Investigating the prognostic and predictive value of the type II cystatin genes in gastric cancer.

BACKGROUND: Accumulating evidence indicates that type II cystatin (CST) genes play a pivotal role in several tumor pathological processes, thereby affecting all stages of tumorigenesis and tumor development. However, the prognostic and predictive value of type II CST genes in GC has not yet been investigated. METHODS: The present study evaluated the expression and prognostic value of type II CST genes in GC by using The Cancer Genome Atlas (TCGA) database and the Kaplan-Meier plotter (KM plotter) online database. The type II CST genes related to the prognosis of GC were then screened out. We then validated the expression and prognostic value of these genes by immunohistochemistry. We also used Database for Annotation, Visualization, and Integrated Discovery (DAVID), Gene Multiple Association Network Integration Algorithm (GeneMANIA), Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), nomogram, genome-wide co-expression analysis, and other bioinformatics tools to analyze the value of type II CST genes in GC and the underlying mechanism. RESULTS: The data from the TCGA database and the KM plotter online database showed that high expression of CST2 and CST4 was associated with the overall survival (OS) of patients with GC. The immunohistochemical expression analysis showed that patients with high expression of CST4 in GC tissues have a shorter OS than those with low expression of CST4 (HR = 1.85,95%CI: 1.13-3.03, P = 0.015). Multivariate Cox regression analysis confirmed that the high expression level of CST4 was an independent prognostic risk factor for OS. CONCLUSIONS: Our findings suggest that CST4 could serve as a tumor marker that affects the prognosis of GC and could be considered as a potential therapeutic target for GC.

[Efficacy and safety of Regan Syrup in treatment of common cold (wind-heat syndrome):a multicenter, randomized, double-blind, double-dummy, placebo and positive drug-controlled, parallel, phase â ¡b clinical trial].

Regan Syrup has the effect of clearing heat, releasing exterior, benefiting pharynx and relieving cough, and previous phase Ⅱ clinical trial showed that the efficacy of Regan Syrup high-dose and low-dose groups was better than that of the placebo group, and there was no statistically significant difference in the safety between the three groups. The present study was conducted to further investigate the efficacy and safety of the recommended dose(20 mL) of Regan Syrup in the treatment of common cold(wind-heat syndrome). Patients who met the inclusion and exclusion criteria were selected and divided into the test group(Regan Syrup+Shufeng Jiedu Capsules placebo), positive drug group(Regan Syrup placebo+Shufeng Jiedu Capsules) and placebo group(Regan Syrup placebo+Shufeng Jiedu Capsules placebo) at a 1∶1∶1 using a block randomization method. The course of treatment was 3 days. A total of 119 subjects were included from six study centers, 39 in the test group, 40 in the positive drug group and 40 in the placebo group. The onset time of antipyretic effect was shorter in the test group than in the placebo group(P≤0.01) and the positive drug group, but the difference between the test group and the positive drug group was not significant. The test group was superior to the positive drug group in terms of fever resolution(P<0.05), and had a shorter onset time of fever resolution than the placebo group, but without obvious difference between the two groups. Compared to the positive drug group, the test group had shortened disappearance time of all symptoms(P≤0.000 1). In addition, the test group was better than the positive drug group and the placebo group in relieving symptoms of sore throat and fever(P<0.05), and in terms of clinical efficacy, the recovery rate of common cold(wind-heat syndrome) was improved in the test group compared to that in the placebo group(P<0.05). On the fourth day after treatment, the total TCM syndrome score in both test group and positive drug group was lower than that in the placebo group(P<0.05). There was no significant difference in the incidence of adverse events between three groups and none of them experienced any serious adverse events related to the study drug. The results indicated that Regan Syrup could shorten the onset time of antipyretic effect, reduce the time of fever resolution, alleviate the symptoms such as sore throat and fever caused by wind-heat cold, reduce the total score of Chinese medicine symptoms, and improve the clinical recovery rate with good safety.

