Identification of Hub Genes Associated with Gastric Cancer via Bioinformatics Analysis and Validation Studies.

Introduction: Hub genes related to the development of gastric cancer (GC) were identified based on bioinformatics methods. This study aimed to identify GC hub genes, explore the expression of genes in GC and their correlation with prognosis, so as to provide strategies for GC diagnosis and targeted therapy. Methods: Two messenger RNA (mRNA) microarray datasets were downloaded from GEO database. These data were combined with TCGA database to obtain common DEGs between GC tissues and normal tissues. GO and KEGG pathway enrichment analysis was performed. Visualized PPI network analysis was performed by Cytoscape to further identify hub genes. GEPIA database was used to evaluate the prognostic value of hub genes. The online software Ualcan was applied to analyze the expression of the prognosis-related genes in cancer tissues and normal tissues from different perspectives of primary GC, TNM stage, nodal metastasis status and tumor grade. Immunohistochemical staining of GC tissues and normal tissues was performed to evaluate the expression of signature genes in GC. Results: Eighty-four common differentially expressed genes (DEGs) in GC were identified. These genes were closely related to the P13K-Akt signal pathway and other signaling pathways. Ten hub genes were identified. Collagen type I alpha 1 (COL1A1) and collagen type IV alpha 1 (COL4A1) were significantly associated with poor prognosis of GC and were all positively correlated with T stage, distant metastasis, and TNM stage of GC. Immunohistochemistry revealed that the expression of these 2 genes was upregulated in GC tissues. These 2 genes expression was negatively related with 5-year survival rate of GC patients. Conclusion: Ten highly expressed hub genes in GC tissue were mined by bioinformatics method. COL1A1 and COL4A1 were significantly associated with the prognosis of GC. This study provided a theoretical basis for the pathogenesis, clinical diagnosis and therapeutic targets of GC.

hsa_circ_0092306 Targeting miR-197-3p Promotes Gastric Cancer Development by Regulating PRKCB in MKN-45 Cells.

Gastric cancer (GC) is one of the most common cancers worldwide and is thus a global cancer burden. Here, we focused on a novel circular RNA hsa_circ_0092306 and explored the potential molecular mechanism to provide a new target for and novel insights into GC treatment. The GEO microarray was mined and analyzed with R software. Sanger sequencing and RNase R assay were applied to verify the identification of hsa_circ_0092306. Quantitative real-time PCR and western blot were performed to measure the mRNA and protein levels. Pull-down and luciferase reporter assays were conducted to confirm the target relationships. Annexin V-PI apoptosis flow cytometry, 3-(4,5Dimethylthiazol- yl)-2,5Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), wound healing, and Transwell assays were applied to detect cell apoptosis, viability, migration, and invasion in MKN-45 cells, respectively. A xenograft in vivo experiment was conducted to confirm the cell experiment results. hsa_circ_0092306 was upregulated in GC tissues and GC cells, and promoted GC development in MKN-45 cells. hsa_circ_0092306 inhibited tumor suppressor miR-197-3p expression but promoted tumor promotor protein kinase C beta (PRKCB) expression in MKN-45 cells. hsa_circ_0092306 and PRKCB had a common target (miR-197-3p) and were negatively related to miR-197-3p expression. hsa_circ_0092306 promoted the development of GC by regulating the pathway of miR-197-3p/PRKCB in MKN-45 cells.

Effect of PLK1 inhibition on cisplatin-resistant gastric cancer cells.

OBJECTIVE: This study aims to investigate the effect of polo-like kinase 1 (PLK1) inhibition on cisplatin (DDP)-resistant gastric cancer (GC) cells. METHODS: The transcriptional level of PLK1 was measured by quantitative reverse-transcription polymerase chain reaction. Expressions of PLK1 and its downstream mediators as well as autophagy-related protein LC3 I/LC3 II were detected by western blot. An 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 5-ethynyl-2'-deoxyuridine immunofluorescent staining were conducted to evaluate the cell viability and replication activity separately. Flow cytometry was carried out to determine the cell cycle status. The GFP-LC3 vector contributed toward tracking the formation and aggregation of autophagosomes. RESULTS: Drug-resistant SGC-7901/DDP cells showed insignificant changes in all phases after DDP treatment, including DNA replication, cell proliferation, cell cycle, and apoptosis, whereas DDP could significantly improve the autophagy level of SGC-7901/DDP as well as PLK1expression. By downregulating the expression of PLK1, both BI2536 andsi-PLK1 enhanced SGC-7901/DDP sensitivity to DDP, suppressing the proliferation and autophagy as well as improving the apoptosis rate. PLK1 inhibition also resulted in the repression of cell division regulators CDC25C and cyclin B1. CONCLUSION: Together, our experimental results illustrated that the DDP resistance of GC cells might be associated with the aberrant overexpression of PLK1. PLK1 inhibition, including si-PLK1 and BI2536 treatment, could restore the chemosensitivity of drug-resistant SGC-7901/DDP cells and enhance the efficacy of DDP, revealing the potential value of PLK1 inhibition in GC chemotherapy.

