells displayed a distinctive profile characterized by increased DNMT1 expression and decreased RB1 expression (p < 0.05). However, upon the introduction of SGI-1027, a notable decrease in DNMT1 levels within GC cells was observed, concomitant with an elevation in RB1 gene expression, with 25 μmol/L SGI-1027 identified as the optimal concentration (p < 0.05). Functional assays demonstrated that SGI-1027-treated GC cells exhibited pronounced features of inhibited proliferation, migration, and invasion when compared to untreated MKN45 cells (p < 0.05). Moreover, in SGI-1027-treated GC cells, the levels of Cyclin D1, Cyclin E1, Cyclin B1, and BCL-2 were significantly reduced, while the expression level of BAX increased (p < 0.05). Notably, the most pronounced impact was observed at 25 μmol/L SGI-1027, further underscoring its regulatory effects on tumor cell behavior (p < 0.05). In animal experiments, the Model group exhibited a substantial increase in tumor volume, with HE staining results indicating extensive necrosis in most gastric tissues and noticeable signs of lung metastasis, accompanied by increased DNMT1 expression and decreased RB1 gene expression. In contrast, the SGI-1027 group displayed a reduction in gastric tumor volume, decreased necrosis, and reduced lung tumor metastasis (p < 0.05). Additionally, the expression of DNMT1 was significantly reduced in SGI-1027-treated GC cells, while RB1 expression increased (p < 0.05), further confirming the inhibitory effects of SGI-1027 on tumor growth and metastasis. SGI-1027 effectively hinders the proliferation and dissemination of GC cells by downregulating DNMT1 and promoting the expression of RB1.

Asialoglycoprotein Receptor 2 Expression in Patients With Locally Advanced Gastric Cancer After Curative Resection.

The asialoglycoprotein receptor 2 gene (ASGR2) encodes a subunit of the asialoglycoprotein receptor, a transmembrane protein, which has recently been reported to be involved in gastric cancer (GC) progression. This study aimed to investigate the clinical significance of ASGR2 expression in GC tissues of patients with locally advanced gastric cancer (LAGC) after curative resection. ASGR2 expression was measured in GC tissues and adjacent normal gastric mucosa in 253 patients with pStage II/III GC who underwent curative resection, by using quantitative polymerase chain reaction. We compared the expression levels in GC tissues and adjacent normal stomach mucosa, and evaluated the relationship of its expression in GC tissues with clinicopathological factors and overall survival (OS). ASGR2 expression was significantly associated with lymph node metastasis and venous invasion. The high ASGR2-expression group demonstrated significantly lower survival than the low expression group (5-year survival 55.5% vs. 72.6%; p=0.009). Furthermore, in multivariate analysis, high ASGR2 expression was an independent factor for poor OS (hazard ratio=2.030; 95% confidence interval=1.318-3.127; p=0.001). ASGR2 expression in GC tissues may be a useful prognostic marker in patients with LAGC after curative resection.

Clinical Significance of Chitinase-3-like Protein 1 Gene Expression in Patients With Locally Advanced Gastric Cancer.

Chitinase-3-like protein 1 (CHI3L1), encoded by CHI3L1, is thought to be involved in growth, invasion, migration, and resistance to chemotherapy in cancer. This study aimed to investigate the clinical significance of CHI3L1 expression as a biomarker in gastric cancer (GC) tissues of patients with locally advanced GC after curative resection. Quantitative polymerase chain reaction (PCR) was used to determined CHI3L1 expression in GC tissues and adjacent normal gastric mucosa of 253 patients with pStage II/III GC who underwent curative resection. We compared the expression levels in GC tissues and adjacent normal gastric mucosa, and examined the relationship between expression in GC tissues and clinicopathological factors and overall survival (OS) in these patients. CHI3L1 expression was significantly associated with lymph-node metastasis and venous invasion. OS rate was significantly lower in the high- than in the low-CHI3L1 expression group (5-year survival 55.5% vs. 72.6%; p=0.009). Furthermore, in multivariate analysis, high CHI3L1 gene expression was an independent factor for poor OS (hazard ratio=2.030; 95% confidence interval=1.318-3.127; p=0.001). In patients with locally advanced GC after curative resection, expression of the CHI3L1 in GC tissue may be a useful prognostic marker.