[Effects of total ginsenosides from Panax ginseng stems and leaves on gut microbiota and short-chain fatty acids metabolism in acute lung injury mice].

This study aimed to investigate the biological effects and underlying mechanisms of the total ginsenosides from Panax ginseng stems and leaves on lipopolysaccharide(LPS)-induced acute lung injury(ALI) in mice. Sixty male C57BL/6J mice were randomly divided into a control group, a model group, the total ginsenosides from P. ginseng stems and leaves normal administration group(61.65 mg·kg~(-1)), and low-, medium-, and high-dose total ginsenosides from P. ginseng stems and leaves groups(15.412 5, 30.825, and 61.65 mg·kg~(-1)). Mice were administered for seven continuous days before modeling. Twenty-four hours after modeling, mice were sacrificed to obtain lung tissues and calculate lung wet/dry ratio. The number of inflammatory cells in bronchoalveolar lavage fluid(BALF) was detected. The levels of interleukin-1ß(IL-1ß), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in BALF were detected. The mRNA expression levels of IL-1ß, IL-6, and TNF-α, and the levels of myeloperoxidase(MPO), glutathione peroxidase(GSH-Px), superoxide dismutase(SOD), and malondialdehyde(MDA) in lung tissues were determined. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in lung tissues. The gut microbiota was detected by 16S rRNA sequencing, and gas chromatography-mass spectrometry(GC-MS) was applied to detect the content of short-chain fatty acids(SCFAs) in se-rum. The results showed that the total ginsenosides from P. ginseng stems and leaves could reduce lung index, lung wet/dry ratio, and lung damage in LPS-induced ALI mice, decrease the number of inflammatory cells and levels of inflammatory factors in BALF, inhibit the mRNA expression levels of inflammatory factors and levels of MPO and MDA in lung tissues, and potentiate the activity of GSH-Px and SOD in lung tissues. Furthermore, they could also reverse the gut microbiota disorder, restore the diversity of gut microbiota, increase the relative abundance of Lachnospiraceae and Muribaculaceae, decrease the relative abundance of Prevotellaceae, and enhance the content of SCFAs(acetic acid, propionic acid, and butyric acid) in serum. This study suggested that the total ginsenosides from P. ginseng stems and leaves could improve lung edema, inflammatory response, and oxidative stress in ALI mice by regulating gut microbiota and SCFAs metabolism.

ITGA5 Promotes Tumor Progression through the Activation of the FAK/AKT Signaling Pathway in Human Gastric Cancer.

Background: ITGA5 is an adhesion molecule that integrates the intracellular structures with the extracellular matrix to perform biological functions. However, ITGA5 is highly expressed in a variety of tumors and is involved in tumor progression by promoting cell proliferation and metastasis. Nevertheless, little research has been performed on its function in gastric cancer. Therefore, the aim of this study was to investigate the role of ITGA5 in gastric cancer, focusing on the mechanism regulating the proliferation, invasion and migration. Methods: The expression of ITGA5 in gastric cancer tissues was assessed by the use of molecular bioinformatics databases and high-throughput sequencing of gastric cancer tissues from patients. Western blot, qPCR, and immunohistochemistry were performed to detect the expression of ITGA5 in samples from gastric cancer patients and gastric cancer cell lines. Furthermore, the ITGA5 gene was silenced and overexpressed in gastric cancer cells, and the effect on proliferation, invasion, migration, and tumorigenic ability was assessed. Results: ITGA5 mRNA and protein expression were upregulated in gastric cancer cell lines and tissues from patients, and its expression was closely associated with tumor size, lymph node metastasis, and TNM stage. In vitro and in vivo experiments showed that ITGA5 silencing resulted in the inhibition of proliferation, invasion, migration, and graft growth of gastric cancer cells; conversely, the overexpression resulted in the promotion of these cell functions. Our results finally showed that the effect of ITGA5 on proliferation, invasion, and migration of gastric cancer cells was performed through the activation of the FAK/AKT pathway. Conclusions: ITGA5 promotes proliferation, invasion, and migration of gastric cancer cells through the activation of FAK/AKT signaling pathway, suggesting that ITGA5 may be potentially considered as a new target in gastric cancer therapy.