Mn12Ac inhibits the migration, invasion and epithelial-mesenchymal transition of lung cancer cells by downregulating the Wnt/ß-catenin and PI3K/AKT signaling pathways.

Lung cancer is the leading cause of global cancer-associated mortality, therefore it is important to reveal the molecular mechanisms of lung cancer progression and to develop novel therapeutic targets. The results of the present study identified that manganese-12 acetate (Mn12Ac) was able to significantly inhibit the migration and invasion of A549 cells. Western blotting demonstrated that treatment with Mn12Ac was able to upregulate E-cadherin, and downregulate N-cadherin and vimentin. It was also identified by a quantitative polymerase chain reaction analysis that Mn12Ac was able to reduce the mRNA expression levels of EMT-associated transcription factors Snail, Slug, Twist-related protein 1 and zinc finger E-box-binding homeobox 1. It was also demonstrated that Mn12Ac was able to reduce the expression levels of Wnt and ß-catenin proteins, and suppress the phosphorylation of phosphoinositide 3-kinase (PI3K) and AKT in A549 cells. Notably, it was revealed that Mn12Ac was able to decrease the mRNA and protein expression levels of programmed death ligand-1. Taken together, the results suggested that Mn12Ac is able to inhibit cell migration, invasion and EMT in lung cancer cells by regulating the Wnt/ß-catenin and PI3K/AKT signaling pathways.

Prader-Willi region non-protein coding RNA 1 suppressed gastric cancer growth as a competing endogenous RNA of miR-425-5p.

Gastric cancer (GC) is one of the major global health problems, especially in Asia. Nowadays, long non-coding RNA (lncRNA) has gained significant attention in the current research climate such as carcinogenesis. This research desires to explore the mechanism of Prader-Willi region non-protein coding RNA 1 (PWRN1) on regulating GC process. Differentially expressed lncRNAs in GC tissues were screened out through microarray analysis. The RNA and protein expression level were detected by quantitative real-time PCR (qRT-PCR) and Western blot. Cell proliferation, apoptosis rate, metastasis abilities were respectively determined by cell counting kit 8 (CCK8), flow cytometry, wound healing, and transwell assay. The luciferase reporter system was used to verify the targetting relationships between PWRN1, miR-425-5p, and phosphatase and tensin homolog (PTEN). RNA-binding protein immunoprecipitation (RIP) assay was performed to prove whether PWRN1 acted as a competitive endogenous RNA (ceRNA) of miR-425-5p Tumor xenograft model and immunohistochemistry (IHC) were developed to study the influence of PWRN1 on tumor growth in vivo Microarray analysis determined that PWRN1 was differently expressed between GC tissues and adjacent tissues. qRT-PCR revealed PWRN1 low expression in GC tissues and cells. Up-regulated PWRN1 could reduce proliferation and metastasis and increase apoptosis in GC cells, while miR-425-5p had reverse effects. The RIP assay indicated that PWRN1 may target an oncogene, miR-425-5p The tumor xenograft assay found that up-regulated PWRN1 suppressed the tumor growth. The bioinformatics analysis, luciferase assay, and Western blot indicated that PWRN1 affected PTEN/Akt/MDM2/p53 axis via suppressing miR-425-5p Our findings suggested that PWRN1 functioned as a ceRNA targetting miR-425-5p and suppressed GC development via p53 signaling pathway.

Manganese-12 acetate suppresses the migration, invasion, and epithelial-mesenchymal transition by inhibiting Wnt/ß-catenin and PI3K/AKT signaling pathways in breast cancer cells.

BACKGROUND: Breast cancer is the leading cause of cancer-related death in the world, and it is of great value to reveal the molecular mechanisms of breast cancer progression and develop new therapeutic targets. METHODS: Transwell assay is used to analyze the migration and invasion of breast cancer cells. Real-time PCR and western blotting assay are applied to detect the expression levels of epithelial-mesenchymal transition markers and the key members of Wnt/ß-catenin and PI3K/AKT signaling pathways. RESULTS: Manganese-12 acetate (Mn12Ac) significantly inhibited the migration and invasion of MCF7 and MDA-MB-231 breast cancer cells. Western blotting assay further showed that Mn12Ac significantly upregulated E-cadherin, and downregulated N-cadherin and vimentin. We further found that Mn12Ac reduced the mRNA expressions of epithelial-mesenchymal transition-associated transcription factors snail, slug, twist1, and ZEB1 using real-time PCR assay. Importantly, we further found that Mn12Ac significantly reduced the Wnt1 and ß-catenin protein expressions, and suppressed the phosphorylation of PI3K and AKT in MCF7 and MDA-MB-231 breast cancer cells. Very interestingly, we also showed that Mn12Ac decreased the mRNA and protein expressions of programmed cell death ligand 1. CONCLUSION: Taken together, our results suggested that Mn12Ac inhibited the migration, invasion, and epithelial-mesenchymal transition by regulating Wnt/ß-catenin and PI3K/AKT signaling pathways in breast cancer.