Circ_0006089 Facilitates Gastric Cancer Progression via Decoying miR- 515-5p and Up-regulating CXCL6.

Gastric cancer (GC) is the most common cancer globally. Recent research has suggested that circular RNAs (circRNAs) play crucial roles in GC tumorigenesis and progression. The present study is performed to clarify the possible mechanism of circRNA has_circ_0006089 (circ_0006089) in GC. The differentially expressed circRNAs were screened out by analyzing the dataset GSE83- 521. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect circ_0006089, miR-515-5p and CXCL6 expression levels in GC tissues and cell lines. CCK-8, BrdU and Transwell assays were adopted to examine the biological function of circ_0006089 in GC cells. The interaction between miR-515-5p and circ_0006089, as well as between CXCL6 and miR-515-5p, was confirmed through bioinformatics, RNA immunoprecipitation (RIP) assay, dual-luciferase reporter gene assay and RNA pull-down assay. Circ_0006089 was significantly upregulated in GC tissues and cells, and miR-515-5p was remarkably downregulated. After knocking down circ_0006089 or overexpressing miR-515-5p, the growth, migration and invasion of GC cells were markedly reduced. In terms of mechanism, miR-515- 5p was verified to be the target of circ_0006089, and CXCL6 was validated as miR-515-5p's downstream target gene. Inhibiting miR-515-5p reversed the inhibitory effect knocking down circ_0006089 had on GC cell proliferation, migration and invasion. Circ_0006089 facilitates the malignant biological behaviors of GC cells via the miR-515- 5p/CXCL6 axis. Circ_0006089 can probably act as one of the important biomarkers and therapeutic targets in GC treatment strategies.

Over-Expression of ARID3B Suppresses Tumor Progression and Predicts Better Prognosis in Patients With Gastric Cancer.

ARID3B (AT-rich interaction domain 3B) has been demonstrated to be associated with the progression and patient prognosis of several human tumors. We conducted the present study to investigate the biological behavior and clinical relevance of ARID3B in gastric cancer (GC). Detection of the expression level in GC tissues and cell lines were performed by Western blot and immunohistochemistry. We also retrospectively analyzed the correlation of ARID3B with clinicopathological characteristics and patient prognosis in gastric cancer. The biological functions of ARID3B in GC cells were further explored by transwell migration assays, wound healing assays and cell proliferation assay. The present study suggested that the expression of ARID3B was significantly lower in GC tissues than in adjacent normal tissues. IHC staining in tissues of 406 GC patients from training and validation sets verified that ARID3B over-expression correlated with clinicopathological features, such as degree of differentiation and clinical stage. Meanwhile, ARID3B was proved to be an independent prognostic factor for GC prognosis. Furthermore, over-expression of ARID3B suppressed proliferation in GC cells according CCK8 assay. We found that over-expression of ARID3B inhibited GC cell migration by transwell assay and wound healing assay. Furthermore, EMT markers were detected in ARID3B over-expression GC cells, which showed that ARID3B may inhibit metastasis of GC cells. Our results firstly revealed that the expression level of ARID3B was closely correlated with clinicopathological features and may serve as an independent prognostic factor for GC patients. More importantly, ARID3B could suppress GC progression, including cell proliferation, migration and metastasis.

High CHAF1A Expression Levels Are Positively-Correlated with PD-L1 Expression and Indicate Poor Prognosis in Gastric Cancer.