NKCC1 promotes proliferation, invasion and migration in human gastric cancer cells via activation of the MAPK-JNK/EMT signaling pathway.

Aims: This study aimed to explore the function of NKCC1 in the proliferation, migration and invasion of Gastric cancer (GC) cells. Materials and Methods: GC data extracted from the database was analyzed using molecular bioinformatics. The expression levels of NKCC1 in tissue samples from GC patients and GC cell lines were determined by Western blotting, qRT-PCR, and immunohistochemistry. Immunofluorescence was used to detect protein localization. The GC cell lines were transfected with NKCC1-shRNA or expression plasmid, and in vitro proliferation, invasion and migration were analyzed by the CCK8, wound healing and transwell tests. Results: The NKCC1 mRNA level was significantly increased in GC tissues than that in normal gastric tissues (P = 0.0195). This phenomenon was further confirmed by the analysis of the TCGA-GTEx database that includes 408 gastric cancer tissues and 211 normal gastric tissues (P < 0.01). Furthermore, the increased level of NKCC1 was significantly correlated with Tumor size (P = 0.039), lymphatic node metastasis (P = 0.035) and tumor stage (P = 0.034). In vitro experiments confirmed that NKCC1 expression was higher in GC cells compared to that in GES-1 cells, and was mainly localized to the cytoplasm and membrane. NKCC1 silencing inhibited GC cell proliferation, invasion, migration and EMT, whereas its overexpression had the opposite effects. Furthermore, NKCC1 overexpression upregulated and activated JNK, and the targeted inhibition of JNK by SP600125 abrogated the pro-metastatic effects of NKCC1. Conclusions: NKCC1 promotes migration and invasion of GC cells by MAPK-JNK/EMT pathway and can be a potential therapeutic target.

Expression and Prognostic Analysis of Integrins in Gastric Cancer.

BACKGROUND: Integrins are involved in the biological process of a variety of cancers, but their importance in the diagnosis and prognosis of gastric cancer (GC) is still unclear. Therefore, this study aimed at exploring the significance of ITG gene expression in GC to evaluate its diagnosis and prognosis. METHODS: GEPIA data were used to evaluate the mRNA expression of ITG genes in GC patients. The prognostic value of these genes was assessed by analyzing their mRNA expression using the Kaplan-Meier curve. The biological function of ITG genes was evaluated by GC tissue sequencing combined with GSEA bioinformatics. Based on the sequencing data, ITGA5 with the largest expression difference was selected for verification, and RT-PCR was used to verify its mRNA expression level in 40 pairs of GC and normal tissues. RESULTS: ITG (A2, A3, A4, A5, A6, A11, AE, AL, AM, AV, AX, B1, B2, B4, B5, B6, and B8) was highly expressed in GC tissues, while ITGA8 was low, compared with their expression in normal tissues. RNA-seq data shows that ITG (A2, A5, A11, AV, and B1) expression was associated with poor prognosis and overall survival. In addition, combined with the results of GC tissue mRNA sequencing, it was further found that the differentially expressed genes in the ITGs genes. ITGA5 was highly expressed in GC tissues compared with its expression in normal tissues, as evaluated by qRT-PCR (P < 0.001) and ROC (P < 0.001, AUC (95% CI) = 0.747 (0.641-0.851)), and confirmed that ITGA5 expression was a potential diagnostic marker for GC. Bioinformatics analysis revealed that the signaling pathway involved in ITGA5 was mainly enriched in focal adhesion, ECM-receptor interaction, and PI3K-AKT and was mainly involved in biological processes such as cell adhesion, extracellular matrix, and cell migration. CONCLUSION: This study suggested that ITGs were associated with the diagnosis and prognosis of GC and discovered the prognostic value and biological role of ITGA5 in GC. Thus, ITGA5 might be used as a potential diagnostic marker for GC.