Navicularines A-C: New diterpenoid alkaloids from Aconitum naviculare and their cytotoxic activities.

Three new bisditerpenoid alkaloids, navicularines A-C (1-3), and three known ones (4-6), were isolated from the ground parts of Aconitum naviculare. Their structures were elucidated by spectroscopic methods. All the new compounds were tested against five cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480). It was found that navicularine B exhibited certain cytotoxic activities in vitro, with IC50 values of 13.50, 18.52, 17.22, 11.18, and 16.36µM, respectively.

Contrasting effects of ethylene biosynthesis on induced plant resistance against a chewing and a piercing-sucking herbivore in rice.

Ethylene is a stress hormone with contrasting effects on herbivore resistance. However, it remains unknown whether these differences are plant- or herbivore-specific. We cloned a rice 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene, OsACS2, whose transcripts were rapidly up-regulated in response to mechanical wounding and infestation by two important pests: the striped stem borer (SSB) Chilo suppressalis and the brown planthopper (BPH) Nilaparvata lugens. Antisense expression of OsACS2 (as-acs) reduced elicited ethylene emission, SSB-elicited trypsin protease inhibitor (TrypPI) activity, SSB-induced volatile release, and SSB resistance. Exogenous application of ACC restored TrypPI activity and SSB resistance. In contrast to SSB, BPH infestation increased volatile emission in as-acs lines. Accordingly, BPH preferred to feed and oviposit on wild-type (WT) plants--an effect that could be attributed to two repellent volatiles, 2-heptanone and 2-heptanol, that were emitted in higher amounts by as-acs plants. BPH honeydew excretion was reduced and natural enemy attraction was enhanced in as-acs lines, resulting in higher overall resistance to BPH. These results demonstrate that ethylene signaling has contrasting, herbivore-specific effects on rice defense responses and resistance against a chewing and a piercing-sucking insect, and may mediate resistance trade-offs between herbivores of different feeding guilds in rice.

The 9-lipoxygenase Osr9-LOX1 interacts with the 13-lipoxygenase-mediated pathway to regulate resistance to chewing and piercing-sucking herbivores in rice.

Oxylipins produced by the 13-lipoxygenase (LOX) have been reported to play an important role in plant defense responses to herbivores. Yet, the role of oxylipins produced by the 9-LOX pathway in this process remains largely unknown. Here we cloned a gene encoding a chloroplast-localized 9-LOX, Osr9-LOX1, from rice. Transcriptional analysis revealed that herbivore infestation, mechanical wounding and jasmonic acid (JA) treatment either repressed or did not enhance the level of Osr9-LOX1 transcripts at early stages but did at later stages, whereas salicylic acid (SA) treatment quickly increased the transcript level of Osr9-LOX1. Antisense expression of Osr9-lox1 (as-r9lox1) decreased the amount of wound-induced (Z)-3-hexenal but increased levels of striped stem borer (SSB)-induced linolenic acid, JA, SA and trypsin protease inhibitors. These changes were associated with increased resistance in rice to the larvae of the SSB Chilo suppressalis. In contrast, although no significant differences were observed in the duration of the nymph stage or the number of eggs laid by female adults between the brown planthopper (BPH) Nilaparvata lugens that fed on as-r9lox1 lines and BPH that fed on wild-type (WT) rice plants, the survival rate of BPH nymphs that fed on as-r9lox1 lines was higher than that of nymphs that fed on WT plants, possibly because of a higher JA level. The results demonstrate that Osr9-LOX1 plays an important role in regulating an herbivore-induced JA burst and cross-talk between JA and SA, and in controlling resistance in rice to chewing and phloem-feeding herbivores.

An EAR-motif-containing ERF transcription factor affects herbivore-induced signaling, defense and resistance in rice.

Ethylene responsive factors (ERFs) are a large family of plant-specific transcription factors that are involved in the regulation of plant development and stress responses. However, little to nothing is known about their role in herbivore-induced defense. We discovered a nucleus-localized ERF gene in rice (Oryza sativa), OsERF3, that was rapidly up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis. Antisense and over-expression of OsERF3 revealed that it positively affects transcript levels of two mitogen-activated protein kinases (MAPKs) and two WRKY genes as well as concentrations of jasmonate (JA), salicylate (SA) and the activity of trypsin protease inhibitors (TrypPIs). OsERF3 was also found to mediate the resistance of rice to SSB. On the other hand, OsERF3 was slightly suppressed by the rice brown planthopper (BPH) Nilaparvata lugens (Stål) and increased susceptibility to this piercing sucking insect, possibly by suppressing H(2)O(2) biosynthesis. We propose that OsERF3 affects early components of herbivore-induced defense responses by suppressing MAPK repressors and modulating JA, SA, ethylene and H(2)O(2) pathways as well as plant resistance. Our results also illustrate that OsERF3 acts as a central switch that gears the plant's metabolism towards an appropriate response to chewing or piercing/sucking insects.