Objective The aim of this study was to analyze the association between the expression of chromatin assembly factor 1 subunit A (CHAF1A) in gastric cancer (GC) and clinicopathological features, disease prognosis, and expression of programmed cell death-ligand 1 (PD-L1). Material and Methods. A total of 140 GC tissue specimens were collected between January 2013 and December 2017. CHAF1A expression in GC and paracancerous tissues was determined. Then, the associations between CHAF1A expression level in the collected tissues and clinicopathological features as well as PD-L1 expression level were investigated. Cox regression analyses were carried out to determine whether CHAF1A is an independent prognostic factor for GC. Finally, the association between CHAF1A expression levels and survival of the GC patients was investigated. Results A significantly higher level of CHAF1A expression in GC tissues was found compared to that in paracancerous tissues (p=0.042). CHAF1A expression level in GC tissues was found to be strongly associated with family history (p=0.005), smoking history (p=0.016), T stage (p=0.001), tumor marker AFP (p=0.017), tumor marker CEA (p=0.027), and PD-L1 expression (p=0.029). CHAF1A expression was also found to be positively correlated to PD-L1 expression (p=0.012). Moreover, high CHAF1A expression levels were found to lead to poor prognosis (p=0.019). Univariate and multivariate analyses all showed that CHAF1A was an independent poorer prognostic factor for gastric cancer (p=0.021, HR = 1.175, 95% CI: 1.090–2.890 for univariate analyses; p=0.014, HR = 2.191, 95% CI:1.170–4.105 for multivariate analyses). A high level of CHAF1A expression was thus found to be an independent risk factor for GC prognosis. Conclusion High CHAF1A expression is associated with poor GC prognosis and positively correlated to PD-L1 expression. Thus, CHAF1A expression level may be used as a novel biomarker for GC diagnosis.

Plasma thioredoxin reductase: a potential diagnostic biomarker for gastric cancer.

To improve the early detection of gastric cancer (GC), there is a growing need for novel and efficient biomarkers. We aimed to evaluate diagnostic value of thioredoxin reductase 1 (TXNRD1), which was found to be over expressed in various malignancies. We found that TXNRD1 has a higher expression level in GC tissues compared with adjacent normal tissues, and high TXNRD1 expression was significantly associated with poor outcomes of GC patients. Next, a total of 1446 cases were collected, with 896 cases in GC, 322 in benign gastric disease and 228 in healthy controls. We noticed plasma thioredoxin reductase (TrxR) level in GC [8.4 (7.1, 9.7) U/ml] was significantly higher than that in benign disease [6.1 (5.4, 7.2) U/ml] or healthy controls [3.7 (1.7, 5.6) U/ml]. Receiver operating characteristic analysis showed that the optimal cutoff value of TrxR activity for GC diagnosis was set at 5.75 U/ml with an area under the curve of 0.945. Moreover, a combined panel of TrxR and routine tumor markers could further elevate the diagnostic efficacy compared to a single biomarker. Finally, by measuring pre- and post-treatment TrxR activity and routine tumor markers, we found the change trend of them was broadly consistent, and plasma TrxR activity was significantly decreased in patients treated with platinum/fluorouracil-based therapy. Our findings recommend plasma TrxR activity combined with tumor markers as effective diagnostic tools for GC patients. As well, plasma TrxR has the potential to monitor therapeutic efficacy.

Circular RNA hsa_circ_0026344 suppresses gastric cancer cell proliferation, migration and invasion via the miR-590-5p/PDCD4 axis.