CLIC1 Promotes the Progression of Gastric Cancer by Regulating the MAPK/AKT Pathways.

BACKGROUND/AIMS: Chloride intracellular channel 1 (CLIC1), which is a member of the chloride channel protein family, is associated with various human tumors. Recent studies have shown that CLIC1 is involved in the occurrence and development of gastric cancer (GC). However, the exact mechanism remains unclear in GC. METHODS: Effects of CLIC1 on the progression of GC in vivo and in vitro and the potential underlying mechanisms have been investigated by analysing 54 patients with GC, as well as human gastric cell lines SGC-7901 and MGC-803, utilizing proteomics, RT-PCR, Western blotting, flow cytometry, Cell invasion and migration assays and xenograft tumor models. RESULTS: Our study shows that CLIC1 knockdown by targeted-siRNA markedly inhibits GC cell invasion and migration and induces apoptosis in vitro. In total, 54 differentially expressed proteins were identified in GC cells SGC-7901 after CLIC1 silencing by isobaric tags for relative isotope labeled and absolute quantitation (iTRAQ) technology, including integrin α1 (ITGα1) and ITGα3. The expression levels of ITGα3, ITGαv, ITGß1 and Bcl-2 mRNA and protein were decreased significantly in GC cells after CLIC1 knockdown; ITGα1 and Fas were upregulated, but the level of survivin was not significantly different. GC growth and metabolism were decreased in vivo after CLIC1 silencing, but apoptosis was markedly increased. Further study showed that the expression levels of ITGα3, ITGαv and ITGß1, as well as AKT-phosphorylation, ERK-phosphorylation and p38-phosphorylation, were reduced in vivo after CLIC1 knockdown, while ITGα1 was upregulated. CONCLUSIONS: We speculate that CLIC1 may play an important role in the progression of GC, and its mechanism may be related to the regulation of integrin family proteins, which leads to the sequential regulation of the PI3K/AKT, MAPK/ERK and MAPK/p38 pathways.

Effects of siRNA-mediated silencing of myeloid cell leukelia-1 on the biological behaviors and drug resistance of gastric cancer cells.

OBJECTIVE: This study was to investigate the effects of siRNA mediated silencing of myeloid cell leukelia-1 (Mcl-1) on the biological behaviors and drug resistance of human drug-resistant gastric cancer (GC) cell lines, and to explore the potential mechanisms. METHODS: siRNA targeting Mcl-1 mRNA were designed and independently transfected into SGC-7901/VCR and SGC-7901/DDP. Cell proliferation and drug sensitivity were examined by MTT assay. Cell apoptosis and cell cycle were detected by flow cytometry. Cell Invasion and migration abilities were detected by transwell chamber assays. The expressions of drug-resistance-related genes and apoptosis-related proteins were detected by quantitative real-time PCR and Western blot assay, respectively. RESULTS: siRNA effectively inhibited the Mcl-1 expression, lowered the proliferation rate (P<0.05), raised the apoptosis rate (P<0.05), and arrested cells in S-phase (P<0.05). After inhibiting Mcl-1, the cell migration and invasion decreased (P<0.05), the resistance to VCR, DDP and 5-Fu was reversed to different extents (P<0.05), TS mRNA expression increased significantly (P<0.05), MDR1 remained unchanged (P>0.05), but DPD and TOP2A decreased significantly (P<0.05). Following Mcl-1 silencing, Bcl-2 was over-expressed in VCR-siRNA group, but the expressions of Fas and survivin reduced markedly (P<0.05); Bcl-2 and Fas expressions decreased significantly in DDP-siRNA group (P<0.05), but survivin expression remained unchanged. CONCLUSION: Mcl-1 is implicated in the proliferation, invasion, apoptosis and drug resistance of GC cells, and may be a promising target for the therapy of GC.