Circular RNA (CircRNA) is a class of non-coding RNA transcripts, with multiple pathophysiological functions. Instead, the mechanism and function of circRNA in gastric cancer (GC) are not fully deciphered. CircRNA_0026344 (circ_0026344), microRNA (miR)-590-5p and programmed cell death 4 (PDCD4) mRNA expression levels in GC tissues and cells were probed by quantitative real-time PCR. Cell viability, migration and aggressiveness were examined by cell counting kit-8 and transwell assays. Additionally, the interplay among circ_0026344, miR-590-5p and PDCD4 was verified with bioinformatics and dual-luciferase reporter gene assay. Western blot was conducted to probe PDCD4 protein expression. Circ_0026344 expression was underexpressed in GC tissues and cells, which was associated with clinicopathological characteristics such as tumour size, tumor-node-metastasis stage and lymph node metastasis. Circ_0026344 overexpression restrained the malignant biological behaviours of GC cells, while circ_0026344 knockdown functioned oppositely. Circ_0026344 could act as a competing endogenous RNA of miR-590-5p to negatively modulate its expression, and this miRNA could mitigate the impact of circ_0026344 on GC cells. In addition, PDCD4 was identified as the downstream target of miR-590-5p, and PDCD4 expression was positively modulated by circ_0026344. Circ_0026344 up-regulates PDCD4 expression via sponging miR-590-5p, thus inhibiting the progression of GC. This study further expounds the underlying molecular mechanism in the GC progression.

Hsa_circ_0001020 Serves as a Potential Biomarker for Gastric Cancer Screening and Prognosis.

Circular RNAs (circRNAs) are an intriguing class of RNAs with covalently closed-loop structures. With characteristics of high stability and disease-specific expression, circRNAs are emerging as ideal targets for cancer therapy. However, the screening utility and clinical value of circRNAs in gastric cancer (GC) remain largely elusive. We detected levels of hsa_circ_0001020 in cell lines and tissue and plasma samples and investigated its clinicopathological correlations. Kaplan-Meier survival curves and regression analyses were used to analyze its prognostic value. Receiver operating characteristic curves and biomarker combinations were examined to verify its screening value. Bioinformatics analysis was also performed to predict potential biological functions. Our tests found that hsa_circ_0001020 was significantly upregulated in GC cell lines, GC tissue samples, and even in plasma. High hsa_circ_0001020 expression levels in GC tissues were significantly associated with distal metastasis and blood carbohydrate antigen 19-9 (CA19-9). GC patients with high hsa_circ_0001020 had a lower overall survival and disease-free survival time than the low levels. Regression analysis suggested that the level of hsa_circ_0001020 expression was an independent prognostic factor for survival time. As a biomarker for GC, hsa_circ_0001020 showed a superior AUC, sensitivity, and specificity than carcinoembryonic antigen and CA19-9, and was suitable for combination with clinical tumor biomarkers. Bioinformatics analysis provided valuable clues for the possible oncogenic pathways of GC, such as the FoxO and p53 signaling pathways. In conclusion, our study found that hsa_circ_0001020 in GC could be a reliable biomarker to screen for GC and predict prognosis.

The stabilization of yes-associated protein by TGFβ-activated kinase 1 regulates the self-renewal and oncogenesis of gastric cancer stem cells.

Gastric cancer (GC) is the most frequent digestive system malignant tumour and the second most common cause of cancer death globally. Cancer stem cell (CSC) is a small percentage of cancer cells in solid tumours that have differentiation, self‐renewal and tumorigenic capabilities. They have an active participation in the initiation, development, metastasis, recurrence and resistance of tumours to chemotherapy and radiotherapy. Gastric cancer stem cells (GCSCs) have been shown to be correlated with GC initiation and metastasis. In this study, we found that TAK1 expression level in GC tissues was significantly increased compared to the adjacent non‐cancerous tissues by RT‐qPCR, Western blot and immunohistochemistry. TAK1 has been identified as a critical molecule that promoted a variety of malignant GC phenotypes both in vivo and in vitro and promoted the self‐renewal of GCSCs. Mechanistically, TAK1 was up‐regulated by IL‐6 and prevented the degradation of yes‐associated protein (YAP) in the cytoplasm by binding to YAP. Thus, TAK1 promoted the SOX2 and SOX9 transcription and the self‐renewal and oncogenesis of GCSCs. Our findings provide insights into the mechanism of self‐renewal and tumorigenesis of TAK1 in GCSCs and have broad implications for clinical therapies.

Proprotein Convertase Subtilisin/Kexin Type 9 Promotes Gastric Cancer Metastasis and Suppresses Apoptosis by Facilitating MAPK Signaling Pathway Through HSP70 Up-Regulation.

ObjectiveTo examine the effect of proprotein convertase subtilisin/kexin type 9 (PCSK9) on gastric cancer (GC) progression and prognosis, and to explore the underlying mechanism.MethodsPCSK9 expression levels in human GC tissues were determined by quantitative real-time PCR, western blotting, and immunohistochemical assay. PCSK9 serum levels were detected by enzyme-linked immunosorbent assay. The relationships of PCSK9 and GC progression and survival were analyzed using the Chi-square test, Kaplan-Meier analysis, and Cox proportional hazards model. The effect of PCSK9 on cell invasion, migration, and apoptosis were determined in human GC cell lines and mouse xenograft model separately using PCSK9 knockdown and overexpression strategies. The PCSK9 interacting molecules, screened by co-immunoprecipitation combined with LC-MS/MS, were identified by immunofluorescence localization and western blotting. Additionally, the mitogen-activated protein kinase (MAPK) pathway was assessed by western blotting.ResultsPCSK9 mRNA and protein levels were significantly elevated in GC tissues compared with the paired normal tissues at our medical center (P < 0.001). Notably, the up-regulation of PCSK9 expression in GC tissues was related to tumor progression and poor survival. GC patients had higher serum levels of PCSK9 than the age-matched healthy controls (P < 0.001); PCSK9 promoted invasive and migratory ability and inhibited apoptosis in GC cells with no apparent affection in cell proliferation. The silencing of PCSK9 reversed these effects, suppressing tumor metastasis in vitro and in vivo. Furthermore, PCSK9 maintained these functions through up-regulating heat shock protein 70 (HSP70), ultimately facilitating the mitogen-activated protein kinase (MAPK) pathway.ConclusionCollectively, our data revealed that high PCSK9 expression levels in GC tissue were correlated with GC progression and poor prognosis and that PCSK9 could promote GC metastasis and suppress apoptosis by facilitating MAPK signaling pathway through HSP70 up-regulation. PCSK9 may represent a novel potential therapeutic target in GC.

JHDM1D-AS1 aggravates the development of gastric cancer through miR-450a-2-3p-PRAF2 axis

AimsTo investigate the molecular function and mechanisms of JHDM1D antisense 1 (JHDM1D-AS1) during gastric cancer (GC) progression. Materials and methodsThe qPCR assay was used to detect the JHDM1D-AS1 and miR-450a-2-3p expression levels in GC tissues and cell lines. Bioinformatics analysis was used for exploring the lncRNA-microRNA-mRNA interaction network. We performed dual-luciferase reporter assay and qPCR assay in order to validate the direct interactions. We explored the JHDM1D-AS1 and miR-450a-2-3p on GC progression by using JHDM1D-AS1 siRNA and miR-450a-2-3p inhibitor; in vitro CCK-8 assay, colony formation assay, and invasion assay were conducted. Further, in vivo animal experiments were performed, and the expression levels of miR-450a-2-3p and PRAF2 in the tumor tissues were detected using qPCR and western blot analysis. Key findingsThe expression levels of JHDM1D-AS1 and miR-450a-2-3p in GC tissues and cell lines were higher and lower as compared to those in the corresponding normal controls, respectively. Moreover, high levels of JHDM1D-AS1 were closely related with metastasis and the GC TNM stage. Functionally, JHDM1D-AS1 depletion caused an obvious reduction in cell proliferation and invasion both in vitro and in vivo, while the addition of miR-450a-2-3p inhibitor could nullify these effects. Mechanically, JHDM1D-AS1 promoted GC progression via the sponging of miR-450a-2-3p in order to increase PRAF2 expression. SignificanceThe present results showed that the increased expression of JHDM1D-AS1 was closely associated with tumor progression of GC. JHDM1D-AS1/miR-450a-2-3p/PRAF2 axis may be a promising target for GC treatment.

Long Non-Coding RNA NCK1-AS1 Serves an Oncogenic Role in Gastric Cancer by Regulating miR-137/NUP43 Axis.

IntroductionLong non-coding RNA (lncRNA) NCK1-AS1 could regulate multiple cancer progression. However, little is known regarding the roles and acting mechanisms of NCK-AS1 in gastric cancer (GC) progression. This work was aimed to explore the relationship between NCK1-AS1 and GC progression to illustrate the mechanisms of NCK1-AS1.MethodsNCK1-AS1 expression level in GC tissues and cells was measured with a quantitative real-time PCR method. In vitro experiments including cell counting kit-8 assay, colony formation assay, wound-healing assay, and transwell invasion assay were employed to detect biological roles of NCK1-AS1 in GC progression. In vivo experiments were performed to analyze the roles of NCK1-AS1 on GC malignant phenotype. Moreover, mechanisms behind the biological roles of NCK1-AS1 in GC were investigated using bioinformatic analysis, luciferase activity reporter assay, RNA immunoprecipitation assay, and rescue experiments.ResultsNCK1-AS1 was found to have elevated expression in GC tissues and cells in comparison with normal counterparts. Loss-of-function experiments showed knockdown of NCK1-AS1 refrained GC cell proliferation, colony formation, migration, and invasion in vitro. Animal experiments showed silence of NCK1-AS1 suppresses tumor growth in vivo. Functionally, NCK1-AS1 serves as a sponge for microRNA-137 (miR-137) to upregulate nucleoporin 43 (NUP43) expression in GC. Rescue experiments proved the carcinogenic role of NCK1-AS1/miR-137/NUP43 axis in GC progression.DiscussionIn conclusion, the NCK1-AS1/miR-137/NUP43 axis was identified that could contribute to GC malignancy behaviors.

Inhibition of gastric cancer cell apoptosis by long noncoding RNA TRPM2-AS via mitogen-activated protein kinase and activators of transduction-3.

Long noncoding RNA TRPM2-AS has emerged as a novel regulator in cancer initiation and progression of various cancers. However, the function and underlying mechanism of TRPM2-AS in the progression of gastric cancer (GC) remain poorly understood. GEO and TCGA databases were used for isolation of differential lncRNA expression. TRPM2-AS expression levels in GC tissues and cells were measured by quantitative polymerase chain reaction method. TRPM2-AS subcellular location was detected by fluorescence in situ hybridization analysis. The functional roles of TRPM2-AS in cells were analyzed by loss and gain function assays. By using bioinformatics and quantitative polymerase chain reaction methods, TRPM2-AS expression levels were proved to be upregulated in GSE70880 dataset, TCGA database, and 26 GC tissues, which was partly induced by SP1. The results of clinical assays showed that TRPM2-AS could be an indicator for early-stage GC diagnosis. Fluorescence in situ hybridization analysis showed that TRPM2-AS was located in both nucleus and cytoplasm. Functional experiments displayed that knockdown of TRPM2-AS inhibited proliferation, migration, and invasion in GC cells. Furthermore, depression of TRPM2-AS suppressed cell growth though promotion of cell apoptosis. The expression levels of cleaved PARP, caspase 9, caspase 3, and Bax were significantly increased in BGC823 with TRPM2-AS knockdown. In addition, knockdown of TRPM2-AS reduced and phosphorylate signal transducer and activator of transcription 3 and increased and phosphorylate p38 mitogen-activated protein kinase. This study demonstrated that SP1-regulated TRPM2-AS is involved in GC cell apoptosis probably via p38 mitogen-activated protein kinase and signal transducer and activator of transcription 3 pathways, indicating that TRPM2-AS might be a potential therapeutic target in GC.

Identification and Expression of Several Circular RNAs and Knockdown of hsa_circ_0005556 Exerts Oncogenic Functions by miR-767-5p in Gastric Cancer.

BackgroundGastric cancer (GC) remains one of the most fatal digestive cancers in the world; nevertheless, its etiology remains vague. With the development of bioinformatics analysis, numerous circular RNAs (circRNAs) have been found to be dysregulated in GC. However, the functions of a large portion of dysregulated circRNAs in GC need further validation. In this study, we aimed to validate the biological functions of circ_0005556, which was previously identified to be dysregulated in GC.Material/MethodsLevels of circRNAs and miRNAs in GC tissues and cells were estimated by qRT-PCR. The target miRNAs of circ_0005556 were predicted by bioinformatics methods. The interplay between circ_0005556 and miR-767-5p was validated by dual-luciferase reporter and circRNA immunoprecipitation assays. The effects of circ_0005556 and miR-767-5p on GC cell viability, apoptosis, migration, and invasion were assessed by MTT, flow cytometry, wound-healing and in vitro transwell experiments, respectively.ResultsThe upregulation of circ_0005556 was validated by qRT-PCR in GC tissues and cells, and a higher circ_0005556 level indicated a poorer prognosis. miR-767-5p was demonstrated to target circ_0005556 in GC cells, and a negative correlation was found between their expression levels in GC tissues. Knockdown of circ_0005556 promoted miR-767-5p expression in GC cells. Knockdown of circ_0005556 was revealed to repress GC cell viability, invasion, and migration and to promote GC cell apoptosis. Moreover, overexpression of miR-767-5p could significantly augment the repressive impacts of circ_0005556 knockdown on GC cell progression in vitro.ConclusionsThe in vitro knockdown of circ_0005556 remarkably repressed GC cell progression by increasing the expression of miR-767-5p.

Integrative Analysis of Differential lncRNA/mRNA Expression Profiling in Helicobacter pylori Infection-Associated Gastric Carcinogenesis.

Helicobacter pylori (H. pylori) infection is the greatest known risk factor for gastric cancer (GC). Long non-coding RNAs (lncRNAs) are implicated in multiple biological processes. However, their contribution in H. pylori-associated GC remains largely unknown. We performed transcriptome sequencing to investigate differential lncRNA and mRNA expression profiles in gastric AGS cells infected with the H. pylori strain 7.13 or 43504. We identified significantly differentially expressed (SDE) mRNAs and lncRNAs following H. pylori infection. A co-expression network of lncRNAs and mRNAs was constructed via WGCNA analysis. Moreover, several of the most significantly upregulated genes were selected for further validation by qRT-PCR analysis in H. pylori-infected gastric cells and transgenic INS-GAS mice. We finally evaluated these genes in human GC tissues. A total of 158442 genes were identified between uninfected and infected cells. Of these, 298 mRNAs and 73 lncRNAs were consistently differentially expressed following infection with the H. pylori 7.13 and 43504 strains, respectively. The expression levels of most upregulated mRNAs (DDIT4, NDRG1, CHAC1, IL32, RELB, CTH, and SLC7A1) and lncRNAs (lncRNA36068, lncRNA51663, lncRNA49853, lncRNA49852, and FLJ46906) were validated by qRT-PCR analysis. We found that H. pylori infection significantly induced the transcript levels of the coding genes RELB and SLC7A11 in in vitro and in vivo assays, which was supported by their high expression levels in GC tissues. In addition, lncRNA51663 and FLJ46906 were remarkably increased in H. pylori-infected cells and consistently overexpressed in human GC tissues compared to adjacent normal tissues. Our study identified mRNA and lncRNA expression profiles related to H. pylori infection. These results may provide important insights regarding lncRNAs in H. pylori-induced gastric carcinogenesis.